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Effect of mitochondrial stress on epigenetic modifiers

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Early Mitochondrial Stress Alters Epigenetics, Secures Lifelong Health Benefits

GEN 5/3/2016  http://www.genengnews.com/gen-news-highlights/early-mitochondrial-stress-alters-epigenetics-secures-lifelong-health-benefits/81252685/

A little adversity builds character, or so the saying goes. True or not, the saying does seem an apt description of a developmental phenomenon that shapes gene expression. While it knows nothing of character, the gene expression apparatus appears to respond well to short-term mitochondrial stress that occurs early in development. In fact, transient stress seems to result in lasting benefits. These benefits, which include improved metabolic function and increased longevity, have been observed in both worms and mice, and may even occur—or be made to occur—in humans.

Gene expression is known to be subject to reprogramming by epigenetic modifiers, but such modifiers generally affect metabolism or lifespan, not both. A new set of epigenetic modifiers, however, has been found to trigger changes that do just that—both improve metabolism and extend lifespan.

Scientists based at the University of California, Berkeley, and the École Polytechnique Fédérale de Lausanne (EPFL) have discovered enzymes that are ramped up after mild stress during early development and continue to affect the expression of genes throughout the animal’s life. When the scientists looked at strains of inbred mice that have radically different lifespans, those with the longest lifespans had significantly higher expression of these enzymes than did the short-lived mice.

“Two of the enzymes we discovered are highly, highly correlated with lifespan; it is the biggest genetic correlation that has ever been found for lifespan in mice, and they’re both naturally occurring variants,” said Andrew Dillin, a UC Berkeley professor of molecular and cell biology. “Based on what we see in worms, boosting these enzymes could reprogram your metabolism to create better health, with a possible side effect of altering lifespan.”

Details of the work, which appeared online April 29 in the journal Cell, are presented in a pair of papers. One paper (“Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity”) resulted from an effort led by Dillin and the EPFL’s Johan Auwerx. The other paper (“Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt”) resulted from an effort led by Dillin and his UC Berkeley colleague Barbara Meyer.

According to these papers, mitochondrial stress activates enzymes in the brain that affect DNA folding, exposing a segment of DNA that contains the 1500 genes involved in the work of the mitochondria. A second set of enzymes then tags these genes, affecting their activation for much or all of the lifetime of the animal and causing permanent changes in how the mitochondria generates energy.

The first set of enzymes—methylases, in particular LIN-65—add methyl groups to the DNA, which can silence promoters and thus suppress gene expression. By also opening up the mitochondrial genes, these methylases set the stage for the second set of enzymes—demethylases, in this case jmjd-1.2 and jmjd-3.1—to ramp up transcription of the mitochondrial genes. When the researchers artificially increased production of the demethylases in worms, all the worms lived longer, a result identical to what is observed after mitochondrial stress.

“By changing the epigenetic state, these enzymes are able to switch genes on and off,” Dillin noted. This happens only in the brain of the worm, however, in areas that sense hunger or satiety. “These genes are expressed in neurons that are sensing the nutritional status of the animal, and these signals emanate out to the periphery to change peripheral metabolism,” he continued.

When the scientists profiled enzymes in short- and long-lived mice, they found upregulation of these genes in the brains of long-lived mice, but not in other tissues or in the brains of short-lived mice. “These genes are expressed in the hypothalamus, exactly where, when you eat, the signals are generated that tell you that you are full. And when you are hungry, signals in that region tell you to go and eat,” Dillin explained said. “These genes are all involved in peripheral feedback.”

Among the mitochondrial genes activated by these enzymes are those involved in the body’s response to proteins that unfold, which is a sign of stress. Increased activity of the proteins that refold other proteins is another hallmark of longer life.

These observations suggest that the reversal of aging by epigenetic enzymes could also take place in humans.

“It seems that, while extreme metabolic stress can lead to problems later in life, mild stress early in development says to the body, ‘Whoa, things are a little bit off-kilter here, let’s try to repair this and make it better.’ These epigenetic switches keep this up for the rest of the animal’s life,” Dillin stated.

 

Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity

Carsten Merkwirth6, Virginija Jovaisaite6, Jenni Durieux,…., Reuben J. Shaw, Johan Auwerx, Andrew Dillin

Highlights
  • H3K27 demethylases jmjd-1.2 and jmjd-3.1 are required for ETC-mediated longevity
  • jmjd-1.2 and jmjd-3.1 extend lifespan and are sufficient for UPRmt activation
  • UPRmt is required for increased lifespan due to jmjd-1.2 or jmjd-3.1 overexpression
  • JMJD expression is correlated with UPRmt and murine lifespan in inbred BXD lines

Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPRmt), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPRmt signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPRmt induction, while gain of function is sufficient to extend lifespan in a UPRmt-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPRmt signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations.

Figure thumbnail fx1

 

Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt
Ye Tian, Gilberto Garcia, Qian Bian, Kristan K. Steffen, Larry Joe, Suzanne Wolff, Barbara J. Meyer, Andrew Dillincorrespondence
http://dx.doi.org/10.1016/j.cell.2016.04.011             Publication stage: In Press Corrected Proof
Highlights
  • LIN-65 accumulates in the nucleus in response to mitochondrial stress
  • Mitochondrial stress-induced chromatin changes depend on MET-2 and LIN-65
  • LIN-65 and DVE-1 exhibit interdependence in nuclear accumulation
  • met-2 and atfs-1 act in parallel to affect mitochondrial stress-induced longevity

Organisms respond to mitochondrial stress through the upregulation of an array of protective genes, often perpetuating an early response to metabolic dysfunction across a lifetime. We find that mitochondrial stress causes widespread changes in chromatin structure through histone H3K9 di-methylation marks traditionally associated with gene silencing. Mitochondrial stress response activation requires the di-methylation of histone H3K9 through the activity of the histone methyltransferase met-2 and the nuclear co-factor lin-65. While globally the chromatin becomes silenced by these marks, remaining portions of the chromatin open up, at which point the binding of canonical stress responsive factors such as DVE-1 occurs. Thus, a metabolic stress response is established and propagated into adulthood of animals through specific epigenetic modifications that allow for selective gene expression and lifespan extension

 Siddharta Mukherjee’s Writing Career Just Got Dealt a Sucker Punch
Author: Theral Timpson

Siddharha Mukherjee won the 2011 Pulitzer Prize in non-fiction for his book, The Emperor of All Maladies.  The book has received widespread acclaim among lay audience, physicians, and scientists alike.  Last year the book was turned into a special PBS series.  But, according to a slew of scientists, we should all be skeptical of his next book scheduled to hit book shelves this month, The Gene, An Intimate History.

Publishing an article on epigenetics in the New Yorker this week–perhaps a selection from his new book–Mukherjee has waltzed into one of the most active scientific debates in all of biology: that of gene regulation, or epigenetics.

Jerry Coyne, the evolutionary biologist known for keeping journalists honest, has published a two part critique of Mukherjee’s New Yorker piece.  The first part–wildly tweeted yesterday–is a list of quotes from Coyne’s colleagues and those who have written in to the New Yorker, including two Nobel prize winners, Wally Gilbert and Sidney Altman, offering some very unfriendly sentences.

Wally Gilbert: “The New Yorker article is so wildly wrong that it defies rational analysis.”

Sidney Altman:  “I am not aware that there is such a thing as an epigenetic code.  It is unfortunate to inflict this article, without proper scientific review, on the audience of the New Yorker.”

The second part is a thorough scientific rebuttal of the Mukherjee piece.  It all serves as a great drama about one of the most contested ideas in biology and also as a cautionary tale to journalists, even experienced writers such as Mukherjee, about the dangers of wading into scientific arguments.  Readers may remember that a few years ago, science writer, David Dobbs, similarly skated into the same topic with his piece, Die, Selfish Gene, Die, and which raised a similar shitstorm, much of it from Coyne.

Mukherjee’s mistake is in giving credence to only one side of a very fierce debate–that the environment causes changes in the genome which can be passed on; another kind of evolution–as though it were settled science.   Either Mukherjee, a physicisan coming off from a successful book and PBS miniseries on cancer, is setting himself up as a scientist, or he has been a truly naive science reporter.   If he got this chapter so wrong, what does it mean about an entire book on the gene?

Coyne quotes one of his colleagues who raised some questions about the New Yorker’s science reporting, one particular question we’ve been asking here at Mendelspod.  How do we know what we know?  Does science now have an edge on any other discipline for being able to create knowledge?

Coyne’s colleague is troubled by science coverage in the New Yorker, and goes so far as to write that the New Yorker has been waging a “war on behalf of cultural critics and literary intellectuals against scientists and technologists.”

From my experience, it’s not quite that tidy.  First of all, the New Yorker is the best writing I read each week.  Period.  Second, I haven’t found their science writing to have the slant claimed in the quote above.  For example, most other mainstream outlets–including the New York Times with the Amy Harmon pieces–have given the anti-GMO crowd an equal say in the mistaken search for a “balance” on whether GMOs are harmful.  (Remember John Stewart’s criticism of Fox News?  That they give a false equivalent between two sides even when there is no equivalent on the other side?)

But the New Yorker has not fallen into this trap on GMOs and most of their pieces on the topic–mainly by Michael Specter–have been decidedly pro science and therefore decided pro GMO.

So what led Mukherjee to play scientist as well as journalist?  There’s no question about whether I enjoy his prose.  His writing beautifully whisks me away so that I don’t feel that I’m really working to understand.  There is a poetic complexity that constantly brings different threads effortlessly together, weaving them into the same light.  At one point he uses the metaphor of a web for the genome, with the epigenome being the stuff that sticks to the web.  He borrows the metaphor from the Hindu notion of “being”, or jaal.

“Genes form the threads of the web; the detritus that adheres to it transforms every web into a singular being.”

There have been a few writers on Twitter defending Mukherjee’s piece.  Tech Review’s Antonio Regalado called Coyne and his colleagues “tedious literalists” who have an “issue with epigenetic poetry.”

At his best, Mukherjee can take us down the sweet alleys of his metaphors and family stories with a new curiosity for the scientific truth.  He can hold a mirror up to scientists, or put the spotlight on their work.   At their worst, Coyne and his scientific colleagues can reek of a fear of language and therefore metaphor.  The always outspoken scientist and author, Richard Dawkins, who made his name by personifying the gene, was quick to personify epigentics in a tweet:   “It’s high time the 15 minutes of underserved fame for “epigenetics” came to an overdue end.”  Dawkins is that rare scientist who has consistently been as comfortable with rhetoric and language as he is with data.

Hats off to Coyne who reminds us that a metaphor–however lovely–does not some science make. If Mukherjee wants to play scientist, let him create and gather data. If it’s the role of science journalist he wants, let him collect all the science he can before he begins to pour it into his poetry.

 

Same but Different  

How epigenetics can blur the line between nature and nurture.

Annals of Science MAY 2, 2016 ISSUE     BY

The author’s mother (right) and her twin are a study in difference and identity. CREDIT: PHOTOGRAPH BY DAYANITA SINGH FOR THE NEW YORKER

October 6, 1942, my mother was born twice in Delhi. Bulu, her identical twin, came first, placid and beautiful. My mother, Tulu, emerged several minutes later, squirming and squalling. The midwife must have known enough about infants to recognize that the beautiful are often the damned: the quiet twin, on the edge of listlessness, was severely undernourished and had to be swaddled in blankets and revived.

The first few days of my aunt’s life were the most tenuous. She could not suckle at the breast, the story runs, and there were no infant bottles to be found in Delhi in the forties, so she was fed through a cotton wick dipped in milk, and then from a cowrie shell shaped like a spoon. When the breast milk began to run dry, at seven months, my mother was quickly weaned so that her sister could have the last remnants.
Tulu and Bulu grew up looking strikingly similar: they had the same freckled skin, almond-shaped face, and high cheekbones, unusual among Bengalis, and a slight downward tilt of the outer edge of the eye, something that Italian painters used to make Madonnas exude a mysterious empathy. They shared an inner language, as so often happens with twins; they had jokes that only the other twin understood. They even smelled the same: when I was four or five and Bulu came to visit us, my mother, in a bait-and-switch trick that amused her endlessly, would send her sister to put me to bed; eventually, searching in the half-light for identity and difference—for the precise map of freckles on her face—I would realize that I had been fooled.

But the differences were striking, too. My mother was boisterous. She had a mercurial temper that rose fast and died suddenly, like a gust of wind in a tunnel. Bulu was physically timid yet intellectually more adventurous. Her mind was more agile, her tongue sharper, her wit more lancing. Tulu was gregarious. She made friends easily. She was impervious to insults. Bulu was reserved, quieter, and more brittle. Tulu liked theatre and dancing. Bulu was a poet, a writer, a dreamer.

….. more

Why are identical twins alike? In the late nineteen-seventies, a team of scientists in Minnesota set out to determine how much these similarities arose from genes, rather than environments—from “nature,” rather than “nurture.” Scouring thousands of adoption records and news clips, the researchers gleaned a rare cohort of fifty-six identical twins who had been separated at birth. Reared in different families and different cities, often in vastly dissimilar circumstances, these twins shared only their genomes. Yet on tests designed to measure personality, attitudes, temperaments, and anxieties, they converged astonishingly. Social and political attitudes were powerfully correlated: liberals clustered with liberals, and orthodoxy was twinned with orthodoxy. The same went for religiosity (or its absence), even for the ability to be transported by an aesthetic experience. Two brothers, separated by geographic and economic continents, might be brought to tears by the same Chopin nocturne, as if responding to some subtle, common chord struck by their genomes.

One pair of twins both suffered crippling migraines, owned dogs that they had named Toy, married women named Linda, and had sons named James Allan (although one spelled the middle name with a single “l”). Another pair—one brought up Jewish, in Trinidad, and the other Catholic, in Nazi Germany, where he joined the Hitler Youth—wore blue shirts with epaulets and four pockets, and shared peculiar obsessive behaviors, such as flushing the toilet before using it. Both had invented fake sneezes to diffuse tense moments. Two sisters—separated long before the development of language—had invented the same word to describe the way they scrunched up their noses: “squidging.” Another pair confessed that they had been haunted by nightmares of being suffocated by various metallic objects—doorknobs, fishhooks, and the like.

The Minnesota twin study raised questions about the depth and pervasiveness of qualities specified by genes: Where in the genome, exactly, might one find the locus of recurrent nightmares or of fake sneezes? Yet it provoked an equally puzzling converse question: Why are identical twins different? Because, you might answer, fate impinges differently on their bodies. One twin falls down the crumbling stairs of her Calcutta house and breaks her ankle; the other scalds her thigh on a tipped cup of coffee in a European station. Each acquires the wounds, calluses, and memories of chance and fate. But how are these changes recorded, so that they persist over the years? We know that the genome can manufacture identity; the trickier question is how it gives rise to difference.

….. more

But what turns those genes on and off, and keeps them turned on or off? Why doesn’t a liver cell wake up one morning and find itself transformed into a neuron? Allis unpacked the problem further: suppose he could find an organism with two distinct sets of genes—an active set and an inactive set—between which it regularly toggled. If he could identify the molecular switches that maintain one state, or toggle between the two states, he might be able to identify the mechanism responsible for cellular memory. “What I really needed, then, was a cell with these properties,” he recalled when we spoke at his office a few weeks ago. “Two sets of genes, turned ‘on’ or ‘off’ by some signal.”

more…

“Histones had been known as part of the inner scaffold for DNA for decades,” Allis went on. “But most biologists thought of these proteins merely as packaging, or stuffing, for genes.” When Allis gave scientific seminars in the early nineties, he recalled, skeptics asked him why he was so obsessed with the packing material, the stuff in between the DNA.  …. A skein of silk tangled into a ball has very different properties from that same skein extended; might the coiling or uncoiling of DNA change the activity of genes?

In 1996, Allis and his research group deepened this theory with a seminal discovery. “We became interested in the process of histone modification,” he said. “What is the signal that changes the structure of the histone so that DNA can be packed into such radically different states? We finally found a protein that makes a specific chemical change in the histone, possibly forcing the DNA coil to open. And when we studied the properties of this protein it became quite clear that it was also changing the activity of genes.” The coils of DNA seemed to open and close in response to histone modifications—inhaling, exhaling, inhaling, like life.

Allis walked me to his lab, a fluorescent-lit space overlooking the East River, divided by wide, polished-stone benches. A mechanical stirrer, whirring in a corner, clinked on the edge of a glass beaker. “Two features of histone modifications are notable,” Allis said. “First, changing histones can change the activity of a gene without affecting the sequence of the DNA.” It is, in short, formally epi-genetic, just as Waddington had imagined. “And, second, the histone modifications are passed from a parent cell to its daughter cells when cells divide. A cell can thus record ‘memory,’ and not just for itself but for all its daughter cells.”

…..

 

 

The New Yorker screws up big time with science: researchers criticize the Mukherjee piece on epigenetics

Jerry Coyne
https://whyevolutionistrue.wordpress.com/2016/05/05/the-new-yorker-screws-up-big-time-with-science-researchers-criticize-the-mukherjee-piece-on-epigenetics/

Abstract: This is a two part-post about a science piece on gene regulation that just appeared in the New Yorker. Today I give quotes from scientists criticizing that piece; tomorrow I’ll present a semi-formal critique of the piece by two experts in the field.

esterday I gave readers an assignment: read the new New Yorkerpiece by Siddhartha Mukherjee about epigenetics. The piece, called “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”) was brought to my attention by two readers, both of whom praised it.  Mukherjee, a physician, is well known for writing the Pulitzer-Prize-winning book (2011) The Emperor of All Maladies: A Biography of Cancer. (I haven’t read it yet, but it’s on my list.)  Mukherjee has a new book that will be published in May: The Gene: An Intimate History. As I haven’t seen it, the New Yorker piece may be an excerpt from this book.

Everyone I know who has read The Emperor of All Maladies gives it high praise. I wish I could say the same for Mukherjee’s New Yorker piece. When I read it at the behest of the two readers, I found his analysis of gene regulation incomplete and superficial. Although I’m not an expert in that area, I knew that there was a lot of evidence that regulatory proteins called “transcription factors”, and not “epigenetic markers” (see discussion of this term tomorrow) or modified histones—the factors emphasized by Mukherjee—played hugely important roles in gene regulation. The speculations at the end of the piece about “Lamarckian evolution” via environmentally induced epigenetic changes in the genome were also unfounded, for we have no evidence for that kind of adaptive evolution. Mukherjee does, however, mention that lack of evidence, though I wish he’d done so more strongly given that environmental modification of DNA bases is constantly touted as an important and neglected factor in evolution.

Unbeknownst to me, there was a bit of a kerfuffle going on in the community of scientists who study gene regulation, with many of them finding serious mistakes and omissions in Mukherjee’s piece.  There appears to have been some back-and-forth emailing among them, and several wrote letters to the New Yorker, urging them to correct the misconceptions, omissions, and scientific errors in “Same but different.” As I understand it, both Mukherjee and the New Yorker simply batted these criticisms away, and, as far as I know, will not publish any corrections.  So today and tomorrow I’ll present the criticisms here, just so they’ll be on the record.

Because Mukherjee writes very well, and because even educated laypeople won’t know the story of gene regulation revealed over the last few decades,  they may not see the big lacunae in his piece. It is, then,  important to set matters straight, for at least we should know what science has told us about how genes are turned on and off. The criticism of Mukherjee’s piece, coming from scientists who really are experts in gene regulation, shows a lack of care on the part of Mukherjee and theNew Yorker: both a superficial and misleading treatment of the state of the science, and a failure of the magazine to properly vet this piece (I have no idea whether they had it “refereed” not just by editors but by scientists not mentioned in the piece).

Let me add one thing about science and the New Yorker. I believe I’ve said this before, but the way the New Yorker treats science is symptomatic of the “two cultures” problem. This is summarized in an email sent me a while back by a colleague, which I quote with permission:

The New Yorker is fine with science that either serves a literary purpose (doctors’ portraits of interesting patients) or a political purpose (environmental writing with its implicit critique of modern technology and capitalism). But the subtext of most of its coverage (there are exceptions) is that scientists are just a self-interested tribe with their own narrative and no claim to finding the truth, and that science must concede the supremacy of literary culture when it comes to anything human, and never try to submit human affairs to quantification or consilience with biology. Because the magazine is undoubtedly sophisticated in its writing and editing they don’t flaunt their postmodernism or their literary-intellectual proprietariness, but once you notice it you can make sense of a lot of their material.

. . . Obviously there are exceptions – Atul Gawande is consistently superb – but as soon as you notice it, their guild war on behalf of cultural critics and literary intellectuals against scientists, technologists, and analytic scholars becomes apparent.

…. more

Researchers criticize the Mukherjee piece on epigenetics: Part 2

Trigger warning: Long science post!

Yesterday I provided a bunch of scientists’ reactions—and these were big names in the field of gene regulation—to Siddhartha Mukherjee’s ill-informed piece in The New Yorker, “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”). Today, in part 2, I provide a sentence-by-sentence analysis and reaction by two renowned researchers in that area. We’ll start with a set of definitions (provided by the authors) that we need to understand the debate, and then proceed to the critique.

Let me add one thing to avoid confusion: everything below the line, including the definition (except for my one comment at the end) was written by Ptashne and Greally.

by Mark Ptashne and John Greally

Introduction

Ptashne is The Ludwig Professor of Molecular Biology at the Memorial Sloan Kettering Cancer Center in New York. He wrote A Genetic Switch, now in its third edition, which describes the principles of gene regulation and the workings of a ‘switch’; and, with Alex Gann, Genes and Signals, which extends these principles and ideas to higher organisms and to other cellular processes as well.  John Greally is the Director of the Center for Epigenomics at the Albert Einstein College of Medicine in New York.

 

The New Yorker  (May 2, 2016) published an article entitled “Same But Different” written by Siddhartha Mukherjee.  As readers will have gathered from the letters posted yesterday, there is a concern that the article is misleading, especially for a non-scientific audience. The issue concerns our current understanding of “gene regulation” and how that understanding has been arrived at.

First some definitions/concepts:

Gene regulation refers to the “turning on and off of genes”.  The primary event in turning a gene “on” is to transcribe (copy) it into messenger RNA (mRNA). That mRNA is then decoded, usually, into a specific protein.  Genes are transcribed by the enzyme called RNA polymerase.

Development:  the process in which a fertilized egg (e.g., a human egg) divides many times and eventually forms an organism.  During this process, many of the roughly 23,000 genes of a human are turned “on” or “off” in different combinations, at different times and places in the developing organism. The process produces many different cell types in different organs (e.g. liver and brain), but all retain the original set of genes.

Transcription factors: proteins that bind to specific DNA sequences near specific genes and turn transcription of those genes on and off. A transcriptional ‘activator’, for example, bears two surfaces: one binds a specific sequence in DNA, and the other binds to, and thereby recruits to the gene, protein complexes that include RNA polymerase. It is widely acknowledged that the identity of a cell in the body depends on the array of transcription factors present in the cell, and the cell’s history.  RNA molecules can also recognize specific genomic sequences, and they too sometimes work as regulators.  Neither transcription factors nor these kinds of RNA molecules – the fundamental regulators of gene expression and development – are mentioned in the New Yorker article.

Signals:  these come in many forms (small molecules like estrogen, larger molecules (often proteins such as cytokines) that determine the ability of transcription factors to work.  For example, estrogen binds directly to a transcription factor (the estrogen receptor) and, by changing its shape, permits it to bind DNA and activate transcription.

Memory”:  a dividing cell can (often does) produce daughters that are identical, and that express identical genes as does the mother cell.  This occurs because the transcription factors present in the mother cell are passively transmitted to the daughters as the cell divides, and they go to work in their new contexts as before.  To make two different daughters, the cell must distribute its transcription factors asymmetrically.

Positive Feedback: An activator can maintain its own expression by  positive feedback.  This requires, simply, that a copy of the DNA sequence to which the activator binds is  present  near its own gene. Expression of the activator  then becomes self-perpetuating.  The activator (of which there now are many copies in the cell) activates  other target genes as it maintains its own expression. This kind of ‘memory circuit’, first described  in  bacteria, is found in higher organisms as well.  Positive feedback can explain how a fully differentiated cell (that is, a cell that has reached its developmental endpoint) maintains its identity.

Nucleosomes:  DNA in higher organisms (eukaryotes) is wrapped, like beads on a string, around certain proteins (called histones), to form nucleosomes.  The histones are subject to enzymatic modifications: e.g., acetyl, methyl, phosphate, etc. groups can be added to these structures. In bacteria there are no nucleosomes, and the DNA is more or less ‘naked’.

“Epigenetic modifications: please don’t worry about the word ”epigenetic”; it is misused in any case. What Mukherjee refers to by this term are the histone modifications mentioned above, and a modification to DNA itself: the addition of methyl groups. Keep in mind that the organisms that have taught us the most about development – flies (Drosophila) and worms (C. elegans)—do not have the enzymes required for DNA methylation. That does not mean that DNA methylation cannot do interesting things in humans, for example, but it is obviously not at the heart of gene regulation.

Specificity Development requires the highly specific sequential turning on and off of sets of genes.  Transcription factors and RNA supply this specificity, but   enzymes that impart modifications to histones  cannot: every nucleosome (and hence every gene) appears the same to the enzyme.  Thus such enzymes cannot pick out particular nucleosomes associated with particular genes to modify.  Histone modifications might be imagined to convey ‘memory’ as cells divide – but there are no convincing indications that this happens, nor are there molecular models that might explain why they would have the imputed effects.

Analysis and critique of Mukherjee’s article

The picture we have just sketched has taken the combined efforts of many scientists over 50 years to develop.  So what, then, is the problem with the New Yorker article?

There are two: first, the picture we have just sketched, emphasizing the primary role of transcription factors and RNA, is absent.  Second, that picture is replaced by highly dubious speculations, some of which don’t make sense, and none of which has been shown to work as imagined in the article.

(Quotes from the Mukherjee article are indented and in plain text; they are followed by comments, flush left and in bold, by Ptashne and Greally.)

In 1978, having obtained a Ph.D. in biology at Indiana University, Allis began to tackle a problem that had long troubled geneticists and cell biologists: if all the cells in the body have the same genome, how does one become a nerve cell, say, and another a blood cell, which looks and functions very differently?

The problems referred to were recognized long before 1978.  In fact, these were exactly the problems that the great French scientists François Jacob and Jacques Monod took on in the 1950s-60s.  In a series of brilliant experiments, Jacob and Monod showed that in bacteria, certain genes encode products that regulate (turn on and off) specific other genes.  Those regulatory molecules turned out to be proteins, some of which respond to signals from the environment.  Much of the story of modern biology has been figuring out how these proteins – in bacteria and in higher organisms  – bind to and regulate specific genes.  Of note is that in higher organisms, the regulatory proteins look and act like those in bacteria, despite the fact that eukaryotic DNA is wrapped in nucleosomes  whereas bacterial DNA is not.   We have also learned that certain RNA molecules can play a regulatory role, a phenomenon made possible by the fact that RNA molecules, like regulatory proteins, can recognize specific genomic sequences.

In the nineteen-forties, Conrad Waddington, an English embryologist, had proposed an ingenious answer: cells acquired their identities just as humans do—by letting nurture (environmental signals) modify nature (genes). For that to happen, Waddington concluded, an additional layer of information must exist within a cell—a layer that hovered, ghostlike, above the genome. This layer would carry the “memory” of the cell, recording its past and establishing its future, marking its identity and its destiny but permitting that identity to be changed, if needed. He termed the phenomenon “epigenetics”—“above genetics.”

This description greatly misrepresents the original concept.  Waddington argued that development proceeds not by the loss (or gain) of genes, which would be a “genetic” process, but rather that some genes would be selectively expressed in specific and complex cellular patterns as development proceeds.  He referred to this intersection of embryology (then called “epigenesis”) and genetics as “epigenetic”.We now understand that regulatory proteins work in combinations to turn on and off genes, including their own genes, and that sometimes the regulatory proteins respond to signals sent by other cells.  It should be emphasized that Waddington never proposed any “ghost-like” layer of additional information hovering above the gene.  This is a later misinterpretation of a literal translation of the term epigenetics, with “epi-“ meaning “above/upon” the genetic information encoded in DNA sequence.  Unfortunately, this new and pervasive definition encompasses all of transcriptional regulation and is of no practical value.

…..more

By 2000, Allis and his colleagues around the world had identified a gamut of proteins that could modify histones, and so modulate the activity of genes. Other systems, too, that could scratch different kinds of code on the genome were identified (some of these discoveries predating the identification of histone modifications). One involved the addition of a chemical side chain, called a methyl group, to DNA. The methyl groups hang off the DNA string like Christmas ornaments, and specific proteins add and remove the ornaments, in effect “decorating” the genome. The most heavily methylated parts of the genome tend to be dampened in their activity.

It is true that enzymes that modify histones have been found—lots of them.  A striking problem is that, after all this time, it is not at all clear what the vast majority of these modifications do.  When these enzymatic activities are eliminated by mutation of their active sites (a task substantially easier to accomplish in yeast than in higher organisms) they mostly have little or no effect on transcription.  It is not even clear that histones are the biologically relevant substrates of most of these enzymes.  

 In the ensuing decade, Allis wrote enormous, magisterial papers in which a rich cast of histone-modifying proteins appear and reappear through various roles, mapping out a hatchwork of complexity. . . These protein systems, overlaying information on the genome, interacted with one another, reinforcing or attenuating their signals. Together, they generated the bewildering intricacy necessary for a cell to build a constellation of other cells out of the same genes, and for the cells to add “memories” to their genomes and transmit these memories to their progeny. “There’s an epigenetic code, just like there’s a genetic code,” Allis said. “There are codes to make parts of the genome more active, and codes to make them inactive.”

By ‘epigenetic code’ the author seems to mean specific arrays of nucleosome modifications, imparted over time and cell divisions, marking genes for expression.  This idea has been tested in many experiments and has been found not to hold.

….. and more

 

Larry H. Bernstein, MD, FCAP

I hope that this piece brings greater clarity to the discussion.  I have heard the use of the term “epigenetics” for over a decade.  The term was never so clear.  I think that the New Yorker article was a reasonable article for the intended audience.  It was not intended to clarify debates about a mechanism for epigenetic based changes in evolutionary science.  I think it actually punctures the “classic model” of the cell depending only on double stranded DNA and transcription, which deflates our concept of the living cell.  The concept of epigenetics was never really formulated as far as I have seen, and I have done serious work in enzymology and proteins at a time that we did not have the technology that exists today.  I have considered with the critics that protein folding, protein misfolding, protein interactions with proximity of polar and nonpolar groups, and the regulatory role of microRNAs that are not involved in translation, and the evolving concept of what is “dark (noncoding) DNA” lend credence to the complexity of this discussion.  Even more interesting is the fact that enzymes (and isoforms of enzymes) have a huge role in cellular metabolic differences and in the function of metabolic pathways.  What is less understood is the extremely fast reactions involved in these cellular reactions.  These reactions are in my view critical drivers.  This is brought out by Erwin Schroedinger in the book What is Life? which infers that there can be no mathematical expression of life processes.

 

 

 

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New Insights into mtDNA, mitochondrial proteins, aging, and metabolic control

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Newly discovered proteins may protect against age-related illnesses  

The proteins could play a key role in the aging process and the onset of diseases linked to older age

BY Beth Newcomb   APRIL 13, 2016

http://news.usc.edu/98412/newly-discovered-proteins-may-protect-against-agings-illnesses/

http://news.usc.edu/files/2016/04/20160208_PinchasCohen_web-824×549.jpg

Pinchas Cohen led a team that identified tiny proteins that appear to play a role in controlling how the body ages. (Photo/Beth Newcomb)

A group of six newly discovered proteins may help to divulge secrets of how we age, potentially unlocking insights into diabetes, Alzheimer’s, cancer and other aging-related diseases.

The tiny proteins appear to play several big roles in our bodies’ cells, from decreasing the amount of damaging free radicals and controlling the rate at which cells die to boosting metabolism and helping tissues throughout the body respond better to insulin. The naturally occurring amounts of each protein decrease with age, leading researchers to believe that they play an important role in the aging process and the onset of diseases linked to older age.

The research team led by Pinchas Cohen, dean of the USC Davis School of Gerontology, identified the tiny proteins for the first time and observed their surprising origin from organelles in the cell called mitochondria and their game-changing roles in metabolism and cell survival. This latest finding builds upon prior research by Cohen and his team that uncovered two significant proteins, humanin and MOTS-c, hormones that appear to have significant roles in metabolism and diseases of aging.

Unlike most other proteins, humanin and MOTS-c are encoded in mitochondria, the structure within cells that produces energy from food, instead of in the cell’s nucleus where most genes are contained.

Key functions

Mitochondria have their own small collection of genes, which were once thought to play only minor roles within cells but now appear to have important functions throughout the body. Cohen’s team used computer analysis to see if the part of the mitochondrial genome that provides the code for humanin was coding for other proteins as well. The analysis uncovered the genes for six new proteins, which were dubbed small humanin-like peptides, or SHLPs, 1 through 6 (the name of this hardworking group of proteins is appropriately pronounced “schlep”).

After identifying the six SHLPs and successfully developing antibodies to test for several of them, the team examined both mouse tissues and human cells to determine their abundance in different organs as well as their functions. The proteins were distributed quite differently among organs, which suggests that the proteins have varying functions based on where they are in the body.

Of particular interest is SHLP 2, Cohen said. The protein appears to have profound insulin-sensitizing, anti-diabetic effects as well as potent neuro-protective activity that may emerge as a strategy to combat Alzheimer’s disease. He added that SHLP 6 is also intriguing, with a unique ability to promote cancer cell death and thus potentially target malignant diseases.

“Together with the previously identified mitochondrial peptides, the newly recognized SHLP family expands the understanding of the mitochondria as an intracellular signaling organelle that communicates with the rest of the body to regulate metabolism and cell fate,” Cohen said. “The findings are an important advance that will be ripe for rapid translation into drug development for diseases of aging.”

The study first appeared online in the journal Aging on April 10. Cohen’s research team included collaborators from the Albert Einstein College of Medicine; the findings have been licensed to the biotechnology company CohBar for possible drug development.

The research was supported by a Glenn Foundation Award and National Institutes of Health grants to Cohen (1P01AG034906, 1R01AG 034430, 1R01GM 090311, 1R01ES 020812) and an Ellison/AFAR postdoctoral fellowship to Kelvin Yen. Study authors Laura Cobb, Changhan Lee, Nir Barzilai and Pinchas Cohen are consultants and stockholders of CohBar Inc.

Feature: The man who wants to beat back aging

By Stephen S. Hall Sep. 16, 201

Nir Barzilai hopes to persuade FDA to bless the proposed anti-aging trial, which is unconventional in its goals and design.
http://www.sciencemag.org/news/2015/09/feature-man-who-wants-beat-back-aging

On a blazingly hot morning this past June, a half-dozen scientists convened in a hotel conference room in suburban Maryland for the dress rehearsal of what they saw as a landmark event in the history of aging research. In a few hours, the group would meet with officials at the U.S. Food and Drug Administration (FDA), a few kilometers away, to pitch an unprecedented clinical trial—nothing less than the first test of a drug to specifically target the process of human aging.

“We think this is a groundbreaking, perhaps paradigm-shifting trial,” said Steven Austad, chairman of biology at the University of Alabama, Birmingham, and scientific director of the American Federation for Aging Research (AFAR). After Austad’s brief introductory remarks, a scientist named Nir Barzilai tuned up his PowerPoint and launched into a practice run of the main presentation.

Barzilai is a former Israeli army medical officer and head of a well-known study of centenarians based at the Albert Einstein College of Medicine in the Bronx, New York. To anyone who has seen the ebullient scientist in his natural laboratory habitat, often in a short-sleeved shirt and always cracking jokes, he looked uncharacteristically kempt in a blue blazer and dress khakis. But his practice run kept hitting a historical speed bump. He had barely begun to explain the rationale for the trial when he mentioned, in passing, “lots of unproven, untested treatments under the category of anti-aging.” His colleagues pounced.

“Nir,” interrupted S. Jay Olshansky, a biodemographer of aging from the University of Illinois, Chicago. The phrase “anti-aging … has an association that is negative.”

“I wouldn’t dignify them by calling them ‘treatments,’” added Michael Pollak, director of cancer prevention at McGill University in Montreal, Canada. “They’re products.”

Barzilai, a 59-year-old with a boyish mop of gray hair, wore a contrite grin. “We know the FDA is concerned about this,” he conceded, and deleted the offensive phrase.

Then he proceeded to lay out the details of an ambitious clinical trial. The group—academics all—wanted to conduct a double-blind study of roughly 3000 elderly people; half would get a placebo and half would get an old (indeed, ancient) drug for type 2 diabetes called metformin, which has been shown to modify aging in some animal studies. Because there is still no accepted biomarker for aging, the drug’s success would be judged by an unusual standard—whether it could delay the development of several diseases whose incidence increases dramatically with age: cardiovascular disease, cancer, and cognitive decline, along with mortality. When it comes to these diseases, Barzilai is fond of saying, “aging is a bigger risk factor than all of the other factors combined.”

But the phrase “anti-aging” kept creeping into the rehearsal, and critics kept jumping in. “Okay,” Barzilai said with a laugh when it came up again. “Third time, the death penalty.”

The group’s paranoia about the term “anti-aging” captured both the audacity of the proposed trial and the cultural challenge of venturing into medical territory historically associated with charlatans and quacks. The metformin initiative, which Barzilai is generally credited with spearheading, is unusual by almost any standard of drug development. The people pushing for the trial are all academics, none from industry (although Barzilai is co-founder of a biotech company, CohBar Inc., that is working to develop drugs targeting age-related diseases). The trial would be sponsored by the nonprofit AFAR, not a pharmaceutical company. No one stood to make money if the drug worked, the scientists all claimed; indeed, metformin is not only generic, costing just a few cents a dose, but belongs to a class of drugs that has been part of the human apothecary for 500 years. Patient safety was unlikely to be an issue; millions of diabetics have taken metformin since the 1960s, and its generally mild side effects are well-known.

Finally, the metformin group insisted they didn’t need a cent of federal money to proceed (although they do intend to ask for some). Nor did they need formal approval from FDA to proceed. But they very much wanted the agency’s blessing. By recognizing the merit of such a trial, Barzilai believes,  FDA would make aging itself a legitimate target for drug development.

By the time the scientists were done, the rehearsal—which was being filmed for a television documentary—had the feel of a pep rally. They spoke with unguarded optimism. “What we’re talking about here,” Olshansky said, “is a fundamental sea change in how we look at aging and disease.” To Austad, it is “the key, potentially, to saving the health care system.”

As the group piled into a van for the drive to FDA headquarters, there was more talk about setting precedents and opening doors. So it was a little disconcerting when Austad led the delegation up to the main entrance of FDA—and couldn’t get the door open.   ……

Mitochondrial Peptides Found in a Preclinical Study Seen to Control Cell Metabolism

APRIL 13, 2016 BY MAGDALENA KEGEL  IN NEWS.
http://mitochondrialdiseasenews.com/2016/04/13/newly-discovered-mitochondrial-factors-might-give-insights-into-aging/

 

CohBar, a developer of mitochondria-based therapeutics, announced that preclinical research by its academic collaborators has found small humanin-like peptides (SHLPs) that can control metabolism and cell survival. The findings have implications for age-related diseases such as Alzheimer’s and cancer.

The study, “Naturally Occurring Mitochondrial-derived Peptides are Age-dependent Regulators of Apoptosis, Insulin Sensitivity, and Inflammatory Markers,” was the result of a joint effort between researchers at the University of Southern California (USC) and theInstitute for Aging Research at the Albert Einstein College of Medicine of Yeshiva University. The study was published in the journal Aging.

Researchers discovered the SHLPs by examining the genome of mitochondria with the help of a bioinformatics approach, which identified six peptides. The team then verified the presence of the factors and explored their function in laboratory animals.

CohBar, who have the exclusive license to develop SHLPs into therapeutics, works closely with its academic partners to explore the peptides in preclinical models.

While it was previously believed that mitochondria only have 37 genes, research has revealed that the mitochondrial genome is far more versatile, potentially harboring a multitude of new genes, which can encode peptides acting as cellular signaling factors. The peptides, it has turned out, have shown neuroprotective and anti-inflammatory effects, and act to protect cells in disease-modifying ways in preclinical models of aging.

CohBar’s goal is to bring these peptides to the market as therapies for age-related diseases, such as obesity, type 2 diabetes, cancer, atherosclerosis and neurodegenerative disorders.

“Together with the previously described mitochondrial-derived peptides humanin and MOTS-c, the SHLP family expands our understanding of the role that these peptides play in intracellular signaling throughout the body to regulate both metabolism and cell survival,” Pinchas Cohen, dean of the USC Leonard Davis School of Gerontology, founder and director of CohBar, and the study’s senior author, said in a press release. “These findings further illustrate the enormous potential that mitochondria-based therapeutics could have on treating age-associated diseases like Alzheimer’s and cancer.”

“The pre-clinical evidence continues to confirm that these peptides represent a new class of naturally occurring metabolic regulators,” added Simon Allen, CohBar’s CEO. “They form the foundation of our pipeline of first-in-class treatments for age-related diseases, and we are committed to rapidly advancing them through pre-clinical and clinical activities as we move forward.”

Naturally occurring mitochondrial-derived peptides are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory markers

Laura J. Cobb1,5, Changhan Lee2, Jialin Xiao2, Kelvin Yen2, Richard G. Wong2, Hiromi K. Nakamura1, ….., Derek M. Huffman4, Junxiang Wan2, Radhika Muzumdar3, Nir Barzilai4 , and Pinchas Cohen2
http://www.impactaging.com/papers/v8/n4/full/100943.html

Mitochondria are key players in aging and in the pathogenesis of age-related diseases. Recent mitochondrial transcriptome analyses revealed the existence of multiple small mRNAs transcribed from mitochondrial DNA (mtDNA). Humanin (HN), a peptide encoded in the mtDNA 16S ribosomal RNA region, is a neuroprotective factor. An in silico search revealed six additional peptides in the same region of mtDNA as humanin; we named these peptides small humanin-like peptides (SHLPs). We identified the functional roles for these peptides and the potential mechanisms of action. The SHLPs differed in their ability to regulate cell viability in vitro. We focused on SHLP2 and SHLP3 because they shared similar protective effects with HN. Specifically, they significantly reduced apoptosis and the generation of reactive oxygen species, and improved mitochondrial metabolism in vitro. SHLP2 and SHLP3 also enhanced 3T3-L1 pre-adipocyte differentiation. Systemic hyperinsulinemic-euglycemic clamp studies showed that intracerebrally infused SHLP2 increased glucose uptake and suppressed hepatic glucose production, suggesting that it functions as an insulin sensitizer both peripherally and centrally. Similar to HN, the levels of circulating SHLP2 were found to decrease with age. These results suggest that mitochondria play critical roles in metabolism and survival through the synthesis of mitochondrial peptides, and provide new insights into mitochondrial biology with relevance to aging and human biology.

Human mitochondrial DNA (mtDNA) is a double-stranded, circular molecule of 16,569 bp and contains 37 genes encoding 13 proteins, 22 tRNAs, and 2 rRNAs. Recent mitochondrial transcriptome analyses revealed the existence of small RNAs derived from mtDNA [1]. In 2001, Nishimoto and colleagues identified humanin (HN), a 24-amino-acid peptide encoded from the 16S ribosomal RNA (rRNA) region of mtDNA. HN is a potent neuroprotective factor capable of antagonizing Alzheimer’s disease (AD)-related cellular insults [2]. HN is a component of a novel retrograde signaling pathway from the mitochondria to the nucleus, which is distinct from mitochondrial signaling pathways, such as the SIRT4-AMPK pathway [3]. HN-dependent cellular protection is mediated in part by interacting with and antagonizing pro-apoptotic Bax-related peptides [4] and IGFBP-3 (IGF binding protein 3) [5].

Because of their involvement in energy production and free radical generation, mitochondria likely play a major role in aging and age-related diseases [68]. In fact, improvement of mitochondrial function has been shown to ameliorate age-related memory loss in aged mice [9]. Recent studies have shown that HN levels decrease with age, suggesting that HN could play a role in aging and age-related diseases, such as Alzheimer’s disease (AD), atherosclerosis, and diabetes. Along with lower HN levels in the hypothalamus, skeletal muscle, and cortex of older rodents, the circulating levels of HN were found to decline with age in both humans and mice [10]. Notably, circulating HN levels were found to be (i) significantly higher in long-lived Ames dwarf mice but lower in short-lived growth hormone (GH) transgenic mice, (ii) significantly higher in a GH-deficient cohort of patients with Laron syndrome, and (iii) reduced in mice and humans treated with GH or IGF-1 (insulin-like growth factor 1) [11]. Age-dependent declines in the circulating HN levels may be due to higher levels of reactive oxygen species (ROS) that contribute to atherosclerosis development. Using mouse models of atherosclerosis, it was found that HN-treated mice had a reduced disease burden and significant health improvements [12,13]. In addition, HN improved insulin sensitivity, suggesting clinical potential for mitochondrial peptides in diseases of aging [10]. The discovery of HN represents a unique addition to the spectrum of roles that mitochondria play in the cell [14,15]. A second mitochondrial-derived peptide (MDP), MOTS-c (mitochondrial open reading frame of the 12S rRNA-c), has also been shown to have metabolic effects on muscle and may also play a role in aging [16].

We further investigated mtDNA for the presence of other MDPs. Recent technological advances have led to the identification of small open reading frames (sORFs) in the nuclear genomes ofDrosophila[17,18] and mammals [19,20]. Therefore, we attempted to identify novel sORFs using the following approaches: 1) in silico identification of potential sORFs; 2) determination of mRNA expression levels; 3) development of specific antibodies against these novel peptides to allow for peptide detection in cells, organs, and plasma; 4) elucidating the actions of these peptides by performing cell-based assays for mitochondrial function, signaling, viability, and differentiation; and 5) delivering these peptides in vivo to determine their systemic metabolic effects. Focusing on the 16S rRNA region of the mtDNA where the humanin gene is located, we identified six sORFs and named them small humanin-like peptides (SHLPs) 1-6. While surveying the biological effects of SHLPs, we found that SHLP2 and SHLP3 were cytoprotective; therefore, we investigated their effects on apoptosis and metabolism in greater detail. Further, we showed that circulating SHLP2 levels declined with age, similar to HN, suggesting that SHLP2 is involved in aging and age-related disease progression.

SHLP2 and SHLP3 regulate the expression of metabolic and inflammatory markers

Epidemiological studies have demonstrated that increased levels of mediators of inflammation and acute-phase reactants, such as fibrinogen, C-reactive protein (CRP), and IL-6, correlate with the incidence of type 2 diabetes mellitus (T2DM) [3436]. In humans, anti-inflammatory drugs, such as aspirin and sodium salicylate, reduce fasting plasma glucose levels and ameliorate the symptoms of T2DM. In addition, anti-diabetic drugs, such as fibrates [37] and thiazolidinediones [38], have been found to lower some markers of inflammation. SHLP2 increased the levels of leptin, which is known to improve insulin sensitivity, but had no effect on the levels of the pro-inflammatory cytokines IL-6 and MCP-1. SHLP3 significantly increased the leptin levels, but also elevated IL-6 and MCP-1 levels, which could explain the lack of an in vivo insulin-sensitizing effect of SHLP3. The mechanism by which SHLPs regulate the expression of metabolic and inflammatory markers remains unclear and needs to be further investigated. Furthermore, SHLPs have different effects on inflammatory marker expression, suggesting differential regulation and function of individual SHLPs.

SHLP2 in aging

Mitochondria have been implicated in increased lifespan in several life-extending treatments [39,40]; however, it is not known whether the relationship is correlative or causative [40]. Additionally, it is well known that hormone levels change with aging. For example, levels of aldosterone, calcitonin, growth hormone, and IGF-I decrease with age. Circulating HN levels decline with age in humans and rodents, specifically in the hypothalamus and skeletal muscle of older rats. These changes parallel increases in the incidence of age-associated diseases such as AD and T2DM. The decline in circulating SHLP2 levels with age (Fig. 6), the anti-oxidative stress function of SHLP2 (Fig. 3C), and its neuroprotective effect (Fig. 6B) indicate that SHLP2 has a role in the regulation of aging and age-related diseases.

Conclusion

By analyzing the mitochondrial transcriptome, we found that sORFs from mitochondrial DNA encode functional peptides. We identified many mRNA transcripts within 13 protein-coding mitochondrial genes [1]. Such previously underappreciated sORFs have also been described in the nuclear genome [41]. The MDPs we describe here may represent retrograde communication signals from the mitochondria to the nucleus and may explain important aspects of mitochondrial biology that are implicated in health and longevity.

Larry, John Walker is working on mt proteins dynamics. His rotor – stator mechanism in ATPase synthase, a ‘complex’ that biologist accepted as energy generator is likely wrong. I was suppose to have met him in Germany few years ago. Energy in biological systems has nothing to do with heat. Heat is an outcome of a reaction, meaning that IR spectra accordingly to wave theory is a source of information memorized in water interference with carbon open systems within protein and glyo-proteins complexes as well as genome space-time outcomes. Physically speaking from a pure perspective of science ATP is highly unstable form of phosphate ‘chains’. It cannot hold energy, it is actually in contrary, it is like a resonator, trapping negativity, thus functioning as space propeller by expanding carbon skeleton of protein ‘machines’ Now, we don’t know what is ‘aging’ in a pure physical sense, except that we observe structural changes in what we call complexes. We we know is that proteins are not stationary structures, but highly dynamic forms of matter, seemingly occupying discrete and relative spaces. A piece of mt ATP ase could be discovered in the nucleus as transcription factor. Our notion of operational space in terms of electro dynamics from a motor – stator perspective is now translated toward defining semi conducting and supracoductive strings. The reality of which is so much more fascinating and beautiful as time progresses overally. There are spaces where time does not change, and there are spaces where time walks, and there are spaces, where time flies, and there are spaces where time runs. Amazing, indeed! The story of aging gets a lot deeper that science could even imagine, probably to roots of immortal energy- spaces. We know that matter is transient, that is nearly all living matter, replenishes of about 3 to 7 weeks.

Take a glass full of some kind of liquid, you know the mass of the glass and the mass of the liquid (say wine, beer, water, or milk) You also know to an approximate reality the composition of both. Now lift the glass full of liquid and let it break on a surface of your choice. Depending on the surface pieces of the glass would travel differential from a center projected by the vertical axis of your hand. What technology does today is recollecting those pieces and modelling them to fit in a form again that would resemble a holding device, a glass. The liquid we don’t know exactly how it spilled due the nature of its absorbancy of both surface physics and physical ‘state’ properties. Thus we can say how much approximate energy we have held thinking of m/z as time flight objectives. Each technology can read 1D and approximate the 2D, absolutely lacking computational methodology for 3D dynamic reality. Many scientists confuse space and volume. Volume is a one dimensional characteristic! So is crystalography! BY taking quantum chemical method computing principles following imaginative rules we could approach 2D, however , that is not enough to define 3D. Time we use as a reference frame of clocks we have invented in order to keep track of a sense to observable ‘change’ . But remember, time is absolute and parallel in continuity while energy is discrete , coming in quantum packages, realization of accumulated information. Information is highly redundant we see, so annotating information is an objective to modern days simulations that could predict outcomes of possible parallel realities we call worlds. One could ‘jump’ from one reality to another through guidance of light and water, but what remains unsolved is why people make mistakes, constantly by accusing in name of greed and power , or disobedience of commandments of the Lord!

On Thu, Apr 21, 2016 at 3:41 AM, Leaders in Pharmaceutical Business Intelligence (LPBI) Group wrote:

> larryhbern posted: “New Insights into mtDNA, mitochondrial proteins, > aging, and metabolic control Larry H. Bernstein, MD, FCAP, Curator LPBI > Newly discovered proteins may protect against age-related illnesses The > proteins could play a key role in the ” >

 

Metabolic features of the cell danger response
– Mitochondria in Health and Disease

Mitochondrion  Volume 16, May 2014, Pages 7–17     doi:10.1016/j.mito.2013.08.006

 

Highlights

  •  The Cell Danger Response (CDR) is defined in terms of an ancient metabolic response to threat.
  •  The CDR encompasses inflammation, innate immunity, oxidative stress, and the ER stress response.
  •  The CDR is maintained by extracellular nucleotide (purinergic) signaling.
  •  Abnormal persistence of the CDR lies at the heart of many chronic diseases.
  •  Antipurinergic therapy (APT) has proven effective in many chronic disorders in animal models

The cell danger response (CDR) is the evolutionarily conserved metabolic response that protects cells and hosts from harm. It is triggered by encounters with chemical, physical, or biological threats that exceed the cellular capacity for homeostasis. The resulting metabolic mismatch between available resources and functional capacity produces a cascade of changes in cellular electron flow, oxygen consumption, redox, membrane fluidity, lipid dynamics, bioenergetics, carbon and sulfur resource allocation, protein folding and aggregation, vitamin availability, metal homeostasis, indole, pterin, 1-carbon and polyamine metabolism, and polymer formation. The first wave of danger signals consists of the release of metabolic intermediates like ATP and ADP, Krebs cycle intermediates, oxygen, and reactive oxygen species (ROS), and is sustained by purinergic signaling. After the danger has been eliminated or neutralized, a choreographed sequence of anti-inflammatory and regenerative pathways is activated to reverse the CDR and to heal. When the CDR persists abnormally, whole body metabolism and the gut microbiome are disturbed, the collective performance of multiple organ systems is impaired, behavior is changed, and chronic disease results. Metabolic memory of past stress encounters is stored in the form of altered mitochondrial and cellular macromolecule content, resulting in an increase in functional reserve capacity through a process known as mitocellular hormesis. The systemic form of the CDR, and its magnified form, the purinergic life-threat response (PLTR), are under direct control by ancient pathways in the brain that are ultimately coordinated by centers in the brainstem. Chemosensory integration of whole body metabolism occurs in the brainstem and is a prerequisite for normal brain, motor, vestibular, sensory, social, and speech development. An understanding of the CDR permits us to reframe old concepts of pathogenesis for a broad array of chronic, developmental, autoimmune, and degenerative disorders. These disorders include autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD), asthma, atopy, gluten and many other food and chemical sensitivity syndromes, emphysema, Tourette’s syndrome, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), epilepsy, suicidal ideation, organ transplant biology, diabetes, kidney, liver, and heart disease, cancer, Alzheimer and Parkinson disease, and autoimmune disorders like lupus, rheumatoid arthritis, multiple sclerosis, and primary sclerosing cholangitis.

The double face of mitochondrial dysfunction

Dmitry Knorre, Anna Zyrina, and Fedor Severin

pp 420-420

Full text | PDF

 

 

Flawed Mitochondrial DNA Could Undermine Stem Cell Therapies

http://www.genengnews.com/gen-news-highlights/flawed-mitochondrial-dna-could-undermine-stem-cell-therapies/81252622/

http://www.genengnews.com/Media/images/GENHighlight/thumb_Apr18_2016_OHSU_ConfocalMicroscopyImage7543773219.jpg

This is a confocal microscopy image of human fibroblasts derived from embryonic stem cells. The nuclei appear in blue, while smaller and more numerous mitochondria appear in red. [Shoukhrat Mitalipov]

Mutations in our mitochondrial DNA tend to be inconspicuous, but they can become more prevalent as we age. They can even vary in frequency from cell to cell. Naturally, some cells will be relatively compromised because they happen to have a higher percentage of mutated mitochondrial DNA. Such cells make a poor basis for stem cell lines. They should be excluded. But how?

To answer this question, a team of scientists scrutinized skin fibroblasts, blood cells, and induced pluripotent stem cells (iPSCs) for mitochondrial genome integrity. When the scientists tested the samples for mitochondrial DNA mutations, the levels of mutations appeared low. But when the scientists sequenced the iPS cell lines, they found higher numbers of mitochondrial DNA mutations, particularly in cells from patients over 60.

The scientists were led by Shoukhrat Mitalipov, Ph.D., director of the Center for Embryonic Cell and Gene Therapy at Oregon Health & Science University, and Taosheng Huang, M.D., a medical geneticist and director of the Mitochondrial Medicine Program at Cincinnati Children’s Hospital. The Mitalipov/Huang-led team also found higher percentages of mitochondria containing mutations within a cell. The higher the load of mutated mitochondrial DNA in a cell, the more compromised the cell’s function.

Since each iPSC line is created from a different cell, each line may contain different types of mitochondrial DNA mutations and mutation loads. To choose the least damaged line, the authors recommend screening multiple lines per patient. “It’s a good idea to check the iPS clones for mitochondrial DNA mutations and make sure you pick a good cell line,” said Dr. Huang.

This recommendation appeared April 14 in the journal Cell Stem Cell, in an article entitled, “Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.” This article holds that mitochondrial genome integrity is a vital readout in assessing the proficiency of patient-derived regenerative products destined for clinical applications.

“We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues,” wrote the article’s authors. “The frequency of mtDNA defects in iPSCs increased with age, and many mutations were nonsynonymous or resided in RNA coding genes and thus can lead to respiratory defects.”

Potential therapies using stem cells hold tremendous promise for treating human disease. However, defects in the mitochondria could undermine the iPS cells’ ability to repair damaged tissue or organs.

“If you want to use iPS cells in a human, you must check for mutations in the mitochondrial genome,” declared Dr. Huang. “Every single cell can be different. Two cells next to each other could have different mutations or different percentages of mutations.”

Prior to the creation of a therapeutic iPS cell line, a collection of cells is taken from the patient. These cells will be tested for mutations. If the tester uses Sanger sequencing, older technology that is not as sensitive as newer next-generation sequencing, any mutation that occurs in less than 20% of the sample will go undetected. But mitochondrial DNA mutations might occur in less than 20% of mitochondria in the pooled cells. As a result, mutation rates have not been well understood. “These mitochondrial mutations are actually hidden,” explained Dr. Mitalipov.

The mitochondrial genome is relatively small, containing just 37 genes, so screening should be feasible using next generation sequencing, Dr. Mitalipov added. “It should be relatively cheap and do-able.”

Dr. Mitalipov also commented on a more general point, the implications of the current study on illuminating the mechanisms of age-related disease: “Pathogenic mutations in our mitochondrial DNA have long been thought to be a driving force in aging and age-onset diseases, though clear evidence was missing. This foundational knowledge of how cells are damaged in the natural process of aging may help to illuminate the role of mutated mitochondria in degenerative disease.”

New Mitalipov paper on stem cell mitochondria: challenge for IPS cell field?

http://www.ipscell.com/tag/age-related-accumulation-of-somatic-mitochondrial-dna-mutations-in-adult-derived-human-ipscs/

A new paper from Shoukhrat Mitalipov’s lab on stem cell mitochondria points to a pattern whereby induced pluripotent stem (IPS) cells tend to have more problems if they are from older patients.

What does this paper mean for the stem cell field and could it impact more specifically the clinical applications of IPS cells?

http://i0.wp.com/www.ipscell.com/wp-content/uploads/2016/04/Mitalipov-mitochondrial-mutations.jpg?resize=300%2C295

Graphical Abstract, Kang, et al. 2016

The new paper Kang, et al is entitled “Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs”.

This paper reminds us of the very important realities that mitochondria are key players in stem cell function and that mitochondria have their own genomes that impact that function. A lot of us don’t think about mitochondria and their genome as often as we should.

The paper came to three major scientific conclusions (this from the Highlights section of the paper and also see the graphical abstract for a visual sense of the results overall):

  • Human iPSC clones derived from elderly adults show accumulation of mtDNA mutations
  • Fewer mtDNA mutations are present in ESCs and iPSCs derived from younger adults
  • Accumulated mtDNA mutations can impact metabolic function in iPSCs

Importantly the team looked at IPS cells derived from both blood and skin cells and found that the former were less likely to have mitochondrial mutations.

This study suggests that those teams producing or working with human IPS cells (hIPSCs) should be screening the different lines for mitochondrial mutations. This excellent piece from Sara Reardon on the Mitalipov paper quotes IPS cell expert Jeanne Loring on this very point:

“It’s one of those things most of us don’t think about,” says Jeanne Loring, a stem-cell biologist at the Scripps Research Institute in La Jolla, California. Her lab is working towards using iPS cells to treat Parkinson’s disease, and Loring now plans to go back and examine the mitochondria in her cell lines. She suspects that it will be fairly easy for researchers to screen cells for use in therapies.”

Mitalipov goes further and suggests that his team’s new findings could support the use of human embryonic stem cells (hESC) derived by somatic cell nuclear transfer (SCNT) which would be expected to have mitochondria with fewer mutations. However, as Loring points out in the Reardon article, SCNT is really difficult to successfully perform and only a few labs in the world can do it at present. In that context, working with hIPSC and adding on the additional layer of mitochondrial DNA mutation screening could be more practical.

New York stem cell researcher Dieter Egli, however, is quoted that hIPSC have other differences with hESC as well such as epigenetic differences and he’s quoted in the Reardon piece, “It’s going to be very hard to find a cell line that’s perfect.”

One might reasonably ask both Egli and oneself, “What is a perfect cell line”?

In the end the best approach for use of human pluripotent stem cells of any kind is going to involve a balance between practicality of production and the potentially positive or negative traits of those cells as determined by rigorous validation screening.

With this new paper we’ve just learned more about another layer of screening that is needed. An interesting question is whether adult stem cells such as mesenchymal stromal/stem cells (MSC) also should be screened for mitochondrial mutations. They are often produced from patients who are getting up there in years. I hope that someone will publish on that too.

As to pluripotent cells, I expect that sometimes the best lines, meaning those most perfect for a given clinical application, will be hIPSC (autologous or allogeneic in some instances) and in other cases they may be hESC made from leftover IVF embryos. If SCNT-derived hESC can be more widely produced in an affordable manner and they pass validation as well then those (sometimes called NT-hESC) may also come into play clinically. So far that hasn’t happened for the SCNT cells, but it may over time.   …..

 Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs

Eunju Kang, Xinjian Wang, Rebecca Tippner-Hedges, …, Don P. Wolf, Taosheng Huang, Shoukhrat Mitalipov
http://www.cell.com/cell-stem-cell/pdfExtended/S1934-5909(16)00067-9

In Brief Mitalipov, Huang, and colleagues show that human iPSCs derived from older adults carry more mitochondrial DNA mutations than those derived from younger individuals. Defects in metabolic function caused by mtDNA mutations suggest careful screening of hiPSC clones for mutational load before clinical application.

Highlights

  1. Human iPSC clones derived from elderly adults show accumulation of mtDNA mutations
  2. Fewer mtDNA mutations are present in ESCs and iPSCs derived from younger adults
  3. Accumulated mtDNA mutations can impact metabolic function in iPSCs

Kang et al., 2016, Cell Stem Cell 18, 1–12 May 5, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.stem.2016.02.005

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24–72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

Induced pluripotent stem cells (iPSCs) offer an unlimited source for autologous cell replacement therapies to treat age-associated degenerative diseases. Aging is generally characterized by increased DNA damage and genomic instability (Garinis et al., 2008; Lombard et al., 2005); thus, iPSCs derived from elderly subjects may harbor point mutations and larger genomic rearrangements. Indeed, iPSCs display increased chromosome aberrations (Mayshar et al., 2010), subchromosomal copy number variations (CNVs) (Abyzov et al., 2012; Laurent et al., 2011), and exome mutations (Johannesson et al., 2014), compared to natural embryonic stem cell (ESC) counterparts (Ma et al., 2014). The rate of mtDNA mutations is believed to be at least 10- to 20-fold higher than that observed in the nuclear genome (Wallace, 1994), and often both mutated and wild-type mtDNA (heteroplasmy) can coexist in the same cell (Rossignol et al., 2003). Large deletions are most frequently observed mtDNA abnormalities in aged post-mitotic tissues such as brain, heart, and muscle (Bender et al., 2006; Bua et al., 2006; Corral-Debrinski et al., 1992; Cortopassi et al., 1992; Mohamed et al., 2006) and have been implicated in aging and diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and diabetes (Larsson, 2010; Lin and Beal, 2006; Petersen et al., 2003; Wallace, 2005). In addition, mtDNA point mutations were reported in some tumors and replicating tissues (Chatterjee et al., 2006; Ju et al., 2014; Michikawa et al., 1999; Taylor et al., 2003). However, the extent of mtDNA defects in proliferating peripheral tissues commonly used for iPSC induction, such as skin and blood, is thought to be low and limited to common non-coding variants (Schon et al., 2012; Yao et al., 2015). Accumulation of mtDNA variants in these tissues with age was insignificant (Greaves et al., 2010; Hashizume et al., 2015). Several point mutations were identified in iPSCs generated from the newborn foreskin fibroblasts, although most of these variants were non-coding, common for the general population, and did not affect their metabolic activity (Prigione et al., 2011). Somatic mtDNA mutations may be under-reported secondary to the level of sample interrogation. …..

Figure 2. mtDNA Mutations in Skin Fibroblasts, Blood, and the iPSCs of a 72-YearOld B Subject (A) Sixteen mutations at low heteroplasmy levels were detected in the DNA of PF, while a panel of ten FiPSC lines carried nine mutations, including four that were homoplasmic. Gray rectangles define the mutations shared between PF and FiPSCs. (B) Venn diagram showing only one mutation in FiPSCs shared with PF. (C) All ten FiPSC lines carried between one and five high-heteroplasmy (>15%) mutations. (D) Mutation distribution in whole blood and BiPSCs was similar to that in PF and FiPSCs. Six mutations at low-heteroplasmy levels were observed in blood, while BiPSC lines displayed 21 mutations, including four over the 80% heteroplasmy level. (E) Venn diagram showing four mutations in BiPSCs shared with whole blood and the 17 novel variants. (F) Distribution of mutations in individual BiPSC lines. See also Figures S2 and S3; Table S1; Table S3, sheet 2; and Table S4, sheet 1   ….

Figure 4. Transmission and Distribution of Somatic mtDNA Mutations to iPSCs (A) A total of 112 mtDNA mutations were discovered in parental cells (PF, CF, and blood) from 11 subjects. Of these, 39 variants (35%) were found in corresponding 130 iPSC lines. Among non-transmitted, transmitted, and novel mutations in iPSCs, comparable percentages of variants (68%, 69%, and 79%, respectively) were coding mutations in protein, rRNA, or tRNA genes. This suggests that most pathogenic mutations do not affect iPSC induction. However, certain coding mutations including in ND3, ND4L, and 14 tRNA genes were not detected in iPSCs, suggesting possible pathogenicity. n, the number of mtDNA mutations. Blue font genes were detected in parental cells. (B–D) A total of 80 high heteroplasmic (>15%) variants were detected in the present study in 130 FiPSC or BiPSC lines from 11 subjects. (B) The majority of these variants (76%) were non-synonymous or frame-shift mutations in protein-coding genes or affected rRNA and tRNA genes. (C) More than half of the mutations (56%) were never reported in a database containing whole mtDNA sequences from 26,850 healthy subjects representing the general human population (http://www.mitomap.org/MITOMAP). (D) Most mutations (90%) were never reported in a database containing sequences from healthy subjects with corresponding mtDNA haplotypes. freq., frequent. See also Figure S5 and Tables S3 and S4. ….

sjwilliamspa

Mutations will accumulate over age in mitochondrial DNA, however the current study has the difficulty that the authors could not use patient-age-matched controls, in essence they could only compare induced pluripotent stem cells derived from different patients. This could confound the results but the result with higher frequency of mutation in mtDNA in cells reprogrammed from younger patients is interesting but might limit the ability of autologous regenerative therapy in older patients. However reprogramming, although the method not mentioned here although I am assuming by transfection with lentivirus is a rough procedure, involving multiple dedifferentiation steps. Therefore it is very understandable that cells obtained from elderly patients would respond less favorably to such a rough reprogramming regimen, especially if it produced a higher degree of ROS, which has been shown to alter mtDNA. This is why I feel it is more advantageous to obtain a stem cell population from fat cells and forgo the Oct4, htert, reprogramming with lentiviral vectors.

 

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Insight on Cell Senescence

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Granule exocytosis mediates immune surveillance of senescent cells

A Sagiv1 , A Biran1 , M Yon2,3, J Simon2,4, SW Lowe2,4 and V Krizhanovsky1,2
Oncogene (2013) 32, 1971–1977    http://www.nature.com/onc/journal/v32/n15/pdf/onc2012206a.pdf

Senescence is a stable cell cycle arrest program that contributes to tumor suppression, organismal aging and certain wound healing responses. During liver fibrosis, for example, hepatic stellate cells initially proliferate and secrete extracellular matrix components that produce fibrosis; however, these cells eventually senesce and are cleared by immune cells, including natural killer (NK) cells. Here, we examine how NK cells target senescent cells and assess the impact of this process on liver fibrosis. We show that granule exocytosis, but not death-receptor-mediated apoptosis, is required for NK-cell-mediated killing of senescent cells. This pathway bias is due to upregulation of the decoy death receptor, Dcr2, an established senescence marker that attenuates NK-mediated cell death. Accordingly, mice with defects in granule exocytosis accumulate senescent stellate cells and display more liver fibrosis in response to a fibrogenic agent. Our results thus provide new insights into the immune surveillance of senescent cells and reveal how granule exocytosis has a protective role against liver fibrosis. Oncogene (2013) 32, 1971–1977; http://dx.doi.org:/10.1038/onc.2012.206

 

Senescence is accompanied by phenotypic and transcriptional changes that identify senescent cells in vitro and in vivo. For example, senescent cells display a large and flat morphology in vitro and upregulate a senescence-associated b-galactosidase (SA-b-gal).9 Senescent cells often display global changes in chromatin structure10 that are associated with downregulation of cell cycle genes and components of the extracellular matrix and upregulation of immune modulators and matrix degrading enzymes.4 Comparative analyses of gene expression data have produced some markers that appear specific for senescence,11 including the p15ink4b cyclin-dependent kinase inhibitor and the decoy receptor 2 (Dcr2, formally TNFRSF10D). Although p15ink4b likely contributes to the senescence-associated cell cycle arrest,12 whether decoy receptors or some other senescence markers actively participate in the program remains unknown.

Senescence acts through a coordinated program involving cell autonomous and cell nonautonomous components.13 In a cell autonomous manner, the Rb and p53 tumor suppressor pathways act to produce the stable cell cycle arrest that is the hallmark of senescence.1 These proteins are activated by, or activate, cyclindependent kinase inhibitors, such as p15ink4b, p16ink4a and p21, which lead to stable suppression of E2F target genes.10,14 Secreted proteins, regulated at least partially by NF-kB, enhance cell cycle arrest and are largely responsible for mediating the impact of senescent cells on tissue biology.15–17 These factors can attract immune cells, including natural killer (NK) cells, triggering the recognition and ultimate clearance of the senescent cells from tumors or tissue.4,18 Such mechanisms may be necessary to prevent the long-term damage that might be produced by senescent cells, and to facilitate tissue repair and homeostasis.

The mechanisms whereby NK cells eliminate senescent cells from tissues are not known. NK cells rely on two independent mechanisms to eliminate a variety of external and internal threats, including tumor cells.19,20 The ligands on the surface of NK cells, TRAIL and FAS ligand (FasL) bind corresponding receptors on target cells leading to caspase activation and cell death—a process that can be exquisitely controlled though the expression of various positive and negative regulators.21,22 NK cells can also eliminate target cells through granule exocytosis, a process involving the production of perforin and granzyme (A, B) containing granules, which are secreted from the NK cell upon interaction with the target cell.21,23 Perforin is responsible for perforating the cell membrane and thus enabling granzyme release into the target cells where it can induce cell death by both caspase-dependent and independent pathways.24 B

Here, we set out to understand how NK cells eliminate senescent cells from tissues and the implications of such mechanisms on liver fibrosis. Our results indicate that the granule exocytosis, and not death-receptor-mediated apoptosis, is essential for the NK-mediated surveillance of the senescent cells and that disruption of this pathway leads to the accumulation of senescent cells in damaged livers and increased fibrosis. Our study thus provides the key biological and mechanistic insights into the immune surveillance of senescent cells.

Figure 1. NK cells preferentially recognize senescent cells in a wide range of target:effector cell ratios. Senescent or growing IMR-90 fibroblasts were co-incubated with YT cells for 12 h at the indicated ratios and cytotoxicity was determined. The graphs represent the average and the s.e. of triplicate measurements from at least three independent experiments. *Po0.005.

Figure 2. Caspases are dispensable for NK-mediated cell killing of senescent cells. Senescent or growing IMR-90 fibroblasts were incubated for 12 h with either 2 or 10 nM FasL (a). Caspase inhibitors Z-VAD-FMK or Z-IEDT-FMK were added at the concentration of 10 mM as indicated. Cytotoxicity was determined at the end of the coincubation period. Senescent or growing IMR-90 fibroblasts were coincubated with YT cells for 12 h in the presence of 10 mM of caspase inhibitors Z-VAD-FMK or Z-IEDT-FMK and then the cytotoxicity was determined (b). The graphs represent the average and the s.e. of triplicate measurements from at least three independent experiments. *Po0.05, ***Po0.001.

Figure 3. Granule exocytosis pathway is required for NK-cell-mediated killing of senescent cells. Senescent and growing IMR-90 fibroblasts (a, c) or HSCs (b) were co-incubated with YT cells for 12 h (a, b) or with primary NK cells for 2 h (c). Cytotoxicity assays were performed either in the presence of 100 nM granule exocytosis inhibitor, CMA or following pre-incubation of the YT or primary NK cells with 25 mM Granzyme B inhibitor 3,4-DCI. The graphs represent the average and the the s.e. of triplicate measurements from at least three independent experiments. *Po0.01, **Po0.001, ***Po0.0001.

Figure 4. Dcr2 attenuates killing of senescent cells through the death receptor pathway. Dcr2 expression level in senescent and growing IMR- 90 fibroblasts (a, b) and human HSCs (c, d) were evaluated by quantitative RT–PCR analysis (a, c) and immunoblotting (b, d). Dcr2-deficient senescent IMR-90 cells were incubated with either 10 or 100 ng/ml TRAIL and cytotoxicity was determined (e), and Dcr2 knockdown confirmed (f). Senescent IMR-90 cells with siDcr2 or siControl were incubated with YT cells for 12 h and cytotoxicity was determined (g). In the parallel approach IMR-90 cells were infected with short hairpin RNA (shRNA) targeting Dcr2 (shDcr2) or control shRNA targeting luciferase (shLuci) and induced to senescence by etoposide treatment. Dcr2 protein level was assessed by immunoblot (h). The cells were co-incubated for 12 h with YT cells and cytotoxicity was determined (i). The graphs represent the average and the s.e. of triplicate measurements from at least four independent experiments *Po0.05, **Po0.001, ***Po0.0001.

Figure 5. Perforin promotes senescent cell clearance and limits liver fibrosis. Perforin knockout (Prf / ) and wt mice were treated with CCl4 to induce fibrosis. H&E and Sirius red staining show liver morphology and accumulation of fibrotic scar following the treatment (a). Morphometric analysis of Sirius red stained, entire liver sections (b). Expression of markers of activated HSCs, aSMA and Colagen1a, and senescence marker p15ink4b were tested by immunoblotting of whole-liver extracts (c). Four mice of each genotype are shown. SA-b-gal staining identified accumulation of senescent cells along the fibrotic scar areas in the livers (d). The presence of SA-b-gal-positive cells was quantified in the entire liver sections (e). At least five mice of each genotype were used for the analysis in B and E; **Po0.001, ***Po0.0001.

…..

NK-cell-mediated clearance of senescent cells is one component of the coordinated process whereby cellular senescence limits the extent of liver fibrosis and facilitates wound repair.4,18 Recent studies also suggest that senescent cell clearance by immune cells promotes tumor regression in established tumors.18 Our results demonstrate that the granule exocytosis pathway, but not the death receptor pathway, is necessary for the specific killing of senescent fibroblasts and stellate cells by NK cells and participates in the clearance of senescent activated HSCs to limit liver fibrosis. Therefore, NK-cell-mediated cytotoxicity through granule exocytosis contributes to immune surveillance of senescent cells in vitro and in vivo.

In addition to the granule exocytosis pathway, most cytotoxic lymphocytes engage the death receptor pathway to eliminate target cells. This pathway is widely used by NK cells in the liver.21 NK cell express high levels of the death receptor ligand TRAIL upon activation with IL-2,26 are suggested to participate in the surveillance of the HSCs,35 and protect against tumor development following chemical carcinogenesis.36 Given this, we were surprised that death-receptor-mediated cytotoxicity was dispensable for the immune surveillance of senescent cells. Consistent with these findings, an anti-TRAIL antibody failed to inhibit immune system-mediated tumor clearance following p53 restoration in a liver carcinoma model18 (W Xue and SWL, unpublished data). Of course, we cannot rule out the possibility that death receptor pathways contribute to senescent cell clearance in other settings.

Why does granule exocytosis, and not the death-receptor signaling, mediate NK-cell surveillance of senescent cells? Mechanistically, this appears partly because of the accumulation of Dcr2 during senescence, which occurs in fibroblasts, certain epithelial cells11,18 and, as shown here, also senescent activated HSCs. Dcr2 can bind death-receptor ligands, with higher affinity to TRAIL, but as it lacks the activation domain it prevents downstream signaling through the death receptor pathway31,37 and, therefore, can protect senescent cells from death-receptorligand-mediated killing. Another decoy receptor, Dcr3, has higher affinity to FASL.38 However, in contrast to Dcr2, Dcr3 is a secreted receptor and is much less likely to have a role in direct interaction between senescent and NK cells. Although previously considered merely a senescence marker, our results establish a functional role for Dcr2 in protecting senescent cells from cytotoxicity through the death receptor pathway induced by NK cells and possibly other cells as well. The biological rationale for this regulation remains unclear, but may serve to prevent autoimmunity following short-term tissue damage.

In addition to blocking the death receptor pathway, senescent cells may also stimulate NK cells to induce the perforin-mediated killing. Senescent cells upregulate expression of several ligands of NK-cell receptor NKG2D4,39 and ICAM-1, the ligand of NK-cell receptor LFA-1.40 Studies suggest that activation of the NKG2D receptor induces granule exocytosis to eliminate cancer cells, a process that might be reinforced by signaling from LFA-1.41 In this manner, ligands upregulated in senescent cells might activate multiple NK-cell receptors to trigger granule exocytosis.

The role of granule exocytosis in the surveillance of senescent cells has important ramifications for understanding and treating wound healing and cancer. Indeed, we show that the immune clearance of senescent activated HSCs has a significant impact on the pathophysiology of liver fibrosis in which the granule exocytosis pathway has been previously implicated.42,43 Beyond the liver, immune surveillance of senescent cells might have a significant role in other fibrosis-related pathological conditions.

Still, the most prevalent conditions where senescence has been studied to date involve cancer and aging.3,9 Senescent cells accumulate with age and contribute to functional decline of multiple tissues7,9 while perforin-mediated granule exocytosis diminishes at that time.47,48 Separate studies suggest that the integrity of the granule exocytosis pathway can modulate a variety of cancer phenotypes.49,50 Though definitive proof will require further testing, we speculate that the granule exocytosis pathway contributes to immune surveillance of senescent cells in each of these conditions. In principle, pharmacological modulation of this pathway, as has been recently described using IL21,51 might increase the clearance of senescent cells from premalignant, damaged or aged tissues to limit carcinogenesis and the decline in tissue function accompanying the accumulation of senescent cells.

REFERENCES 1 Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997; 88: 593–602.

2 Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 2002; 109: 335–346.

3 Narita M, Lowe SW. Senescence comes of age. Nat Med 2005; 11: 920–922.

4 Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C et al. Senescence of activated stellate cells limits liver fibrosis. Cell 2008; 134: 657–667.

5 Jun JI, Lau LF. The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nat Cell Biol 2010; 12: 676–685.

6 Pitiyage GN, Slijepcevic P, Gabrani A, Chianea YG, Lim KP, Prime SS et al. Senescent mesenchymal cells accumulate in human fibrosis by a telomereindependent mechanism and ameliorate fibrosis through matrix metalloproteinases. J Pathol 2011; 223: 604–617.

7 Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 2011; 479: 232–236.

8 Kang TW, Yevsa T, Woller N, Hoenicke L, Wuestefeld T, Dauch D et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 2011; 479: 547–551.

9 Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995; 92: 9363–9367.

10 Narita M, Nunez S, Heard E, Narita M, Lin AW, Hearn SA et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 2003; 113: 703–716

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Heat Shock Proteins (HSP) and Molecular Chaperones

Curator: Larry H. Bernstein, MD, FCAP

 

HSP

 

Report on the VIIth International Symposium on Heat Shock Proteins in Biology & Medicine

The major themes of this meeting were: new properties of heat shock proteins (HSPs) and heat shock factor (HSF) and role in the etiology of cancer, molecular chaperones in aging, extracellular HSPs in inflammation and immunity, role of heat shock and the heat shock response in immunity and cancer, protein aggregation disorders and HSP expression, and Hsp70 in blood cell differentiation.
This symposium was the seventh symposium in a series presided over by Dr Stuart Calderwood aimed at exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes. The biochemistry and ultrastructure of molecular chaperones was not emphasized, as these topics are well represented at other meetings. The major themes were: new properties of heat shock proteins (HSPs) and heat shock factor (HSF) and role in the etiology of cancer, molecular chaperones in aging, extracellular HSPs in inflammation and immunity, role of heat shock and the heat shock response in immunity and cancer, protein aggregation disorders and HSP expression, and Hsp70 in blood cell differentiation. This report gives a thematic overview and does not include all the topics presented.
NEW PROPERTIES OF HSPS AND HSF, AND ROLE IN THE ETIOLOGY OF CANCER 

One of the exciting aspects of the meeting involved advances made in understanding the biology of Hsp90. In recent years, we have understood the molecular chaperone activities of Hsp90 mostly in terms of its biochemistry, cooperative interactions with cochaperones. However, Dr Len Neckers (NCI/NIH), the conference keynote speaker, has opened up new areas in our understanding of this chaperone by characterizing the role of posttranslational modification (PTM) in terms of phosphorylation, acetylation, and sumoylation in Hsp90 biology. One particularly intriguing possibility is that altered signaling mechanisms characteristic of cancer may target such PTMs, and this could contribute to the “addiction to chaperones” observed in malignant cells. (Also discussed later by Dr Mehdi Mollapour, SUNY Upstate Medical University).

In addition, interesting differences in properties of the two Hsp90 isoforms have been detected. Dr Wei Li (University of Southern California) has shown that Hsp90a can be released into the extracellular environment and there take part in cell regulation, mediating for instance wound healing effects. In addition, proteomic studies carried out by Thomas Prince (NCI/NIH) in the Neckers lab indicate that Hsp90β may be more dedicated to “housekeeping” molecular chaperone functions while Hsp90α may play more glamorous roles in cell regulation. These distinctions might not be anticipated based on the rather minimal sequence differences between the Hsp90s but offer keen insights into the biology of this chaperone. Finally, Dr Tim Haystead (Duke University) discussed the approach of targeting ectopically expressed Hsp90 for imaging and treatment.

Another PTM with implications in the stress response is the modification of intracellular proteins by monosaccharides of O-linked β-N- acetylglucosamine (O-GlcNAc). Dr Natasha Zachara (Johns Hopkins University School of Medicine) discussed targets for this modification and roles in cytoprotection.

The poster session was also rich in Hsp90 studies, mostly from the Neckers lab—presentations by Kristin Beebe et al. (NCI/NIH) Posttranslational modification state of Hsp90 differentially affects binding of small molecule inhibitors; Toshiki Kijima et al. (NCI/NIH), Defined interactions between HSF1 and Hsp90; T. Prince et al. (NCI/NIH) Hsp90 and tyrosine kinase inhibitors: A synergistic approach towards combating cancer; Andrew W. Truman (University of Chicago)Quantitive ptoteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperonedependent regulation of ribonucleotide reductase. Inhibition of Hsp90 via Cdomain induces temporally distinct phosphorylation patterns; and Diana M. Dunn (SUNY Upstate Medical University)Phosphorylation of human Hsp90 threonine 115 modulates chaperone function and drug sensitivity.

Hsp70 is also emerging as a factor in cell regulation, exhibiting properties beyond a narrow role in chaperoning. Dr Michael Sherman (Boston University) showed a key role for Hsp72 in mammary cancer, and this property did not seem to depend on alterations in protein folding. Instead, Hsp72 appeared to function through its co-chaperone Bag3, a major regulatory molecule in cell signaling. In addition, a presentation by Stuart Calderwood (Harvard Medical School) that included work by Jianlin Gong showed that Hsp72 is required for tumor initiation and metastasis in murine spontaneous breast cancer. These effects appeared to be partially mediated through regulation of expression of the protoooncogene cMet, a key player in invasion and metastasis in cancer. We anticipate advances in understanding of the roles of individual members of the Hsp70 family, as is currently emerging for Hsp90. The prospect of targeting Hsp70 with small molecule inhibitors was elegantly discussed by Maureen Murphy (The Wistar Institute), who introduced a novel class of drugs that could selectively kill cancer cells by inhibiting Hsp70 function. In a related topic, Dr Mathias P. Mayer (University of Heidelberg) showed a detailed analysis of the activities of inhibitors targeting various domains in Hsp70.

Dr Takanori Eguchi (Harvard Medical School) then described his studies showing an unconventional role for the extracellular protease MMP3 as a nuclear protein that could trigger molecular chaperone synthesis (HspA7) in mammary cancer. Interestingly, a role in cancer for the Hsp70 co-chaperone Hsp40 was also shown by Dr Jane Trepel (NCI/NIH).

One presentation that stood apart was that of Dr Carmen Garrido (INSERM U866) who has shown very impressive studies indicating a key role for Hsp70 in hematopoiesis, acting through the factor GATA1. This role appeared to depend on nuclear localization of Hsp70, and Dr Garrido is attempting to study the role of PTM, particularly phosphorylation in this function/localization of Hsp70. This continued the theme of HSP PTM and regulation in the cell.

MOLECULAR CHAPERONES IN AGING

A symposium on molecular chaperones in aging was organized by Dr Shelley Buffenstein (University of Texas Health Science Center San Antonio). This symposium featured some fascinating studies on the naked mole rat (NMR), a rodent with a remarkable lifespan based on size (32 years compared to 3 years in the comparably sized mouse). This has permitted comparative biology studies that have uncovered important aspects of the aging process in mammals. Dr Buffenstein showed that one aspect of the proteotoxic response was enhanced in NMR—proteasome activity that was resistant to oxidative stress as well as conventional proteasome inhibitors. Such proteasome resistance appeared to be conferred by Hsp70 and Hsp40. Karl Rodriguez (also from the UTHSC San Antonio) stressed the importance of Hsp25 in the longevity of NMR. This small HSP is expressed to very high levels in this organism. Kenneth B. Storey (Carleton University) finally gave an encyclopedic presentation entitled “HeatShock Proteins and Hypometabolism in Nature”, discussing the multiple roles of chaperones in hibernation and other processes involving a step down in metabolism.

PROTEIN AGGREGATION DISORDERS AND HSP EXPRESSION

Michael Sherman (Boston University) chaired a lively and highly diverse session on protein aggregation disorders and HSPs. Gary Jones (Maynooth University) discussed his studies on the roles of Hsp104, Hsp70, and Hsp40 in prion propagation in yeast, concentrating on Hsp70. The Hsp complex was able to dissolve prions in yeast. Daniel Kaganovich (Hebrew University) then continued in a yeast theme, discussing a further strategy for resolving proteotoxic stress involving asymmetric cell division in which damaged proteins and mitochondria remain with the mother cell after mitosis. Nava Zaarur (Boston University) then discussed the role of aggresome particles in resolving aggregated proteins, in this case in eucaryotes. Alberto Macario (University of Maryland School of Medicine) discussed the role of chaperonins in proteotoxic disorders dealing with the effects of a pathogenic mutation of human CCT5 on its intrinsic properties. Dr Elaine C. Lee (University of Connecticut) discussed another type of stress. She showed significant roles for chaperones in osmotic stress responses of Caenorhabditis elegans models of polyglutamine diseases.

EXTRACELLULAR HSPS, INFLAMMATION, AND IMMUNITY

Although it is now generally accepted that HSPs can escape the confines of the cell, many questions still remain regarding their extracellular properties, particularly with regard to their immune effects. These questions include: whether HSPs are mostly immunostimulatory or immunosuppressive, whether they can induce sterile inflammation, and what structures on the immune cells recognize the HSPs. Dr Cristina Bonorino (Pontifícia Universidade Católica do Rio Grande do Sul) chaired a symposium “HSP as modulators of immunity: prokaryotic meets eukaryotic” featuring presentations by Robert Binder (University of Pittsburgh), Eckhart R. Podack (University of Miami), Renata Pasqualini (University of New Mexico Medical School), and Cristina Bonorino. In short, the talks indicated that while the prokaryotic chaperone DNA-PK can be immunosppressive and prolong the lifetime of transplanted tissues and reduce the morbidity of arthritis (Drs Bonorino and Kamal Moudgil (University of Maryland School of Medicine)), HSPs can also be immunostimulatory and act as cancer vaccines when associated with cancer antigens (Drs Binder and Podack). In the discussion, it was stressed that these effects may be related to HSP dose, with low doses of HSP antigen complex favoring immunity while higher doses may lead to immunoregulatory effects (Dr Binder). Most parties agreed that much future study is required to resolve all these issues. It was also suggested, inspired by the presentation of Dr Neckers, that HSP PTMs might also be playing roles in shading the immune effects of HSPs (Dr Bonorino). In the next session, Drs Shawn Wang (Virginia Commonwealth University School of Medicine) and John Subjeck (Roswell Park Cancer Institute) discussed the molecular foundations of their highly effective large HSP vaccines that are now in clinical trial for tumor immunotherapy. They indicated that the high avidity for antigen of the larger HSPs might be key for effectiveness. Although the nature of HSP receptors is still not fully resolved, Ayesha Murshid (Harvard Medical School) made a strong case for the scavenger receptor SREC-I as a key molecule in the effects of HSPs on immune cells. As many of the HSPs are in large families, it has not been clear whether all members of Hsp90 or Hsp70 can function outside the cell. Dr Wei Li (University of Southern California) showed that HSP90 family member Hsp90α is the major secreted factor while Dr John Williams (University of Chester) showed potent extracellular effects for human HSP70 isoform HSPA1A. Extracellular roles are not restricted to Hsp90, and Edward O’Brien (Libin Cardiovascular Institute of Alberta/University of Calgary) discussed the extracellular role of heat shock protein 27 (HSPB1) in inflammatory vascular disease. Another lively issue is whether HSPs are released as free proteins, packaged in exosomes, or whether both forms co-exist. This issue was discussed by Monika Fleshner (University of Colorado) and Antonio De Maio (University of California San Diego). Dr De Maio brought up the interesting scenario of Hsp70 binding directly to lipid membranes and perhaps forming membrane channels (Ryan White, University of Maryland).

HSPs are evidently not the only types of stress proteins that can function in the extracellular milieu, as indicated by Dr Michael A. Lynes (University of Connecticut). In a presentation entitled Therapeutic manipulation of the stress response during inflammatory disease, Dr Lynes showed a significant role for extracellular metallothionen in inflammatory bowel disease. Along those lines, Dr George Perdrizet (University of California San Diego) discussed the use of hyperbaric oxygen for enhanced wound healing in diabetic neuropathy, showing impressive clinical findings.

see more at — doi:  10.1007/s12192-014-0562-z

Lens Intermediate Filaments

 The ocular lens assembles two separate Intermediate Filament systems sequentially with differentiation. Canonical 8–11 nm IFs composed of Vimentin are assembled in lens epithelial cells and younger fiber cells, while the fiber cell-specific Beaded Filaments are switched on as fiber cell elongation initiates. Some of the key features of both filament systems are reviewed. Actin filaments and microtubules are essential to the most elemental functions of eukaryotic cells. These filamentous structures are assembled from proteins derived from small, highly conserved gene families. Though tissue specificity exists in the expression of some actin and tubulin family members, they are generally expressed in a ubiquitous manner, and are required for eukaryotic cell survival and replication. In contrast, the family of proteins that comprise the cytoplasmic Intermediate Filaments (IFs) is one of the largest in the human genome with greater than 60 members. IFs are generally not required for cell survival, and are absent from single cell eukaryotes, suggesting a more recent appearance on the evolutionary stage, and a less-essential role in the biology of the cell.
The IF family also differs sharply from actins and tubulins in that there is great variation in both size and sequence among the IF proteins, with sequence identity falling below 30% between more distant members of the human IF family. However, despite the large number of IF proteins available for the construction of IFs, any given cell typically expresses only 1–3 IF proteins, with expression tightly restricted to cell type and state of differentiation. This suggests a considerable degree of cell-specific specialization.
While IF proteins show considerable sequence and size variation, they are unified into a family on the basis of three major features:
1. Predicted domain structure (see figure 1): Algorithms that predict coiled-coil structure show a common predicted domain structure consisting of a) head and tail domains which are quite variable in both size and sequence, and b) a central rod domain where the size (~310 amino acids) and predicted secondary structure is strongly conserved. The rod domain consists of large regions of alpha helical structure (coil domains) interrupted by short non-helical regions (“linkers”) that connect the coil domains. The size, number, and placement of linkers and coils are well-conserved. Moreover, the coil domains exhibit a heptad repeat pattern where the 1, 4 positions in the heptad are dominated by hydrophobic amino acids. Because the 1,4 positions are aligned on one  side of the helix, they form a largely hydrophobic “stripe” that runs along one side of the alpha helix. This stripe mediates the dimerization of two matched coil domains. The hydrophobic stripe gently twists around the axis of the helix, giving rise to a supercoiling of two alpha helices, hence the “coiled-coil”.
The requisite hydrophobicity at the 1, 4 positions of the heptad can be conferred by any of several amino acids, thus the central rod domain of IF proteins, while showing conserved secondary structure, also exhibits a generally high degree of sequence variation. The exceptions to this are two short motifs found at either end of the central rod domain, commonly called the Helix Initiation Motif (HIM) and the Helix termination Motif (HTM). At these two sites the primary sequence among IF proteins is well conserved. Not surprisingly, the HIM and HTM motifs are intolerant of mutations, with the majority of known IF diseases arising from point mutations in these sites (http://www.interfil.org).
2. Conserved gene structure: Sequence analysis of IF proteins has allowed clustering of IF proteins into several major classes. Sequence conservation in the rod domain is high within a class (typically greater than 70%) but low between classes. Analysis of the IF genes shows that there is conservation of gene structure as well within the IF family, with the number and placement of introns and exons well conserved, especially in the central rod domain. The degree of gene structure conservation correlates well with the degree of primary sequence conservation, and reinforces the grouping of IF proteins into classes on the basis of primary sequence.
The Type I and II IF classes are called cytokeratins, and these comprise the IFs of epithelia. These begin assembly as an obligate heterodimer of one Type I and one Type II cytokeratin. The Type III IF proteins include vimentin, desmin, GFAP, and peripherin, and these are commonly found in tissues of mesenchymal origin. While Type III IF proteins can heterodimerize, they are more commonly found as homomeric filaments. The Type IV IF proteins are the neurofilament proteins Heavy (NFH), Medium (NFM) and Light (NFL), which assemble collectively into the IFs of neurons.
3. IF proteins form 8–11 nm diameter IFs. Ultimately, despite the differences in head/ tail size and sequence, and variation in the rod domain sequence, all cytoplasmic IF proteins typically assemble into 8–11 nm IFs.
The mechanism by which vimentin is removed as the cell transitions to the organelle-free state is unknown. In cells undergoing mitosis, vimentin IFs are routinely dismantled by phosphorlylation (Inagaki, Nishi et al. 1987), a modification that causes the relatively stable IF polymer break up into smaller subunits, thought to be tetramers. These are subsequently reassembled after cell division is complete. However, vimentin in lens fiber cells appears to removed, and not simply dismantled. IFs are known to be among the first targets of calcium activated proteases (calpains) in cells that are damaged, and many investigators have demonstrated the calcium-activated degradation of both vimentin and BFs in lens(Yoshida, Murachi et al. 1984; Truscott, Marcantonio et al. 1990; Marcantonio and Duncan 1991; Bettelheim, Qin et al. 1995; Andersson, Sjostrand et al. 1996; Sanderson, Marcantonio et al. 1996; Sanderson, Marcantonio et al. 2000). The dismantling of organelles implies a potential release of calcium from organelles in which it is otherwise routinely sequestered. Whether this release occurs, and whether it alters cytoplasmic calcium levels to a degree that would activate those calpains present in the fiber cell is not known.
Caspases activated in the apoptotic cascade can target conserved sites in IF proteins(Caulin, Salvesen et al. 1997). The elimination of organelles from the fiber cell represents an incomplete apoptotic event, and thus those enzymes responsible for organelle elimination may also represent a viable mechanism for explanation of vimentin’s suggested disappearance(Oshima 2002; Omary, Coulombe et al. 2004).
The loss or reduction of vimentin levels does not leave the mature lens fiber cell devoid of an IF system, however. In a manner that emulates IF switching seen in stratified epithelia, a second generation IF system is switched on in the lens as vimentin is being switched off. It is here where the story of the lens IF system takes the most unusual turn yet described in the IF field.
The initial recognition that the mature lens fiber cells departed from the IF dogma was made when Maisel and co-workers noted the presence of “Beaded Chain Filaments” or Beaded Filaments (BFs) in an electron microscopic analysis of chick lens homogenates (Maisel and Perry 1972; Maisel 1977; Bradley and Maisel 1978; Bradley, Ireland et al. 1979). These studies noted a clearly filamentous structure that was distinct from thin filaments, microtubules, and IFs, which at that time were emerging as the universal cytoskeletal structures common to essentially all vertebrate cells. Speculation emerged that these structures were thin filaments with bound alpha crystallin particles, or nucleosomes on DNA, but these explanations were ruled out experimentally (Bloemendal, Berbers et al. 1984; Ireland and Maisel 1984).
Consistent with the emerging role of IFAPs in modulating and adapting IF function is the observation that fiber cell vimentin IFs interact with the N Cadherin-gamma catenin complex (Leonard, Chan et al. 2008), lengsin(Wyatt, Gao et al. 2008), MIP(Lindsey Rose, Gourdie et al. 2006), periplakin(Yoon and FitzGerald 2008), tropomodulin(Fischer, Quinlan et al. 2003) and possibly other complexes which are present in lens(Bagchi, Katar et al. 2002; Straub, Boda et al. 2003; Bagchi, Katar et al. 2004). The number of candidate linker proteins demonstrated in lens leads to the expectation that the BF and IF are likely to accomplish multiple functions, and that these may be modulated as differentiation progresses, and as the fiber cell proteome changes, either by expression or proteolysis. Similarly, the small heat shock proteins, whose chaperone function appears essential to IF/BF assembly and maintenance, must be considered as critical parts of the biology of IFs in lens. Mutations in the small heat shock proteins have been shown to precipitate a failure in the IF systems in many tissues, and in lens specifically, and to subsequently emulate IF diseases (FitzGerald and Graham 1991; Nicholl and Quinlan 1994; Carter, Hutcheson et al. 1995; Vicart, Caron et al. 1998; Muchowski, Valdez et al. 1999; Perng, Cairns et al. 1999; Perng, Muchowski et al. 1999; Evgrafov, Mersiyanova et al. 2004; Treweek, Rekas et al. 2005; Song, Hanson et al. 2008). The growing multiplicity of IF interactions underscores the need to expect that failure in “IF function” in the lens can result from failure in a wide spectrum of proteins that affect assembly, phosphorylation, proteolytic modification, stability, removal, or linkage to other cellular structures, and that “IF failure” is likely to show considerable variability in phenotype.

Morphological characterization of the AlphaA- and AlphaB-crystallin double knockout mouse lens    Edited by Harry Maisel

Daniel L BoyleLarry TakemotoJames P Brady and Eric F Wawrousek
BMC Ophthalmology 2003; 3:3   http://dx.doi.org:/10.1186/1471-2415-3-3

Background: One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). Methods: Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. Results: Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. Conclusion: These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.

Dogfish a-Crystallin Sequences COMPARISON WITH SMALL HEAT SHOCK PROTEINS AND SCHISTOSOMA EGG ANTIGEN*

Wilfried W. de JongS, Jack A. M. Leunissen, Pieter J. M. Leenen, Anneke Zweers, and Marlies Versteeg
J BIOL CHEM  1988; 263(11):5141-5149

The amino acid sequences of the a-crystallin A and B chains of the dogfish, Squalus acanthias, have been determined. Comparison with a-crystallins from other species reveals that charged amino acid replacements have been strongly avoided in the evolution of this lens protein. The homology of a-crystallins with the small heat shock proteins is pronounced throughout the major part of the proteins, starting from the position of the first intron in the a-crystallin genes, but is also detectable in the amino-terminal sequences of human, Xenopus, and Drosophila small heat shock proteins. In addition, a remarkable short sequence similarity is present only in the amino termini of dogfish aB and Drosophila HSP22. The Schistosoma egg antigen p40 turns out to have a tandomly repeated region of homology with the common sequence domain of a-crystallins and small heat shock proteins. Comparison of hydropathy profiles indicates the conservation of conformation of the common domains in these three families of proteins. Construction of phylogenetic trees suggests that the aA and aB genes apparently originated from a single ancestral small heat shock protein gene and indicates that introns have been lost during the evolution of the heat shock protein.

Acknowledgment – Maisel, H. (ed) (1985) The Ocular Lens, Marcel Dekker, New. York

 

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Protein profiling in cancer and metabolic diseases

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Deep Protein Profiling Key

Company has encouraged by two recent reports that emphasise the importance of protein profiling to improve outcomes in cancer treatment.

http://www.technologynetworks.com/Proteomics/news.aspx?ID=190145

Proteome Sciences plc has strongly encouraged by two recent reports that emphasise the importance of protein profiling to improve outcomes in cancer treatment. These highlight the growing need for more detailed, personal assessment of protein profiles to improve the management of cancer treatment.

In the first study two groups from University College London and Cancer Research UK demonstrated that genetic mutations in cancer can lead to changes in the proteins on the cell surface1. These are new sequences which are seen as foreign by the body’s immune system and, with appropriate immunotherapy, the level of response in lung cancer was greatly enhanced.

However many of the patients with these types of mutations unfortunately still did not respond which highlighted the need for deeper analysis of the protein expression in tumours in order to better appreciate the mechanisms that contribute to treatment failure.

The second study, led by Professor Nigel Bundred of Manchester University, reported that use of two drugs that act on the same breast cancer target, an over-expressing protein called Her-2, were able to eradicate detectable tumours in around 10% of those treated in just 11 days, with 87% of those treated having a proteomic change indicating cells had stopped growing and/or cell death had increased2.

Whilst these results appear very promising it is worth noting that the over-expressing Her-2 target is only present in about 20% of breast tumours meaning this combination therapy was successful in clearing tumours in just 2% of the total breast cancer population.

Dr. Ian Pike, Chief Operating Officer of Proteome Sciences commented, “Both these recent studies should rightly be recognised as important steps forward towards better cancer treatment. However, in order to overcome the limitations of current drug therapy programs, a much deeper and more comprehensive analysis of the complex protein networks that regulate tumour growth and survival is required and will be essential to achieve a major advance in the battle to treat cancer.

“Our SysQuant® workflows provide that solution. As an example, in pancreatic cancer3 we have successfully mapped the complex network of regulatory processes and demonstrate the ability to devise personalised treatment combinations on an individual basis for each patient. A retrospective study with SysQuant® to predict response to the targeted drug Sorafenib in liver cancer is in process and we are planning further prospective trials to guide personalised treatment selection in liver cancer.

“We are already delivering systems-wide biology solutions through SysQuant® and TMTcalibrator™ programs to our clients that are generating novel biological data and results using more sensitive profiling that are helping them to better understand their drug development programs and to provide new biomarkers for tracking patient response in clinical trials.

“We are strongly positioned to deliver more comprehensive analysis of proteins and cellular pathways across other areas of disease and in particular to extend the use of SysQuant® with other leading cancer research groups in liver and other cancers.”

Proteome Sciences has also expanded its offering in personalised medicine through the use of its TMTcalibrator™ technology to uniquely identify protein biomarkers that reveal active cancer and other disease processes in body fluid samples. The importance of these ‘mechanistic’ biomarkers is that they are essential to monitor that drugs are being effective and that they can be used as early biomarkers of disease recurrence.

Using SysQuant® and TMTcalibrator™, Proteome Sciences can deliver more comprehensive analysis and provide unparalleled levels of sensitivity and breadth of coverage of the proteome, enabling faster, more efficient drug development and more accurate disease diagnosis.

 

Discovering ‘Outlier’ Enzymes

Researchers at TSRI and Salk Institute have discovered ‘Outlier’ enzymes that could offer new targets to treat type 2 diabetes and inflammatory disorders.

A team led by scientists at The Scripps Research Institute (TSRI) and the Salk Institute for Biological Studies have discovered two enzymes that appear to play a role in metabolism and inflammation—and might someday be targeted with drugs to treat type 2 diabetes and inflammatory disorders. The discovery is unusual because the enzymes do not bear a resemblance—in their structures or amino-acid sequences—to any known class of enzymes.

The team of scientists nevertheless identified them as “outlier” members of the serine/threonine hydrolase class, using newer techniques that detect biochemical activity. “A huge fraction of the human ‘proteome’ remains uncharacterized, and this paper shows how chemical approaches can be used to uncover proteins of a given functionality that have eluded classification based on sequence or predicted structure,” said co-senior author Benjamin F. Cravatt, chair of TSRI’s Department of Chemical Physiology.

“In this study, we found two genes that control levels of lipids with anti-diabetic and anti-inflammatory activity, suggesting exciting targets for diabetes and inflammatory diseases,” said co-senior author Alan Saghatelian, who holds the Dr. Frederik Paulsen Chair at the Salk Institute. The study, which appeared as a Nature Chemical Biology Advance Online Publication on March 28, 2016, began as an effort in the Cravatt laboratory to discover and characterize new serine/threonine hydrolases using fluorophosphonate (FP) probes—molecules that selectively bind and, in effect, label the active sites of these enzymes.

Pulling FP-binding proteins out of the entire proteome of test cells and identifying them using mass spectrometry techniques, the team matched nearly all to known hydrolases. The major outlier was a protein called androgen-induced gene 1 protein (AIG1). The only other one was a distant cousin in terms of sequence, a protein called ADTRP. “Neither of these proteins had been characterized as an enzyme; in fact, there had been little functional characterization of them at all,” said William H. Parsons, a research associate in the Cravatt laboratory who was co-first author of the study.

Experiments on AIG1 and ADTRP revealed that they do their enzymatic work in a unique way. “It looks like they have an active site that is novel—it had never been described in the literature,” said Parsons. Initial tests with panels of different enzyme inhibitors showed that AIG1 and ADTRP are moderately inhibited by inhibitors of lipases—enzymes that break down fats and other lipids. But on what specific lipids do these newly discovered outlier enzymes normally work?

At the Salk Institute, the Saghatelian laboratory was investigating a class of lipids it had discovered in 2014. Known as fatty acid esters of hydroxy fatty acids (FAHFAs), these molecules showed strong therapeutic potential. Saghatelian and his colleagues had found that boosting the levels of one key FAHFA lipid normalizes glucose levels in diabetic mice and also reduces inflammation.

“[Ben Cravatt’s] lab was screening panels of lipids to find the ones that their new enzymes work on,” said Saghatelian, who is a former research associate in the Cravatt laboratory. “We suggested they throw FAHFAs in there—and these turned out to be very good substrates.” The Cravatt laboratory soon developed powerful inhibitors of the newly discovered enzymes, and the two labs began working together, using the inhibitors and genetic techniques to explore the enzymes’ functions in vitro and in cultured cells.

Co-first author Matthew J. Kolar, an MD-PhD student, performed most of the experiments in the Saghatelian lab. The team concluded that AIG1 and ADTRP, at least in the cell types tested, appear to work mainly to break down FAHFAs and not any other major class of lipid. In principle, inhibitors of AIG1 and ADTRP could be developed into FAHFA-boosting therapies.

“Our prediction,” said Saghatelian, “is that if FAHFAs do what we think they’re doing, then using an enzyme inhibitor to block their degradation would make FAHFA levels go up and should thus reduce inflammation as well as improve glucose levels and insulin sensitivity.” The two labs are now collaborating on further studies of the new enzymes—and the potential benefits of inhibiting them—in mouse models of diabetes, inflammation and autoimmune disease.

“One of the neat things this study shows,” said Cravatt, “is that even for enzyme classes as well studied as the hydrolases, there may still be hidden members that, presumably by convergent evolution, arrived at that basic enzyme mechanism despite sharing no sequence or structural homology.”

Other co-authors of the study, “AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs,” were Siddhesh S. Kamat, Armand B. Cognetta III, Jonathan J. Hulce and Enrique Saez, of TSRI; and co-senior author Barbara B. Kahn of Beth Israel Deaconess Medical Center and Harvard Medical School

 

New Weapon Against Breast Cancer

Molecular marker in healthy tissue can predict a woman’s risk of getting the disease, research says.

Harvard Stem Cell Institute (HSCI) researchers at Dana-Farber Cancer Institute (DFCI) and collaborators at Brigham and Women’s Hospital (BWH) have identified a molecular marker in normal breast tissue that can predict a woman’s risk for developing breast cancer, the leading cause of death in women with cancer worldwide.

The work, led by HSCI principal faculty member Kornelia Polyak and Rulla Tamimi of BWH, was published in an early online release and in the April 1 issue of Cancer Research.

The study builds on Polyak’s earlier research finding that women already identified as having a high risk of developing cancer — namely those with a mutation called BRCA1 or BRCA2 — or women who did not give birth before their 30s had a higher number of mammary gland progenitor cells.

In the latest study, Polyak, Tamimi, and their colleagues examined biopsies, some taken as many as four decades ago, from 302 participants in the Nurses’ Health Study and the Nurses’ Health Study II who had been diagnosed with benign breast disease. The researchers compared tissue from the 69 women who later developed cancer to the tissue from the 233 women who did not. They found that women were five times as likely to develop cancer if they had a higher percentage of Ki67, a molecular marker that identifies proliferating cells, in the cells that line the mammary ducts and milk-producing lobules. These cells, called the mammary epithelium, undergo drastic changes throughout a woman’s life, and the majority of breast cancers originate in these tissues.

Doctors already test breast tumors for Ki67 levels, which can inform decisions about treatment, but this is the first time scientists have been able to link Ki67 to precancerous tissue and use it as a predictive tool.

“Instead of only telling women that they don’t have cancer, we could test the biopsies and tell women if they were at high risk or low risk for developing breast cancer in the future,” said Polyak, a breast cancer researcher at Dana-Farber and co-senior author of the paper.

“Currently, we are not able to do a very good job at distinguishing women at high and low risk of breast cancer,” added co-senior author Tamimi, an associate professor at the Harvard T.H. Chan School of Public Health and Harvard Medical School. “By identifying women at high risk of breast cancer, we can better develop individualized screening and also target risk reducing strategies.”

To date, mammograms are the best tool for the early detection, but there are risks associated with screening. False positive and negative results and over-diagnosis could cause psychological distress, delay treatment, or lead to overtreatment, according to the National Cancer Institute (NCI).

Mammography machines also use low doses of radiation. While a single mammogram is unlikely to cause harm, repeated screening can potentially cause cancer, though the NCI writes that the benefits “nearly always outweigh the risks.”

“If we can minimize unnecessary radiation for women at low risk, that would be good,” said Tamimi.

Screening for Ki67 levels would “be easy to apply in the current setting,” said Polyak, though the researchers first want to reproduce the results in an independent cohort of women.

 

AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs

William H ParsonsMatthew J Kolar, …., Barbara B KahnAlan Saghatelian & Benjamin F Cravatt

Nature Chemical Biology 28 March 2016                    http://dx.doi.org:/10.1038/nchembio.2051

Enzyme classes may contain outlier members that share mechanistic, but not sequence or structural, relatedness with more common representatives. The functional annotation of such exceptional proteins can be challenging. Here, we use activity-based profiling to discover that the poorly characterized multipass transmembrane proteins AIG1 and ADTRP are atypical hydrolytic enzymes that depend on conserved threonine and histidine residues for catalysis. Both AIG1 and ADTRP hydrolyze bioactive fatty acid esters of hydroxy fatty acids (FAHFAs) but not other major classes of lipids. We identify multiple cell-active, covalent inhibitors of AIG1 and show that these agents block FAHFA hydrolysis in mammalian cells. These results indicate that AIG1 and ADTRP are founding members of an evolutionarily conserved class of transmembrane threonine hydrolases involved in bioactive lipid metabolism. More generally, our findings demonstrate how chemical proteomics can excavate potential cases of convergent or parallel protein evolution that defy conventional sequence- and structure-based predictions.

Figure 1: Discovery and characterization of AIG1 and ADTRP as FP-reactive proteins in the human proteome.

 

http://www.nature.com/nchembio/journal/vaop/ncurrent/carousel/nchembio.2051-F1.jpg

(a) Competitive ABPP-SILAC analysis to identify FP-alkyne-inhibited proteins, in which protein enrichment and inhibition were measured in proteomic lysates from SKOV3 cells treated with FP-alkyne (20 μM, 1 h) or DMSO using the FP-biotin…

 

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Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesteras-1 inhibitors

Proc Natl Acad Sci U S A. 2011 Apr 26; 108(17): 6811–6816.    doi:  10.1073/pnas.1015248108
National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-β-lactams (ABLs), as potent (IC50 values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.

Protein phosphorylation is a pervasive and dynamic posttranslational protein modification in eukaryotic cells. In mammals, more than 500 protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues on proteins (1). A much more limited number of phosphatases are responsible for reversing these phosphorylation events (2). For instance, protein phosphatase 2A (PP2A) and PP1 are thought to be responsible together for > 90% of the total serine/threonine phosphatase activity in mammalian cells (3). Specificity is imparted on PP2A activity by multiple mechanisms, including dynamic interactions between the catalytic subunit (C) and different protein-binding partners (B subunits), as well as a variety of posttranslational chemical modifications (2, 4). Within the latter category is an unusual methylesterification event found at the C terminus of the catalytic subunit of PP2A that is introduced and removed by a specific methyltransferase (leucine carbxoylmethyltransferase-1 or LCMT1) (5, 6) and methylesterase (protein phosphatase methylesterase-1 or PME-1) (7), respectively (Fig. 1A). PP2A carboxymethylation (hereafter referred to as “methylation”) has been proposed to regulate PP2A activity, at least in part, by modulating the binding interaction of the C subunit with various regulatory B subunits (810). A predicted outcome of these shifts in subunit association is the targeting of PP2A to different protein substrates in cells. PME-1 has also been hypothesized to stabilize inactive forms of nuclear PP2A (11), and recent structural studies have shed light on the physical interactions between PME-1 and the PP2A holoenzyme (12).

There were several keys to the success of our probe development effort. First, screening for inhibitors of PME-1 benefited from the fluopol-ABPP technology, which circumvented the limited throughput of previously described substrate assays for this enzyme. Second, we were fortunate that the NIH compound library contained several members of the ABL class of small molecules. These chiral compounds, which represent an academic contribution to the NIH library, occupy an unusual portion of structural space that is poorly accessed by commercial compound collections. Although at the time of their original synthesis (23) it may not have been possible to predict whether these ABLs would show specific biological activity, their incorporation into the NIH library provided a forum for screening against many proteins and cellular targets, culminating in their identification as PME-1 inhibitors. We then used advanced chemoproteomic assays to confirm the remarkable selectivity displayed by ABLs for PME-1 across (and beyond) the serine hydrolase superfamily. That the mechanism for PME-1 inhibition involves acylation of the enzyme’s conserved serine nucleophile (Fig. 3) suggests that exploration of a more structurally diverse set of ABLs might uncover inhibitors for other serine hydrolases. In this way, the chemical information gained from a single high-throughput screen may be leveraged to initiate probe development programs for additional enzyme targets.

Projecting forward, this research provides an example of how public small-molecule screening centers can serve as a portal for spawning academic collaborations between chemical biology and synthetic chemistry labs. By continuing to develop versatile high-throughput screens and combining them with a small-molecule library of expanding structural diversity conferred by advanced synthetic methodologies, academic biologists and chemists are well-positioned to collaboratively deliver pharmacological probes for a wide range of proteins and pathways in cell biology.

 

New weapon against breast cancer

Molecular marker in healthy tissue can predict a woman’s risk of getting the disease, research says

April 6, 2016 | Popular
BRC_Cancer605

 

New Group of Aging-Related Proteins Discovered

http://www.genengnews.com/gen-news-highlights/new-group-of-aging-related-proteins-discovered/81252599/

Scientists have discovered a group of six proteins that may help to divulge secrets of how we age, potentially unlocking new insights into diabetes, Alzheimer’s, cancer, and other aging-related diseases.

The proteins appear to play several roles in our bodies’ cells, from decreasing the amount of damaging free radicals and controlling the rate at which cells die to boosting metabolism and helping tissues throughout the body respond better to insulin. The naturally occurring amounts of each protein decrease with age, leading investigators to believe that they play an important role in the aging process and the onset of diseases linked to older age.

The research team led by Pinchas Cohen, M.D., dean and professor of the University of Southern California Leonard Davis School of Gerontology, identified the proteins and observed their origin from mitochondria and their game-changing roles in metabolism and cell survival. This latest finding builds upon prior research by Dr. Cohen and his team that uncovered two significant proteins, humanin and MOTS-c, hormones that appear to have significant roles in metabolism and diseases of aging.

Unlike most other proteins, humanin and MOTS-c are encoded in mitochondria. Dr. Cohen’s team used computer analysis to see if the part of the mitochondrial genome that provides the code for humanin was coding for other proteins as well. The analysis uncovered the genes for six new proteins, which were dubbed small humanin-like peptides, or SHLPs, 1 through 6 (pronounced “schlep”).

After identifying the six SHLPs and successfully developing antibodies to test for several of them, the team examined both mouse tissues and human cells to determine their abundance in different organs as well as their functions. The proteins were distributed quite differently among organs, which suggests that the proteins have varying functions based on where they are in the body. Of particular interest is SHLP 2, according to Dr. Cohen.  The protein appears to have insulin-sensitizing, antidiabetic effects as well as neuroprotective activity that may emerge as a strategy to combat Alzheimer’s disease. He added that SHLP 6 is also intriguing, with a unique ability to promote cancer cell death and thus potentially target malignant diseases.

Proteins That May Protect Against Age Related Illnesses Discovered

 

The cell proliferation antigen Ki-67 organises heterochromatin

 Michal Sobecki, 

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.

 

A protein called Ki-67 is only produced in actively dividing cells, where it is located in the nucleus – the structure that contains most of the cell’s DNA. Researchers often use Ki-67 as a marker to identify which cells are actively dividing in tissue samples from cancer patients, and previous studies indicated that Ki-67 is needed for cells to divide. However, the exact role of this protein was not clear. Before cells can divide they need to make large amounts of new proteins using molecular machines called ribosomes and it has been suggested that Ki-67 helps to produce ribosomes.

Now, Sobecki et al. used genetic techniques to study the role of Ki-67 in mice. The experiments show that Ki-67 is not required for cells to divide in the laboratory or to make ribosomes. Instead, Ki-67 alters the way that DNA is packaged in the nucleus. Loss of Ki-67 from mice cells resulted in DNA becoming less compact, which in turn altered the activity of genes in those cells.

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Sleep and memory

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Learning with the Lights Out

Researchers are uncovering the link between sleep and learning and how it changes throughout our lives.

By Jenny Rood | March 1, 2016    http://www.the-scientist.com/?articles.view/articleNo/45335/title/Learning-with-the-Lights-Out

NIGHTY NIGHT: Goffredina Spanò from Jamie Edgin’s University of Arizona lab uses polysomnography to measure sleep in a toddler with Down syndrome.PAMELA SPEDER

By the early 2000s, scientists had found that sleep helps young adults consolidate memory by reinforcing and filing away daytime experiences. But the older adults that Rebecca Spencer was studying at the University of Massachusetts Amherst didn’t seem to experience the same benefit. Spencer wondered if developmental stage altered the relationship between sleep and memory, and chose nearby preschool children as subjects. She found that habitual nappers benefitted the most from daytime rest, largely because their memories decayed the most without a nap. “By staying awake, they have more interference from daytime experiences,” Spencer explains.

Until recently, most of the research into the relationship between memory and sleep has been conducted using young adults or animal models. These studies have suggested that dampened sensory inputs during sleep allow the brain to replay the day’s events during a period relatively free of distracting information, helping to solidify connections and transfer daytime hippocampal memories into long-term storage in the cortex. But how sleep and memory interact at different ages has been an open question.

In children younger than 18 months, learning is thought to occur in the cortex because the hippocampus isn’t yet fully developed. As a result, researchers hypothesize that infants don’t replay memories during sleep, the way adults do. Instead, sleep merely seems to prevent infants from forgetting as much as they would if they were awake. “The net effect is that sleep permits infants to retain more of the redundant details of a learning experience,” says experimental psychologist Rebecca Gómez of the University of Arizona. By the time they are two years old, “we think that children have the brain development that supports an active process of consolidation,” she adds.

At that age, adequate nighttime sleep becomes critical for learning. Toddlers who sleep less than 10 hours display lasting cognitive deficits, even if they catch up on sleep later in their development (Sleep, 30:1213-19, 2007). The effects are particularly strong in children with developmental disorders, who often suffer from sleep disruptions. “Kids with Down syndrome that are sleep-impaired look like they have very large differences in language,” says Jamie Edgin of the University of Arizona who studies sleep and cognition in such children. When comparing Down syndrome children who are sleep deprived with those who sleep normally, she has observed a vocabulary difference of more than 190 words on language tests, even after controlling for behavioral differences.

Understanding the impact of sleep on memory could also help another at-risk group of learners at the other end of the age spectrum. Previous research has suggested that older adults don’t remember newly acquired motor skills as well as young adults do, perhaps because the posttraining stages of the learning process appear diminished. But neuroscientist Maria Korman and her colleagues at the University of Haifa in Israel recently demonstrated that a nap soon after learning can allow the elderly to retain procedural memories just as well as younger people (Neurosci Lett, 606:173-76, 2015). Korman hypothesizes that by shortening the interval between learning and consolidation, the nap prevents intervening experiences from weakening the memory before it solidifies. Overnight sleep might be even better, if the motor skills—in this case a complex sequence of finger and thumb movements on the nondominant hand—are taught late enough in the day, something she is testing now.

Optimizing the timing of sleep and training in the elderly takes advantage of something Korman sees as a positive side of growing old. “As we age, our neural system becomes more aware of the relevance of the task,” Korman says. Unlike young adults who solidify all the information they acquire throughout the day, older people consolidate “those experiences that were tagged by the brain as very important.”

Tests for older adults’ memory acuity are generating new findings about the relationship between sleep and memory at other ages as well. After learning at a conference about a memory test for cognitive impairment and dementia in older adults, neuroscientist Jeanne Duffy of Brigham and Women’s Hospital in Boston wondered if sleep could help strengthen the connection between names and faces. She and her colleagues found that young adults who slept overnight after learning a list of 20 names and faces showed a 12 percent increase in retention when tested 12 hours later compared with subjects who didn’t sleep between training and testing (Neurobiol Learn Mem, 126:31-38, 2015). The findings have “an immediate real-world application,” Duffy says, as they address a common memory concern among people of all ages.

A poll by the National Sleep Foundation found that adolescents have a deficit of nearly two hours of sleep per night during the school week compared with the weekend, suggesting the potential for serious learning impairments, according to Jared Saletin, a postdoctoral sleep researcher at Brown University. In fact, one study found that restricting 13- to 17-year-olds to six and a half hours of sleep a night for five nights reduced the information they absorbed in a school-like setting (J Adolesc Health, 47:523-25, 2010). However, other studies have suggested that four nights of just five hours of sleep didn’t impair 14- to 16-year-olds’ performance on tests of skills and vocabulary (Sleep Med, 12:170-78, 2011). A lack of consistency in study design and the ages of the subjects makes these conflicting results difficult to interpret, Gómez writes in a review, and much remains to be discovered about the true impact of sleep deficits on teenagers’ learning (Trans Issues in Psych Sci, 1:116-25, 2015).

Developing a fuller picture of what happens to memories during sleep—and how best to tweak sleep habits to aid the recall process—could benefit some of society’s most sleep-deprived members of every age. “We need to understand this role of sleep in memory because there is such potential for intervention,” Spencer says. “Now that we have a well-founded concept of what sleep can do for memory, it’s time to put it to the test.”

 

Associations Between Sleep Duration Patterns and Behavioral/Cognitive Functioning at School Entry

Évelyne Touchette, MPs,1,2 Dominique Petit, PhD,1 Jean R. Séguin, PhD,3,4,5 Michel Boivin, PhD,6,7 Richard E. Tremblay, PhD,2,3,4,5,8 and Jacques Y. Montplaisir, MD, CRCP(c), PhD1,5
Sleep. 2007 Sep 1; 30(9): 1213–1219.     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1978413/
See commentary “Sleep and the Developing Brain

The aim of the study was to investigate the associations between longitudinal sleep duration patterns and behavioral/cognitive functioning at school entry.
Design, Setting, and Participants:   Hyperactivity-impulsivity (HI), inattention, and daytime sleepiness scores were measured by questionnaire at 6 years of age in a sample of births from 1997 to 1998 in a Canadian province (N=1492). The Peabody Picture Vocabulary Test – Revised (PPVT-R) was administered at 5 years of age and the Block Design subtest (WISC-III) was administered at 6 years of age. Sleep duration was reported yearly by the children’s mothers from age 2.5 to 6 years. A group-based semiparametric mixture model was used to estimate developmental patterns of sleep duration. The relationships between sleep duration patterns and both behavioral items and neurodevelopmental tasks were tested using weighted multivariate logistic regression models to control for potentially confounding psychosocial factors.  Results:   Four sleep duration patterns were identified: short persistent (6.0%), short increasing (4.8%),10-hour persistent (50.3%), and 11-hour persistent (38.9%). The association of short sleep duration patterns with high HI scores (P=0.001), low PPVT-R performance (P=0.002), and low Block Design subtest performance (P=0.004) remained significant after adjusting for potentially confounding variables.   Conclusions:   Shortened sleep duration, especially before the age of 41 months, is associated with externalizing problems such as HI and lower cognitive performance on neurodevelopmental tests. Results highlight the importance of giving a child the opportunity to sleep at least 10 hours per night throughout early childhood.

Citation: Touchette E; Petit D; Séguin JR; Boivin M; Tremblay RE; Montplaisir JY. Associations between sleep duration patterns and behavioral/cognitive functioning at school entry. SLEEP 2007;30(9):1213-1219.

Nap it or leave it in the elderly: A nap after practice relaxes age-related limitations in procedural memory consolidation

M. Kormana, , Y. DaganbA. Karnib   

 Highlights

•   Elderly individuals gain in practicing a new motor task as do young adults.
•   But elderly individuals fail to show delayed (offline) memory related gains.
•   A post-training nap uncovered robust offline skill consolidation in the elderly.
•   Consolidation processes are preserved in aging but are more stringently controlled.
•   Sleep scheduling can relax age related constraints on mnemonic processes.       
Using a training protocol that effectively induces procedural memory consolidation (PMC) in young adults, we show that older adults are good learners, robustly improving their motor performance during training. However, performance declined over the day, and overnight ‘offline’ consolidation phase performance gains were under-expressed. A post-training nap countered these deficits. PMC processes are preserved but under-engaged in the elderly; sleep can relax some of the age-related constraints on long-term plasticity.

 

A new face of sleep: The impact of post-learning sleep on recognition memory for face-name associations

Leonie Maurera, c, Kirsi-Marja Zittinga, b, Kieran Elliotta, Charles A. Czeislera, b, Joseph M. Rondaa, b, Jeanne F. Duffya, b, ,

Highlights

•   We tested whether sleep influences the accuracy of remembering face-name associations.
•   Presentation and recall were 12 h apart, one time with 8 h sleep and once without.
•   More correct face-name pairs were recalled when there was a sleep opportunity.
•   Sleep duration or sleep stage was not associated with improvement between conditions.     

Sleep has been demonstrated to improve consolidation of many types of new memories. However, few prior studies have examined how sleep impacts learning of face-name associations. The recognition of a new face along with the associated name is an important human cognitive skill. Here we investigated whether post-presentation sleep impacts recognition memory of new face-name associations in healthy adults.

Fourteen participants were tested twice. Each time, they were presented 20 photos of faces with a corresponding name. Twelve hours later, they were shown each face twice, once with the correct and once with an incorrect name, and asked if each face-name combination was correct and to rate their confidence. In one condition the 12-h interval between presentation and recall included an 8-h nighttime sleep opportunity (“Sleep”), while in the other condition they remained awake (“Wake”).

There were more correct and highly confident correct responses when the interval between presentation and recall included a sleep opportunity, although improvement between the “Wake” and “Sleep” conditions was not related to duration of sleep or any sleep stage.

These data suggest that a nighttime sleep opportunity improves the ability to correctly recognize face-name associations. Further studies investigating the mechanism of this improvement are important, as this finding has implications for individuals with sleep disturbances and/or memory impairments.

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The Philosopher’s Stone?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Mitochondria trigger cell aging, researchers discover

How to rejuvenate or prevent aging in human and mice cells
February 5, 2016   http://www.kurzweilai.net/mitochondria-shown-to-trigger-cell-ageing

http://www.kurzweilai.net/images/mitochondria-clearing.jpg

Preventing aging and rejuvenating human and mice cells in the lab (credit: Clara Correia‐Melo et al./EMBO Journal)

An international team of scientists led by João Passos at Newcastle University has for the first time shown thatmitochondria (the “batteries” of the cells) are major triggers for aging, and eliminating them upon the induction of senescence prevents senescence in the aging mouse liver.

As we grow old, cells in our bodies accumulate different types of damage and have increased inflammation, factors that are thought to contribute to the aging process.

As described Feb. 4 in an open-access paper in the EMBO Journal, the team carried out a series of genetic experiments involving human cells grown in the laboratory and succeeded in eliminating the majority, if not all, the mitochondria from aging cells.

Tricking mitochondria

http://www.kurzweilai.net/images/mitochondrion.jpg

Components of a typical mitochondrion (credit: Kelvinsong/Creative Commons)

Cells can normally eliminate faulty mitochondria by a process called mitophagy. The scientists were able to “trick” the cells into inducing this process in a grand scale, until all the mitochondria within the cells were physically removed.

To their surprise, they observed that the aging cells, after losing their mitochondria, showed characteristics similar to younger cells — that is, they became rejuvenated. The levels of inflammatory molecules, oxygen free radicals and expression of genes, which are among the makers of cellular aging, dropped to the level that would be expected in younger cells.

“This is a very exciting and surprising discovery,” said Passos. “We already had some clues that mitochondria played a role in the aging of cells, but scientists around the world have struggled to understand exactly how and to what extent these were involved.”

The team, involving other universities in the UK and the U.S., also deciphered a new mechanism by which mitochondria contribute to aging: mitochondrial biogenesis, the complex process by which mitochondria replicate themselves, is a major driver of cellular aging.

This work was funded by the UK Biotechnology and Biological Sciences Research Council.


Abstract of Mitochondria are required for pro-ageing features of the senescent phenotype

Cell senescence is an important tumour suppressor mechanism and driver of ageing. Both functions are dependent on the development of the senescent phenotype, which involves an overproduction of pro‐inflammatory and pro‐oxidant signals. However, the exact mechanisms regulating these phenotypes remain poorly understood. Here, we show the critical role of mitochondria in cellular senescence. In multiple models of senescence, absence of mitochondria reduced a spectrum of senescence effectors and phenotypes while preserving ATP production via enhanced glycolysis. Global transcriptomic analysis by RNA sequencing revealed that a vast number of senescent‐associated changes are dependent on mitochondria, particularly the pro‐inflammatory phenotype. Mechanistically, we show that the ATM, Akt and mTORC1 phosphorylation cascade integrates signals from the DNA damage response (DDR) towards PGC‐1β‐dependent mitochondrial biogenesis, contributing to a ROS‐mediated activation of the DDR and cell cycle arrest. Finally, we demonstrate that the reduction in mitochondrial content in vivo, by either mTORC1 inhibition or PGC‐1β deletion, prevents senescence in the ageing mouse liver. Our results suggest that mitochondria are a candidate target for interventions to reduce the deleterious impact of senescence in ageing tissues.

 

 

Mayo Clinic researchers extend lifespan by up to 35 percent in mice

February 3, 2016   http://www.kurzweilai.net/mayo-clinic-researchers-extend-lifespan-by-up-to-35-percent-in-mice

Researchers at Mayo Clinic have discovered that senescent cells — cells that no longer divide and accumulate with age — shorten lifespan by as much as 35 percent in normal mice.

Removing these aging cells delays tumor formation, preserves tissue and organ function, and extends lifespan without observed adverse effects, the researchers found, writing Feb. 3 in Nature.

“Cellular senescence is a biological mechanism that functions as an ‘emergency brake’ used by damaged cells to stop dividing,” says Jan van Deursen, Ph.D., Chair of Biochemistry and Molecular biology at Mayo Clinic, and senior author of the paper. “While halting cell division of these cells is important for cancer prevention, it has been theorized that once the ‘emergency brake’ has been pulled, these cells are no longer necessary.”

As the immune system becomes less effective, senescent cells build up and damage adjacent cells, causing chronic inflammation, which is closely associated with frailty and age-related diseases.

Mayo Clinic researchers used a compound called AP20187 to remove senescent cells, which delayed tumor formation and reduced age-related deterioration of several organs, extending mediian lifespan of treated mice by 17 to 35 percent. The mice also had a healthier appearance and less inflammation in fat, muscle and kidney tissue.

The research was supported by the National Institutes of Health, the Paul F. Glenn Foundation, the Ellison Medical Foundation, the Noaber Foundation, and the Mayo Clinic Robert and Arlene Kogod Center on Aging.

Van Deursen is a co-inventor of the technology that has been licensed by Mayo Clinic to Unity Biotechnology. Mayo Clinic and Van Deursen have a financial interest in the technology.

https://youtu.be/w8UHzkXC4HQ

Mayo Clinic | Researchers Extend Lifespan by as Much as 35 Percent in Mice

 

Abstract of Naturally occurring p16Ink4a-positive cells shorten healthy lifespan

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16Ink4a (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16Ink4a-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16Ink4a-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16Ink4a-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

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Beyond tau and amyloid

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

BEYOND AΒ AND TAU: OTHER TOXIC INSULTS AND AD PATHOLOGY

 

Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders.

Berislav V. Zlokovic

Nature Reviews Neuroscience 12, 723-738 (December 2011) |   http:dx.doi.org:/10.1038/nrn3114

The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood–brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal ‘milieu’, which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer’s disease, and highlights therapeutic opportunities relating to these neurovascular deficits.

 

Summary

The neurovascular unit comprises vascular cells (endothelial cells, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (astrocytes, microglia and oliogodendroglia) and neurons.
Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors.
The interactions between endothelial cells and pericytes are crucial for the formation and maintenance of the BBB. Indeed, pericyte deficiency leads to BBB breakdown and extravasation of multiple vasculotoxic and neurotoxic circulating macromolecules, which can contribute to neuronal dysfunction, cognitive decline and neurodegenerative changes.
Alterations in cerebrovascular metabolic functions can also lead to the secretion of multiple neurotoxic and inflammatory factors.
BBB dysfunction and/or breakdown and cerebral blood flow (CBF) reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, blood–spinal cord barrier breakdown and spinal cord hypoperfusion have been reported prior to motor neuron cell death.
Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder have shown diminished amyloid-β clearance from brain tissue. The recognition of amyloid-β clearance pathways opens exciting new therapeutic opportunities for this disease.
‘Multiple-target, multiple-action’ agents will stand a better chance of controlling the complex disease mechanisms that mediate neurodegeneration in disorders such as Alzheimer’s disease than will agents that have only one target. According to the vasculo-neuronal-inflammatory triad model of neurodegenerative disorders, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia all represent important therapeutic targets.

 

Neurons depend on blood vessels for their oxygen and nutrient supplies, and for the removal of carbon dioxide and other potentially toxic metabolites from the brain’s interstitial fluid (ISF). The importance of the circulatory system to the human brain is highlighted by the fact that although the brain comprises ~2% of total body mass, it receives up to 20% of cardiac output and is responsible for ~20% and ~25% of the body’s oxygen consumption and glucose consumption, respectively1. To underline this point, when cerebral blood flow (CBF) stops, brain functions end within seconds and damage to neurons occurs within minutes2.

Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Microvascular deficits diminish CBF and, consequently, the brain’s supply of oxygen, energy substrates and nutrients. Moreover, such deficits impair the clearance of neurotoxic molecules that accumulate and/or are deposited in the ISF, non-neuronal cells and neurons. Recent evidence suggests that vascular dysfunction leads to neuronal dysfunction and neurodegeneration, and that it might contribute to the development of proteinaceous brain and cerebrovascular ‘storage’ disorders. Such disorders include cerebral β-amyloidosis and cerebral amyloid angiopathy (CAA), which are caused by accumulation of the peptide amyloid-β in the brain and the vessel wall, respectively, and are features of Alzheimer’s disease1.

In this Review, I will discuss neurovascular pathways to neurodegeneration, placing a focus on Alzheimer’s disease because more is known about neurovascular dysfunction in this disease than in other neurodegenerative disorders. The article first examines transport mechanisms for molecules to cross the BBB, before exploring the processes that are involved in BBB breakdown at the molecular and cellular levels, and the consequences of BBB breakdown, hypoperfusion, and hypoxia and endothelial metabolic dysfunction for neuronal function. Next, the article reviews evidence for neurovascular changes during normal ageing and neurovascular BBB dysfunction in various neurodegenerative diseases, including evidence suggesting that vascular defects precede neuronal changes. Finally, the article considers specific mechanisms that are associated with BBB dysfunction in Alzheimer’s disease and ALS, and therapeutic opportunities relating to these neurovascular deficits.

The neurovascular unit

The neurovascular unit (NVU) comprises vascular cells (that is, endothelium, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (that is, astrocytes, microglia and oliogodendroglia) and neurons1,2, 13 (Fig. 1). In the NVU, the endothelial cells together form a highly specialized membrane around blood vessels. This membrane underlies the BBB and limits the entry of plasma components, red blood cells (RBCs) and leukocytes into the brain. The BBB also regulates the delivery into the CNS of circulating energy metabolites and essential nutrients that are required for proper neuronal and synaptic function. Non-neuronal cells and neurons act in concert to control BBB permeability and CBF. Vascular cells and glia are primarily responsible for maintenance of the constant ‘chemical’ composition of the ISF, and the BBB and the blood–spinal cord barrier (BSCB) work together with pericytes to prevent various potentially neurotoxic and vasculotoxic macromolecules in the blood from entering the CNS, and to promote clearance of these substances from the CNS1.

In the brain, pial arteries run through the subarachnoid space (SAS), which contains the cerebrospinal fluid (CSF). These vessels give rise to intracerebral arteries, which penetrate into brain parenchyma. Intracerebral arteries are separated from brain parenchyma by a single, interrupted layer of elongated fibroblast-like cells of the pia and the astrocyte-derived glia limitans membrane that forms the outer wall of the perivascular Virchow–Robin space. These arteries branch into smaller arteries and subsequently arterioles, which lose support from the glia limitans and give rise to pre-capillary arterioles and brain capillaries. In an intracerebral artery, the vascular smooth muscle cell (VSMC) layer occupies most of the vessel wall. At the brain capillary level, vascular endothelial cells and pericytes are attached to the basement membrane. Pericyte processes encase most of the capillary wall, and they communicate with endothelial cells directly through synapse-like contacts containing connexins and N-cadherin. Astrocyte end-foot processes encase the capillary wall, which is composed of endothelium and pericytes. Resting microglia have a ‘ramified’ shape and can sense neuronal injury.

Figure 2 | Blood–brain barrier transport mechanisms.

Small lipophilic drugs, oxygen and carbon dioxide diffuse across the blood–brain barrier (BBB), whereas ions require ATP-dependent transporters such as the (Na++K+)ATPase. Transporters for nutrients include the glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)), the lactate transporter monocarboxylate transporter 1 (MCT1) and the L1 and y+ transporters for large neutral and cationic essential amino acids, respectively. These four transporters are expressed at both the luminal and albuminal membranes. Non-essential amino acid transporters (the alanine, serine and cysteine preferring system (ASC), and the alanine preferring system (A)) and excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are located at the abluminal side. The ATP-binding cassette (ABC) efflux transporters that are found in the endothelial cells include multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) and solute carrier organic anion transporter family member 1C1 (OATP1C1). Finally, transporters for peptides or proteins include the endothelial protein C receptor (EPCR) for activated protein C (APC); the insulin receptors (IRs) and the transferrin receptors (TFRs), which are associated with caveolin 1 (CAV1); low-density lipoprotein receptor-related protein 1 (LRP1) for amyloid-β, peptide transport system 1 (PTS1) for encephalins; and the PTS2 and PTS4–vasopressin V1a receptor (V1AR) for arginine vasopressin.

 

Transport across the blood–brain barrier. The endothelial cells that form the BBB are connected by tight and adherens junctions, and it is the tight junctions that confer the low paracellular permeability of the BBB1. Small lipophilic molecules, oxygen and carbon dioxide diffuse freely across the endothelial cells, and hence the BBB, but normal brain endothelium lacks fenestrae and has limited vesicular transport.

The high number of mitochondria in endothelial cells reflects a high energy demand for active ATP-dependent transport, conferred by transporters such as the sodium pump ((Na++K+)ATPase) and the ATP-binding cassette (ABC) efflux transporters. Sodium influx and potassium efflux across the abluminal side of the BBB is controlled by (Na++K+)ATPase (Fig. 2). Changes in sodium and potassium levels in the ISF influence the generation of action potentials in neurons and thus directly affect neuronal and synaptic functions1, 12.

Brain endothelial cells express transporters that facilitate the transport of nutrients down their concentration gradients, as described in detail elsewhere1, 14 (Fig. 2). Glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)) — the BBB-specific glucose transporter — is of special importance because glucose is a key energy source for the brain.

Monocarboxylate transporter 1 (MCT1), which transports lactate, and the L1 and y+ amino acid transporters are expressed at the luminal and abluminal membranes12, 14. Sodium-dependent excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are expressed at the abluminal side of the BBB15 and enable removal of glutamate, an excitatory neurotransmitter, from the brain (Fig. 2). Glutamate clearance at the BBB is essential for protecting neurons from overstimulation of glutaminergic receptors, which is neurotoxic16.

ABC transporters limit the penetration of many drugs into the brain17. For example, multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) controls the rapid removal of ingested toxic lipophilic metabolites17 (Fig. 2). Some ABC transporters also mediate the efflux of nutrients from the endothelium into the ISF. For example, solute carrier organic anion transporter family member 1C1 (OATP1C1) transports thyroid hormones into the brain. MCT8 mediates influx of thyroid hormones from blood into the endothelium18 (Fig. 2).

The transport of circulating peptides across the BBB into the brain is restricted or slow compared with the transport of nutrients19. Carrier-mediated transport of neuroactive peptides controls their low levels in the ISF20, 21, 22, 23, 24 (Fig. 2). Some proteins, including transferrin, insulin, insulin-like growth factor 1 (IGF1), leptin25, 26, 27 and activatedprotein C (APC)28, cross the BBB by receptor-mediated transcytosis (Fig. 2).

Circumventricular organs. Several small neuronal structures that surround brain ventricles lack the BBB and sense chemical changes in blood or the cerebrospinal fluid (CSF) directly. These brain areas are known as circumventricular organs (CVOs). CVOs have important roles in multiple endocrine and autonomic functions, including the control of feeding behaviour as well as regulation of water and salt metabolism29. For example, the subfornical organ is one of the CVOs that are capable of sensing extracellular sodium using astrocyte-derived lactate as a signal for local neurons to initiate neural, hormonal and behavioural responses underlying sodium homeostasis30. Excessive sodium accumulation is detrimental, and increases in plasma sodium above a narrow range are incompatible with life, leading to cerebral oedema (swelling), seizures and death29.

Vascular-mediated pathophysiology

The key pathways of vascular dysfunction that are linked to neurodegenerative diseases include BBB breakdown, hypoperfusion–hypoxia and endothelial metabolic dysfunction (Fig. 3). This section examines processes that are involved in BBB breakdown at the molecular and cellular levels, and explores the consequences of all three pathways for neuronal function and viability.

Figure 3 | Vascular-mediated neuronal damage and neurodegeneration.

a | Blood–brain barrier (BBB) breakdown that is caused by pericyte detachment leads to leakage of serum proteins and focal microhaemorrhages, with extravasation of red blood cells (RBCs). RBCs release haemoglobin, which is a source of iron. In turn, this metal catalyses the formation of toxic reactive oxygen species (ROS) that mediate neuronal injury. Albumin promotes the development of vasogenic oedema, contributing to hypoperfusion and hypoxia of the nervous tissue, which aggravates neuronal injury. A defective BBB allows several potentially vasculotoxic and neurotoxic proteins (for example, thrombin, fibrin and plasmin) to enter the brain. b | Progressive reductions in cerebral blood flow (CBF) lead to increasing neuronal dysfunction. Mild hypoperfusion, oligaemia, leads to a decrease in protein synthesis, whereas more-severe reductions in CBF, leading to hypoxia, cause an array of detrimental effects.


Blood–brain barrier breakdown. Disruption to tight and adherens junctions, an increase in bulk-flow fluid transcytosis, and/or enzymatic degradation of the capillary basement membrane cause physical breakdown of the BBB.

The levels of many tight junction proteins, their adaptor molecules and adherens junction proteins decrease in Alzheimer’s disease and other diseases that cause dementia1, 9, ALS31, multiple sclerosis32 and various animal models of neurological disease8, 33. These decreases might be partly explained by the fact that vascular-associated matrix metalloproteinase (MMP) activity rises in many neurodegenerative disorders and after ischaemic CNS injury34, 35; tight junction proteins and basement membrane extracellular matrix proteins are substrates for these enzymes34. Lowered expression of messenger RNAs that encode several key tight junction proteins, however, has also been reported in some neurodegenerative disorders, such as ALS31.

Endothelial cell–pericyte interactions are crucial for the formation36, 37and maintenance of the BBB33, 38. Pericyte deficiency can lead to a reduction in expression of certain tight junction proteins, including occludin, claudin 5 and ZO1 (Ref. 33), and to an increase in bulk-flow transcytosis across the BBB, causing BBB breakdown38. Both processes can lead to extravasation of multiple small and large circulating macromolecules (up to 500 kDa) into the brain parenchyma33, 38. Moreover, in mice, an age-dependent progressive loss of pericytes can lead to BBB disruption and microvasular degeneration and, subsequently, neuronal dysfunction, cognitive decline and neurodegenerative changes33. In their lysosomes, pericytes concentrate and degrade multiple circulating exogenous39 and endogenous proteins, including serum immunoglobulins and fibrin33, which amplify BBB breakdown in cases of pericyte deficiency.

BBB breakdown typically leads to an accumulation of various molecules in the brain. The build up of serum proteins such as immunoglobulins and albumin can cause brain oedema and suppression of capillary blood flow8, 33, whereas high concentrations of thrombin lead to neurotoxicity and memory impairment40, and accelerate vascular damage and BBB disruption41. The accumulation of plasmin (derived from circulating plasminogen) can catalyse the degradation of neuronal laminin and, hence, promote neuronal injury42, and high fibrin levels accelerate neurovascular damage6. Finally, an increase in the number of RBCs causes deposition of haemoglobin-derived neurotoxic products including iron, which generates neurotoxic reactive oxygen species (ROS)8, 43(Fig. 3a). In addition to protein-mediated vasogenic oedema, local tissue ischaemia–hypoxia depletes ATP stores, causing (Na++K+)ATPase pumps and Na+-dependent ion channels to stop working and, consequently, the endothelium and astrocytes to swell (known as cytotoxic oedema)44. Upregulation of aquaporin 4 water channels in response to ischaemia facilitates the development of cytotoxic oedema in astrocytes45.

Hypoperfusion and hypoxia. CBF is regulated by local neuronal activity and metabolism, known as neurovascular coupling46. The pial and intracerebral arteries control the local increase in CBF that occurs during brain activation, which is termed ‘functional hyperaemia’. Neurovascular coupling requires intact pial circulation, and for VSMCs and pericytes to respond normally to vasoactive stimuli33, 46, 47. In addition to VSMC-mediated constriction and vasodilation of cerebral arteries, recent studies have shown that pericytes modulate brain capillary diameter through constriction of the vessel wall47, which obstructs capillary flow during ischaemia48. Astrocytes regulate the contractility of intracerebral arteries49, 50.

Progressive CBF reductions have increasingly serious consequences for neurons (Fig. 3b). Briefly, mild hypoperfusion — termed oligaemia — affects protein synthesis, which is required for the synaptic plasticity mediating learning and memory46. Moderate to severe CBF reductions and hypoxia affect ATP synthesis, diminishing (Na++K+)ATPase activity and the ability of neurons to generate action potentials9. In addition, such reductions can lower or increase pH, and alter electrolyte balances and water gradients, leading to the development of oedema and white matter lesions, and the accumulation of glutamate and proteinaceous toxins (for example, amyloid-β and hyperphopshorylated tau) in the brain. A reduction of greater than 80% in CBF results in neuronal death2.

The effect of CBF reductions has been extensively studied at the molecular and cellular levels in relation to Alzheimer’s disease. Reduced CBF and/or CBF dysregulation occurs in elderly individuals at high risk of Alzheimer’s disease before cognitive decline, brain atrophy and amyloid-β accumulation10, 46, 51, 52, 53, 54. In animal models, hypoperfusion can induce or amplify Alzheimer’s disease-like neuronal dysfunction and/or neuropathological changes. For example, bilateral carotid occlusion in rats causes memory impairment, neuronal dysfunction, synaptic changes and amyloid-β oligomerization55, leading to accumulation of neurotoxic amyloid-β oligomers56. In a mouse model of Alzheimer’s disease, oligaemia increases neuronal amyloid-β levels and neuronal tau phosphophorylation at an epitope that is associated with Alzheimer’s disease-type paired helical filaments57. In rodents, ischaemia leads to the accumulation of hyperphosphorylated tau in neurons and the formation of filaments that resemble those present in human neurodegenerative tauopathies and Alzheimer’s disease58. Mice expressing amyloid-β precursor protein (APP) and transforming growth factor β1 (TGFβ1) develop deficient neurovascular coupling, cholinergic denervation, enhanced cerebral and cerebrovascular amyloid-β deposition, and age-dependent cognitive decline59.

Recent studies have shown that ischaemia–hypoxia influences amyloidogenic APP processing through mechanisms that increase the activity of two key enzymes that are necessary for amyloid-β production; that is, β-secretase and γ-secretase60, 61, 62, 63. Hypoxia-inducible factor 1α (HIF1α) mediates transcriptional increase in β-secretase expression61. Hypoxia also promotes phosphorylation of tau through the mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase (ERK)) pathway64, downregulates neprilysin — an amyloid-β-degrading enzyme65 — and leads to alterations in the expression of vascular-specific genes, including a reduction in the expression of the homeobox protein MOX2 gene mesenchyme homeobox 2 (MEOX2) in brain endothelial cells5 and an increase in the expression of the myocardin gene (MYOCD) in VSMCs66. In patients with Alzheimer’s disease and in models of this disorder, these changes cause vessel regression, hypoperfusion and amyloid-β accumulation resulting from the loss of the key amyloid-β clearance lipoprotein receptor (see below). In addition, hypoxia facilitates alternative splicing of Eaat2 mRNA in Alzheimer’s disease transgenic mice before amyloid-β deposition67 and suppresses glutamate reuptake by astrocytes independently of amyloid formation68, resulting in glutamate-mediated neuronal injury that is independent of amyloid-β.

In response to hypoxia, mitochondria release ROS that mediate oxidative damage to the vascular endothelium and to the selective population of neurons that has high metabolic activity. Such damage has been suggested to occur before neuronal degeneration and amyloid-β deposition in Alzheimer’s disease69, 70. Although the exact triggers of hypoxia-mediated neurodegeneration and the role of HIF1α in neurodegeneration versus preconditioning-mediated neuroprotection remain topics of debate, mitochondria-generated ROS seem to have a primary role in the regulation of the HIF1α-mediated transcriptional switch that can activate an array of responses, ranging from mechanisms that increase cell survival and adaptation to mechanisms inducing cell cycle arrest and death71. Whether inhibition of hypoxia-mediated pathogenic pathways will delay onset and/or control progression in neurodegenerative conditions such as Alzheimer’s disease remains to be determined.

When comparing the contributions of BBB breakdown and hypoperfusion to neuronal injury, it is interesting to consider Meox2+/− mice. Such animals have normal pericyte coverage and an intact BBB but a substantial perfusion deficit5 that is comparable to that found in pericyte-deficient mice that develop BBB breakdown33 Notably, however, Meox2+/− mice show less pronounced neurodegenerative changes than pericyte-deficient mice, indicating that chronic hypoperfusion–hypoxia alone can cause neuronal injury, but not to the same extent as hypoperfusion–hypoxia combined with BBB breakdown.

Endothelial neurotoxic and inflammatory factors. Alterations in cerebrovascular metabolic functions can lead to the secretion of multiple neurotoxic and inflammatory factors72, 73. For example, brain microvessels that have been isolated from individuals with Alzheimer’s disease (but not from neurologically normal age-matched and young individuals) and brain microvessels that have been treated with inflammatory proteins release neurotoxic factors that kill neurons74, 75. These factors include thrombin, the levels of which increase with the onset of Alzheimer’s disease76. Thrombin can injure neurons directly40and indirectly by activating microglia and astrocytes73. Compared with those from age-matched controls, brain microvessels from individuals with Alzheimer’s disease secrete increased levels of multiple inflammatory mediators, such as nitric oxide, cytokines (for example, tumour necrosis factor (TNF), TGFβ1, interleukin-1β (IL-1β) and IL-6), chemokines (for example, CC-chemokine ligand 2 (CCL2; also known as monocyte chemoattractant protein 1 (MCP1)) and IL-8), prostaglandins, MMPs and leukocyte adhesion molecules73. Endothelium-derived neurotoxic and inflammatory factors together provide a molecular link between vascular metabolic dysfunction, neuronal injury and inflammation in Alzheimer’s disease and, possibly, in other neurodegenerative disorders.

Neurovascular changes

This section examines evidence for neurovascular changes during normal ageing and for neurovascular and/or BBB dysfunction in various neurodegenerative diseases, as well as the possibility that vascular defects can precede neuronal changes.

Age-associated neurovascular changes. Normal ageing diminishes brain circulatory functions, including a detectable decay of CBF in the limbic and association cortices that has been suggested to underlie age-related cognitive changes77. Alterations in the cerebral microvasculature, but not changes in neural activity, have been shown to lead to age-dependent reductions in functional hyperaemia in the visual system in cats78 and in the sensorimotor cortex in pericyte-deficient mice33. Importantly, a recent longitudinal CBF study in neurologically normal individuals revealed that people bearing the apolipoprotein E (APOE) ɛ4allele — the major genetic risk factor for late-onset Alzheimer’s disease79, 80, 81 — showed greater regional CBF decline in brain regions that are particularly vulnerable to pathological changes in Alzheimer’s disease than did people without this allele82.

A meta-analysis of BBB permeability in 1,953 individuals showed that neurologically healthy humans had an age-dependent increase in vascular permeability83. Moreover, patients with vascular or Alzheimer’s disease-type dementia and leucoaraiosis — a small-vessel disease of the cerebral white matter — had an even greater age-dependent increase in vascular permeability83. Interestingly, an increase in BBB permeability in brain areas with normal white matter in patients with leukoaraiosis has been suggested to play a causal part in disease and the development of lacunar strokes84. Age-related changes in the permeability of the blood–CSF barrier and the choroid plexus have been reported in sheep85.

Vascular pathology. Patients with Alzheimer’s disease or other dementia-causing diseases frequently show focal changes in brain microcirculation. These changes include the appearance of string vessels (collapsed and acellular membrane tubes), a reduction in capillary density, a rise in endothelial pinocytosis, a decrease in mitochondrial content, accumulation of collagen and perlecans in the basement membrane, loss of tight junctions and/or adherens junctions3, 4, 5, 6, 9,46, 86, and BBB breakdown with leakage of blood-borne molecules4, 6,7, 9. The time course of these vascular alterations and how they relate to dementia and Alzheimer’s disease pathology remain unclear, as no protocol that allows the development of the diverse brain vascular pathology to be scored, and hence to be tracked with ageing, has so far been developed and widely validated87. Interestingly, a recent study involving 500 individuals who died between the ages of 69 and 103 years showed that small-vessel disease, infarcts and the presence of more than one vascular pathological change were associated with Alzheimer’s disease-type pathological lesions and dementia in people aged 75 years of age87. These associations were, however, less pronounced in individuals aged 95 years of age, mainly because of a marked ageing-related reduction in Alzheimer’s disease neuropathology relative to a moderate but insignificant ageing-related reduction in vascular pathology87.

Accumulation of amyloid-β and amyloid deposition in pial and intracerebral arteries results in CAA, which is present in over 80% of Alzheimer’s disease cases88. In patients who have Alzheimer’s disease with established CAA in small arteries and arterioles, the VSMC layer frequently shows atrophy, which causes a rupture of the vessel wall and intracerebral bleeding in about 30% of these patients89, 90. These intracerebral bleedings contribute to, and aggravate, dementia. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type have accelerated VSMC degeneration resulting in haemorrhagic strokes and dementia91. Duplication of the gene encoding APP causes early-onset Alzheimer’s disease dementia with CAA and intracerebral haemorrhage92.

Early studies of serum immunoglobulin leakage reported that patients with ALS had BSCB breakdown and BBB breakdown in the motor cortex93. Microhaemorrhages and BSCB breakdown have been shown in the spinal cord of transgenic mice expressing mutant variants of human superoxide dismutase 1 (SOD1), which in mice cause an ALS-like disease8, 94, 95. In mice with ALS-like disease and in patients with ALS, BSCB breakdown has been shown to occur before motor neuron degeneration or brain atrophy8, 11, 95.

BBB breakdown in the substantia nigra and the striatum has been detected in murine models of Parkinson’s disease that are induced by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)96, 97, 98. However, the temporal relationship between BBB breakdown and neurodegeneration in Parkinson’s disease is currently unknown. Notably, the prevalence of CAA and vascular lesions increases in Parkinson’s disease99, 100. Vascular lesions in the striatum and lacunar infarcts can cause vascular parkinsonism syndrome101. A recent study reported BBB breakdown in a rat model of Huntington’s disease that is induced with the toxin 3-nitropropionic acid102.

Several studies have established disruption of BBB with a loss of tight junction proteins during neuroinflammatory conditions such as multiple sclerosis and its murine model, experimental allergic encephalitis. Such disruption facilitates leukocyte infiltration, leading to oliogodendrocyte death, axonal damage, demyelination and lesion development32.

Functional changes in the vasculature. In individuals with Alzheimer’s disease, GLUT1 expression at the BBB decreases103, suggesting a shortage in necessary metabolic substrates. Studies using18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) have identified reductions in glucose uptake in asymptomatic individuals with a high risk of dementia104, 105. Several studies have suggested that reduced glucose uptake across the BBB, as seen by FDG PET, precedes brain atrophy104, 105, 106, 107, 108.

Amyloid-β constricts cerebral arteries109. In a mouse model of Alzheimer’s disease, impairment of endothelium-dependent regulation of neocortical microcirculation110, 111 occurs before amyloid-β accumulation. Recent studies have shown that CD36, a scavenger receptor that binds amyloid-β, is essential for the vascular oxidative stress and diminished functional hyperaemia that occurs in response to amyloid-β exposure112. Neuroimaging studies in patients with Alzheimer’s disease have shown that neurovascular uncoupling occurs before neurodegenerative changes10, 51, 52, 53. Moreover, cognitively normal APOE ɛ4 carriers at risk of Alzheimer’s disease show impaired CBF responses to brain activation in the absence of neurodegenerative changes or amyloid-β accumulation54. Recently, patients with Alzheimer’s disease as well as mouse models of this disease with high cerebrovascular levels of serum response factor (SRF) and MYOCD, the two transcription factors that control VSMC differentiation, have been shown to develop a hypercontractile arterial phenotype resulting in brain hypoperfusion, diminished functional hyperaemia and CAA66, 113. More work is needed to establish the exact role of SRF and MYOCD in the vascular dysfunction that results in the Alzheimer’s disease phenotype and CAA.

PET studies with 11C-verapamil, an ABCB1 substrate, have indicated that the function of ABCB1, which removes multiple drugs and toxins from the brain, decreases with ageing114 and is particularly compromised in the midbrain of patients with Parkinson’s disease, progressive supranuclear palsy or multiple system atrophy115. More work is needed to establish the exact roles of ABC BBB transporters in neurodegeneration and whether their failure precedes the loss of dopaminergic neurons that occurs in Parkinson’s disease.

In mice with ALS-like disease and in patients with ALS, hypoperfusion and/or dysregulated CBF have been shown to occur before motor neuron degeneration or brain atrophy8, 116. Reduced regional CBF in basal ganglia and reduced blood volume have been reported in pre-symptomatic gene-tested individuals at risk for Huntington’s disease117. Patients with Huntington’s disease display a reduction in vasomotor activity in the cerebral anterior artery during motor activation118.

Vascular and neuronal common growth factors. Blood vessels and neurons share common growth factors and molecular pathways that regulate their development and maintenance119, 120. Angioneurins are growth factors that exert both vasculotrophic and neurotrophic activities121. The best studied angioneurin is vascular endothelial growth factor (VEGF). VEGF regulates vessel formation, axonal growth and neuronal survival120. Ephrins, semaphorins, slits and netrins are axon guidance factors that also regulate the development of the vascular system121. During embryonic development of the neural tube, blood vessels and choroid plexus secrete IGF2 into the CSF, which regulates the proliferation of neuronal progenitor cells122. Genetic and pharmacological manipulations of angioneurin activity yielded various vascular and cerebral phenotypes121. Given the dual nature of angioneurin action, these studies have not been able to address whether neuronal dysfunction results from a primary insult to neurons and/or whether it is secondary to vascular dysfunction.

Increased levels of VEGF, a hypoxia-inducible angiogenic factor, were found in the walls of intraparenchymal vessels, perivascular deposits, astrocytes and intrathecal space of patients with Alzheimer’s disease, and were consistent with the chronic cerebral hypoperfusion and hypoxia that were observed in these individuals73. In addition to VEGF, brain microvessels in Alzheimer’s disease release several molecules that can influence angiogenesis, including IL-1β, IL-6, IL-8, TNF, TGFβ, MCP1, thrombin, angiopoietin 2, αVβ3 and αVβ5 integrins, and HIF1α73. However, evidence for increased vascularity in Alzheimer’s disease is lacking. On the contrary, several studies have reported that focal vascular regression and diminished microvascular density occur in Alzheimer’s disease4, 5, 73 and in Alzheimer’s disease transgenic mice123. The reason for this discrepancy is not clear. The anti-angiogenic activity of amyloid-β, which accumulates in the brains of individuals with Alzheimer’s disease and Alzheimer’s disease models, may contribute to hypovascularity123. Conversely, genome-wide transcriptional profiling of brain endothelial cells from patients with Alzheimer’s disease revealed that extremely low expression of vascular-restricted MEOX2 mediates aberrant angiogenic responses to VEGF and hypoxia, leading to capillary death5. This finding raises the interesting question of whether capillary degeneration in Alzheimer’s disease results from unsuccessful vascular repair and/or remodelling. Moreover, mice that lack one Meox2 allele have been shown to develop a primary cerebral endothelial hypoplasia with chronic brain hypoperfusion5, resulting in secondary neurodegenerative changes33.

Does vascular dysfunction cause neuronal dysfunction? In summary, the evidence that is discussed above clearly indicates that vascular dysfunction is tightly linked to neuronal dysfunction. There are many examples to illustrate that primary vascular deficits lead to secondary neurodegeneration, including CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts), an hereditary small-vessel brain disease resulting in multiple small ischaemic lesions, neurodegeneration and dementia124; mutations in SLC2A1 that cause dysfunction of the BBB-specific GLUT1 transporter in humans resulting in seizures; cognitive impairment and microcephaly125; microcephaly and epileptiform discharges in mice with genetic deletion of a single Slc2a1allele126; and neurodegeneration mediated by a single Meox2 homebox gene deletion restricted to the vascular system33. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type provide another example showing that primary vascular dysfunction — which in this case is caused by deposition of vasculotropic amyloid-β mutants in the arterial vessel wall — leads to dementia and intracerebral bleeding. Moreover, as reviewed in the previous sections, recent evidence suggests that BBB dysfunction and/or breakdown, and CBF reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, BSCB breakdown and spinal cord hypoperfusion have been reported to occur before motor neuron cell death. Whether neurological changes follow or precede vascular dysfunction in Parkinson’s disease, Huntington’s disease and multiple sclerosis remains less clear. However, there is little doubt that vascular injury mediates, amplifies and/or lowers the threshold for neuronal dysfunction and loss in several neurological disorders.

Disease-specific considerations

This section examines how amyloid-β levels are kept low in the brain, a process in which the BBB has a central role, and how faulty BBB-mediated clearance mechanisms go awry in Alzheimer’s disease. On the basis of this evidence and the findings discussed elsewhere in the Review, a new hypothesis for the pathogenesis of Alzheimer’s disease that incorporates the vascular evidence is presented. ALS-specific disease mechanisms relating to the BBB are then examined.

Alzheimer’s disease. Amyloid-β clearance from the brain by the BBB is the best studied example of clearance of a proteinaceous toxin from the CNS. Multiple pathways regulate brain amyloid-β levels, including its production and clearance (Fig. 4). Recent studies127, 128, 129 have confirmed earlier findings in multiple rodent and non-human primate models demonstrating that peripheral amyloid-β is an important precursor of brain amyloid-β130, 131, 132, 133, 134, 135, 136. Moreover, peripheral amyloid-β sequestering agents such as soluble LRP1 (ref.137), anti-amyloid-β antibodies138, 139, 140, gelsolin and the ganglioside GM1 (Ref. 141), or systemic expression of neprilysin142, 143have been shown to reduce the amyloid burden in Alzheimer’s disease mice by eliminating contributions of the peripheral amyloid-β pool to the total brain pool of this peptide.

Figure 4 | The role of blood–brain barrier transport in brain homeostasis of amyloid-β.

Amyloid-β (Aβ) is produced from the amyloid-β precursor protein (APP), both in the brain and in peripheral tissues. Clearance of amyloid-β from the brain normally maintains its low levels in the brain. This peptide is cleared across the blood–brain barrier (BBB) by the low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 mediates rapid efflux of a free, unbound form of amyloid-β and of amyloid-β bound to apolipoprotein E2 (APOE2), APOE3 or α2-macroglobulin (not shown) from the brain’s interstitial fluid into the blood, and APOE4 inhibits such transport. LRP2 eliminates amyloid-β that is bound to clusterin (CLU; also known as apolipoprotein J (APOJ)) by transport across the BBB, and shows a preference for the 42-amino-acid form of this peptide. ATP-binding cassette subfamily A member 1 (ABCA1; also known as cholesterol efflux regulatory protein) mediates amyloid-β efflux from the brain endothelium to blood across the luminal side of the BBB (not shown). Cerebral endothelial cells, pericytes, vascular smooth muscle cells, astrocytes, microglia and neurons express different amyloid-β-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme (IDE), tissue plasminogen activator (tPA) and matrix metalloproteinases (MMPs), which contribute to amyloid-β clearance. In the circulation, amyloid-β is bound mainly to soluble LRP1 (sLRP1), which normally prevents its entry into the brain. Systemic clearance of amyloid-β is mediated by its removal by the liver and kidneys. The receptor for advanced glycation end products (RAGE) provides the key mechanism for influx of peripheral amyloid-β into the brain across the BBB either as a free, unbound plasma-derived peptide and/or by amyloid-β-laden monocytes. Faulty vascular clearance of amyloid-β from the brain and/or an increased re-entry of peripheral amyloid-β across the blood vessels into the brain can elevate amyloid-β levels in the brain parenchyma and around cerebral blood vessels. At pathophysiological concentrations, amyloid-β forms neurotoxic oligomers and also self-aggregates, which leads to the development of cerebral β-amyloidosis and cerebral amyloid angiopathy.


The receptor for advanced glycation end products (RAGE) mediates amyloid-β transport in brain and the propagation of its toxicity. RAGE expression in brain endothelium provides a mechanism for influx of amyloid-β144, 145 and amyloid-β-laden monocytes146 across the BBB, as shown in Alzheimer’s disease models (Fig. 4). The amyloid-β-rich environment in Alzheimer’s disease and models of this disorder increases RAGE expression at the BBB and in neurons147, 148, amplifying amyloid-β-mediated pathogenic responses. Blockade of amyloid-β–RAGE signalling in Alzheimer’s disease is a promising strategy to control self-propagation of amyloid-β-mediated injury.

Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder149 have shown that diminished amyloid-β clearance occurs in brain tissue in this disease. LRP1 plays an important part in the three-step serial clearance of this peptide from brain and the rest of the body150 (Fig. 4). In step one, LRP1 in brain endothelium binds brain-derived amyloid-β at the abluminal side of the BBB, initiating its clearance to blood, as shown in many animal models151, 152, 153, 154, 155, 156 and BBB models in vitro151, 157,158. The vasculotropic mutants of amyloid-β that have low binding affinity for LRP1 are poorly cleared from the brain or CSF151, 159, 160. APOE4, but not APOE3 or APOE2, blocks LRP1-mediated amyloid-β clearance from the brain and, hence, promotes its retention161, whereas clusterin (also known as apolipoprotein J (APOJ)) mediates amyloid-β clearance across the BBB via LRP2 (Ref. 153). APOE and clusterin influence amyloid-β aggregation162, 163. Reduced LRP1 levels in brain microvessels, perhaps in addition to altered levels of ABCB1, are associated with amyloid-β cerebrovascular and brain accumulation during ageing in rodents, non-human primates, humans, Alzheimer’s disease mice and patients with Alzheimer’s disease66, 151, 152, 164, 165, 166. Moreover, recent work has shown that brain LRP1 is oxidized in Alzheimer’s disease167, and may contribute to amyloid-β retention in brain because the oxidized form cannot bind and/or transport amyloid-β137. LRP1 also mediates the removal of amyloid-β from the choroid plexus168.

In step two, circulating soluble LRP1 binds more than 70% of plasma amyloid-β in neurologically normal humans137. In patients with Alzheimer’s disease or mild cognitive impairment (MCI), and in Alzheimer’s disease mice, amyloid-β binding to soluble LRP1 is compromised due to oxidative changes137, 169, resulting in elevated plasma levels of free amyloid-β isoforms comprising 40 or 42 amino acids (amyloid-β1–40 and amyloid-β1–42). These peptides can then re-enter the brain, as has been shown in a mouse model of Alzheimer’s disease137. Rapid systemic removal of amyloid-β by the liver is also mediated by LRP1 and comprises step three of the clearance process170.

In brain, amyloid-β is enzymatically degraded by neprilysin171, insulin-degrading enzyme172, tissue plasminogen activator173 and MMPs173,174 in various cell types, including endothelial cells, pericytes, astrocytes, neurons and microglia. Cellular clearance of this peptide by astrocytes and VSMCs is mediated by LRP1 and/or another lipoprotein receptor66, 175. Clearance of amyloid-β aggregates by microglia has an important role in amyloid-β-directed immunotherapy176 and reduction of the amyloid load in brain177. Passive ISF–CSF bulk flow and subsequent clearance through the CSF might contribute to 10–15% of total amyloid-β removal152, 153, 178. In the injured human brain, increasing soluble amyloid-β concentrations in the ISF correlated with improvements in neurological status, suggesting that neuronal activity might regulate extracellular amyloid-β levels179.

The role of BBB dysfunction in amyloid-β accumulation, as discussed above, underlies the contribution of vascular dysfunction to Alzheimer’s disease (see Fig. 5 for a model of vascular damage in Alzheimer’s disease). The amyloid hypothesis for the pathogenesis of Alzheimer’s disease maintains that this peptide initiates a cascade of events leading to neuronal injury and loss and, eventually, dementia180, 181. Here, I present an alternative hypothesis — the two-hit vascular hypothesis of Alzheimer’s disease — that incorporates the vascular contribution to this disease, as discussed in this Review (Box 1). This hypothesis states that primary damage to brain microcirculation (hit one) initiates a non-amyloidogenic pathway of vascular-mediated neuronal dysfunction and injury, which is mediated by BBB dysfunction and is associated with leakage and secretion of multiple neurotoxic molecules and/or diminished brain capillary flow that causes multiple focal ischaemic or hypoxic microinjuries. BBB dysfunction also leads to impairment of amyloid-β clearance, and oligaemia leads to increased amyloid-β generation. Both processes contribute to accumulation of amyloid-β species in the brain (hit two), where these peptides exert vasculotoxic and neurotoxic effects. According to the two-hit vascular hypothesis of Alzheimer’s disease, tau pathology develops secondary to vascular and/or amyloid-β injury.

Figure 5 | A model of vascular damage in Alzheimer’s disease.

a | In the early stages of Alzheimer’s disease, small pial and intracerebral arteries develop a hypercontractile phenotype that underlies dysregulated cerebral blood flow (CBF). This phenotype is accompanied by diminished amyloid-β clearance by the vascular smooth muscle cells (VSMCs). In the later phases of Alzheimer’s disease, amyloid deposition in the walls of intracerebral arteries leads to cerebral amyloid angiopathy (CAA), pronounced reductions in CBF, atrophy of the VSMC layer and rupture of the vessels causing microbleeds. b | At the level of capillaries in the early stages of Alzheimer’s disease, blood–brain barrier (BBB) dysfunction leads to a faulty amyloid-β clearance and accumulation of neurotoxic amyloid-β oligomers in the interstitial fluid (ISF), microhaemorrhages and accumulation of toxic blood-derived molecules (that is, thrombin and fibrin), which affect synaptic and neuronal function. Hyperphosphorylated tau (p-tau) accumulates in neurons in response to hypoperfusion and/or rising amyloid-β levels. At this point, microglia begin to sense neuronal injury. In the later stages of the disease in brain capillaries, microvascular degeneration leads to increased deposition of basement membrane proteins and perivascular amyloid. The deposited proteins and amyloid obstruct capillary blood flow, resulting in failure of the efflux pumps, accumulation of metabolic waste products, changes in pH and electrolyte composition and, subsequently, synaptic and neuronal dysfunction. Neurofibrillary tangles (NFTs) accumulate in response to ischaemic injury and rising amyloid-β levels. Activation of microglia and astrocytes is associated with a pronounced inflammatory response. ROS, reactive oxygen species.


Amyotrophic lateral sclerosis. The cause of sporadic ALS, a fatal adult-onset motor neuron neurodegenerative disease, is not known182. In a relatively small number of patients with inherited SOD1 mutations, the disease is caused by toxic properties of mutant SOD1 (Ref. 183). Mutations in the genes encoding ataxin 2 and TAR DNA-binding protein 43 (TDP43) that cause these proteins to aggregate have been associated with ALS182, 184. Some studies have suggested that abnormal SOD1 species accumulate in sporadic ALS185. Interestingly, studies in ALS transgenic mice expressing a mutant version of human SOD1 in neurons, and in non-neuronal cells neighbouring these neurons, have shown that deletion of this gene from neurons does not influence disease progression186, whereas deletion of this gene from microglia186 or astrocytes187 substantially increases an animal’s lifespan. According to an emerging hypothesis of ALS that is based on studies in SOD1 mutant mice, the toxicity that is derived from non-neuronal neighbouring cells, particularly microglia and astrocytes, contributes to disease progression and motor neuron degeneration186, 187, 188, 189, 190, whereas BBB dysfunction might be critical for disease initiation8, 43, 94, 95. More work is needed to determine whether this concept of disease initiation and progression may also apply to cases of sporadic ALS.

Human data support a role for angiogenic factors and vessels in the pathogenesis of ALS. For example, the presence of VEGF variations (which were identified in large meta-analysis studies) has been linked to ALS191. Angiogenin is another pro-angiogenic gene that is implicated in ALS because heterozygous missense mutations in angiogenin cause familial and sporadic ALS192. Moreover, mice with a mutation that eliminates hypoxia-responsive induction of the Vegf gene (Vegfδ/δ mice) develop late-onset motor neuron degeneration193. Spinal cord ischaemia worsens motor neuron degeneration and functional outcome in Vegfδ/δmice, whereas the absence of hypoxic induction of VEGF in mice that develop motor neuron disease from expression of ALS-linked mutant SOD1G93A results in substantially reduced survival191.

Therapeutic opportunities

Many investigators believe that primary neuronal dysfunction resulting from an intrinsic neuronal disorder is the key underlying event in human neurodegenerative diseases. Thus, most therapeutic efforts for neurodegenerative diseases have so far been directed at the development of so-called ‘single-target, single-action’ agents to target neuronal cells directly and reverse neuronal dysfunction and/or protect neurons from injurious insults. However, most preclinical and clinical studies have shown that such drugs are unable to cure or control human neurological disorders2, 181, 183, 194, 195. For example, although pathological overstimulation of glutaminergic NMDA receptors (NMDARs) has been shown to lead to neuronal injury and death in several disorders, including stroke, Alzheimer’s disease, ALS and Huntington’s disease16, NMDAR antagonists have failed to show a therapeutic benefit in the above-mentioned human neurological disorders.

Recently, my colleagues and I coined the term vasculo-neuronal-inflammatory triad195 to indicate that vascular damage, neuronal injury and/or neurodegeneration, and neuroinflammation comprise a common pathological triad that occurs in multiple neurological disorders. In line with this idea, it is conceivable that ‘multiple-target, multiple-action’ agents (that is, drugs that have more than one target and thus have more than one action) will have a better chance of controlling the complex disease mechanisms that mediate neurodegeneration than agents that have only one target (for example, neurons). According to the vasculo-neuronal-inflammatory triad model, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia are all important therapeutic targets.

Here, I will briefly discuss a few therapeutic strategies based on the vasculo-neuronal-inflammatory triad model. VEGF and other angioneurins may have multiple targets, and thus multiple actions, in the CNS120. For example, preclinical studies have shown that treatment of SOD1G93A rats with intracerebroventricular VEGF196 or intramuscular administration of a VEGF-expressing lentiviral vector that is transported retrogradely to motor neurons in SOD1G93A mice197 reduced pathology and extended survival, probably by promoting angiogenesis and increasing the blood flow through the spinal cord as well as through direct neuronal protective effects of VEGF on motor neurons. On the basis of these and other studies, a phase I–II clinical trial has been initiated to evaluate the safety of intracerebroventricular infusion of VEGF in patients with ALS198. Treatment with angiogenin also slowed down disease progression in a mouse model of ALS199.

IGF1 delivery has been shown to promote amyloid-β vascular clearance and to improve learning and memory in a mouse model of Alzheimer’s disease200. Local intracerebral implantation of VEGF-secreting cells in a mouse model of Alzheimer’s disease has been shown to enhance vascular repair, reduce amyloid burden and improve learning and memory201. In contrast to VEGF, which can increase BBB permeability, TGFβ, hepatocyte growth factor and fibroblast growth factor 2 promote BBB integrity by upregulating the expression of endothelial junction proteins121 in a similar way to APC43. However, VEGF and most growth factors do not cross the BBB, so the development of delivery strategies such as Trojan horses is required for their systemic use25.

A recent experimental approach with APC provides an example of a neurovascular medicine that has been shown to favourably regulate multiple pathways in non-neuronal cells and neurons, resulting in vasculoprotection, stabilization of the BBB, neuroprotection and anti-inflammation in several acute and chronic models of the CNS disorders195 (Box 2).

The recognition of amyloid-β clearance pathways (Fig. 4), as discussed above, opens exciting new therapeutic opportunities for Alzheimer’s disease. Amyloid-β clearance pathways are promising therapeutic targets for the future development of neurovascular medicines because it has been shown both in animal models of Alzheimer’s disease1 and in patients with sporadic Alzheimer’s disease149 that faulty clearance from brain and across the BBB primarily determines amyloid-β retention in brain, causing the formation of neurotoxic amyloid-β oligomers56 and the promotion of brain and cerebrovascular amyloidosis3. The targeting of clearance mechanisms might also be beneficial in other diseases; for example, the clearance of extracellular mutant SOD1 in familial ALS, the prion protein in prion disorders and α-synuclein in Parkinson’s disease might all prove beneficial. However, the clearance mechanisms for these proteins in these diseases are not yet understood.

Conclusions and perspectives

Currently, no effective disease-modifying drugs are available to treat the major neurodegenerative disorders202, 203, 204. This fact leads to a question: are we close to solving the mystery of neurodegeneration? The probable answer is yes, because the field has recently begun to recognize that, first, damage to neuronal cells is not the sole contributor to disease initiation and progression, and that, second, correcting disease pathways in vascular and glial cells may offer an array of new approaches to control neuronal degeneration that do not involve targeting neurons directly. These realizations constitute an important shift in paradigm that should bring us closer to a cure for neurodegenerative diseases. Here, I raise some issues concerning the existing models of neurodegeneration and the new neurovascular paradigm.

The discovery of genetic abnormalities and associations by linkage analysis or DNA sequencing has broadened our understanding of neurodegeneration204. However, insufficient effort has been made to link genetic findings with disease biology. Another concern for neurodegenerative research is how we should interpret findings from animal models202. Genetically engineered models of human neurodegenerative disorders in Drosophila melanogaster andCaenorhabditis elegans have been useful for dissecting basic disease mechanisms and screening compounds. However, in addition to having much simpler nervous systems, insects and avascular species do not have cerebrovascular and immune systems that are comparable to humans and, therefore, are unlikely to replicate the complex disease pathology that is found in people.

For most neurodegenerative disorders, early steps in the disease processes remain unclear, and biomarkers for these stages have yet to be identified. Thus, it is difficult to predict whether mammalian models expressing human genes and proteins that we know are implicated in the intermediate or later stages of disease pathophysiology, such as amyloid-β or tau in Alzheimer’s disease7, 181, will help us to discover therapies for the early stages of disease and for disease prevention, because the exact role of these pathological accumulations during disease onset remains uncertain. According to the two-hit vascular hypothesis of Alzheimer’s disease, incorporating vascular factors that are associated with Alzheimer’s disease into current models of this disease may more faithfully replicate dementia events in humans. Alternatively, by focusing on the comorbidities and the initial cellular and molecular mechanisms underlying early neurovascular dysfunction that are associated with Alzheimer’s disease, new models of dementia and neurodegeneration may be developed that do not require the genetic manipulation of amyloid-β or tau expression.

The proposed neurovascular triad model of neurodegenerative diseases challenges the traditional neurocentric view of such disorders. At the same time, this model raises a set of new important issues that require further study. For example, the molecular basis of the neurovascular link with neurodegenerative disorders is poorly understood, in terms of the adhesion molecules that keep the physical association of various cell types together, the molecular crosstalk between different cell types (including endothelial cells, pericytes and astrocytes) and how these cellular interactions influence neuronal activity. Addressing these issues promises to create new opportunities not only to better understand the molecular basis of the neurovascular link with neurodegeneration but also to develop novel neurovascular-based medicines.

The construction of a human BBB molecular atlas will be an important step towards understanding the role of the BBB and neurovascular interactions in health and disease. Achievement of this goal will require identifying new BBB transporters by using genomic and proteomic tools, and by cloning some of the transporters that are already known. Better knowledge of transporters at the human BBB will help us to better understand their potential as therapeutic targets for disease.

Development of higher-resolution imaging methods to evaluate BBB integrity, key transporters’ functions and CBF responses in the microregions of interest (for example, in the entorhinal region of the hippocampus) will help us to understand how BBB dysfunction correlates with cognitive outcomes and neurodegenerative processes in MCI, Alzheimer’s disease and related disorders.

The question persists: are we missing important therapeutic targets by studying the nervous system in isolation from the influence of the vascular system? The probable answer is yes. However, the current exciting and novel research that is based on the neurovascular model has already begun to change the way that we think about neurodegeneration, and will continue to provide further insights in the future, leading to the development of new neurovascular therapies.

References

  1. Zlokovic, B. V. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008).

  2. Moskowitz, M. A., Lo, E. H. & Iadecola, C. The science of stroke: mechanisms in search of treatments. Neuron 67, 181–198 (2010).
    A comprehensive review describing mechanisms of ischaemic injury to the neurovascular unit.

  3. Zlokovic, B. V. Neurovascular mechanisms of Alzheimer’s neurodegeneration. Trends Neurosci. 28, 202–208 (2005).

  4. Brown, W. R. & Thore, C. R. Review: cerebral microvascular pathology in ageing and neurodegeneration. Neuropathol. Appl. Neurobiol. 37, 56–74 (2011).

  5. Wu, Z. et al. Role of the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease. Nature Med. 11, 959–965 (2005).
    A study demonstrating that low expression of MEOX2 in brain endothelium leads to aberrant angiogenesis and vascular regression in Alzheimer’s disease.

  6. Paul, J., Strickland, S. & Melchor, J. P. Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer’s disease. J. Exp. Med. 204, 1999–2008 (2007).
    A study showing BBB breakdown in models of Alzheimer’s disease.

  7. Zipser, B. D. et al. Microvascular injury and blood–brain barrier leakage in Alzheimer’s disease. Neurobiol. Aging 28, 977–986 (2007).

  8. Zhong, Z. et al. ALS-causing SOD1 mutants generate vascular changes prior to motor neuron degeneration. Nature Neurosci. 11, 420–422 (2008).
    A study demonstrating that BSCB defects precede motor neuron degeneration in mice that develop an ALS-like disease.

  9. Kalaria, R. N. Vascular basis for brain degeneration: faltering controls and risk factors for dementia. Nutr. Rev. 68, S74–S87 (2010).

  10. Knopman, D. S. & Roberts, R. Vascular risk factors: imaging and neuropathologic correlates. J. Alzheimers Dis. 20, 699–709 (2010).

  11. Miyazaki, K. et al. Disruption of neurovascular unit prior to motor neuron degeneration in amyotrophic lateral sclerosis. J. Neurosci. Res. 89, 718–728 (2011).

  12. Neuwelt, E. A. et al. Engaging neuroscience to advance translational research in brain barrier biology. Nature Rev. Neurosci. 12, 169–182 (2011).

  13. Guo, S. & Lo, E. H. Dysfunctional cell–cell signaling in the neurovascular unit as a paradigm for central nervous system disease.Stroke 40, S4–S7 (2009).

  14. Redzic, Z. Molecular biology of the blood–brain and the blood–cerebrospinal fluid barriers: similarities and differences. Fluids Barriers CNS 8, 3 (2011).

  15. O’Kane, R. L., Martinez-Lopez, I., DeJoseph, M. R., Vina, J. R. & Hawkins, R. A. Na+-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood–brain barrier. A mechanism for glutamate removal. J. Biol. Chem. 274, 31891–31895 (1999).

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Author affiliations

  1. Department of Physiology and Biophysics, and Center for Neurodegeneration and Regeneration at the Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 1501 San Pablo Street, Los Angeles, California 90089, USA.
    Email: bzlokovi@usc.edu

 

Retromer in Alzheimer disease, Parkinson disease and other neurological disorders.

Scott A. Small and Gregory A. Petsko

Nature Reviews Neuroscience  2015; 16:126-132.   http://dx.doi.org:/10.1038/nrn3896

 

Retromer is a protein assembly that has a central role in endosomal trafficking, and retromer dysfunction has been linked to a growing number of neurological disorders. First linked to Alzheimer disease, retromer dysfunction causes a range of pathophysiological consequences that have been shown to contribute to the core pathological features of the disease. Genetic studies have established that retromer dysfunction is also pathogenically linked to Parkinson disease, although the biological mechanisms that mediate this link are only now being elucidated. Most recently, studies have shown that retromer is a tractable target in drug discovery for these and other disorders of the nervous system.

Yeast has proved to be an informative model organism in cell biology and has provided early insight into much of the molecular machinery that mediates the intracellular transport of proteins1,2. Indeed, the term ‘retromer’ was first introduced in a yeast study in 1998 (Ref. 3). In this study, retromer was referred to as a complex of proteins that was dedicated to transporting cargo in a retrograde direction, from the yeast endosome back to the Golgi.

By 2004, a handful of studies had identified the molecular4 and the functional5, 6 homologies of the mammalian retromer, and in 2005 retromer was linked to its first human disorder, Alzheimer disease (AD)7. At the time, the available evidence suggested that the mammalian retromer might match the simplicity of its yeast homologue. Since then, a dramatic and exponential rise in research focusing on retromer has led to more than 300 publications. These studies have revealed the complexity of the mammalian retromer and its functional diversity in endosomal transport, and have implicated retromer in a growing number of neurological disorders.

New evidence indicates that retromer is a ‘master conductor’ of endosomal sorting and trafficking8. Synaptic function heavily depends on endosomal trafficking, as it contributes to the presynaptic release of neurotransmitters and regulates receptor density in the postsynaptic membrane, a process that is crucial for neuronal plasticity9. Therefore, it is not surprising that a growing number of studies are showing that retromer has an important role in synaptic biology10, 11, 12, 13. These observations may account for why the nervous system seems particularly sensitive to genetic and other defects in retromer. In this Progress article, we briefly review the molecular organization and the functional role of retromer, before discussing studies that have linked retromer dysfunction to several neurological diseases — notably, AD and Parkinson disease (PD).

Function and organization

The endosome is considered a hub for intracellular transport. From the endosome, transmembrane proteins can be actively sorted and trafficked to various intracellular sites via distinct transport routes (Fig. 1a). Studies have shown that the mammalian retromer mediates two of the three transport routes out of endosomes. First, retromer is involved in the retrieval of cargos from endosomes and in their delivery, in a retrograde direction, to the trans-Golgi network (TGN)5,6. Retrograde transport has many cellular functions but, as we describe, it is particularly important for the normal delivery of hydrolases and proteases to the endosomal–lysosomal system. The second transport route in which retromer functions is the recycling of cargos from endosomes back to the cell surface14, 15 (Fig. 1a). It is this transport route that is particularly important for neurons, as it mediates the normal delivery of glutamate and other receptors to the plasma membrane during synaptic remodelling and plasticity10, 11, 12, 13.

Figure 1: Retromer’s endosomal transport function and molecular organization.
Retromer's endosomal transport function and molecular organization.

a | Retromer mediates two transport routes out of endosomes via tubules that extend out of endosomal membranes. The first is the retrograde pathway in which cargo is retrieved from the endosome and trafficked to the trans-Golgi network (TGN). The second is the recycling pathway in which cargo is trafficked back from the endosome to the cell surface. The degradation pathway, which is not mediated by retromer, involves the trafficking of cargo from endosomes to lysosomes for degradation. b | The retromer assembly of proteins can be organized into distinct functional modules, all of which work together as part of retromer’s transport role. The ‘cargo-recognition core’ is the central module of the retromer assembly and comprises a trimer of proteins, in which vacuolar protein sorting-associated protein 26 (VPS26) and VPS29 bind VPS35. The ‘tubulation’ module includes protein complexes that bind the cargo-recognition core and aid in the formation and stabilization of tubules that extend out of endosomes, directing the transport of cargos towards their final destinations. The ‘membrane-recruiting’ proteins recruit the cargo-recognition core to the endosomal membrane. The WAS protein family homologue (WASH) complex of proteins also binds the cargo-recognition core and is involved in endosomal ‘actin remodelling’ to form actin patches, which are important for directing cargos towards retromer’s transport pathways. Retromer cargos includes a range of receptors — which bind the cargo-recognition core — and their ligands. PtdIns3P, phosphatidylinositol-3-phosphate.

As well as extending the endosomal transport routes, recent studies have considerably expanded the number of molecular constituents and what is known about the functional organization of the mammalian retromer. Following this expansion in knowledge of the molecular diversity and organizational complexity, retromer might be best described as a multimodular protein assembly. The protein or group of proteins that make up each module can vary, but each module is defined by its distinct function, and the modules work in unison in support of retromer’s transport role.

Two modules are considered central to the retromer assembly. First and foremost is a trimeric complex that functions as a ‘cargo-recognition core’, which selects and binds to the transmembrane proteins that need to be transported and that reside in endosomal membranes5, 6. This trimeric core comprises vacuolar protein sorting-associated protein 26 (VPS26), VPS29 and VPS35; VPS35 functions as the core’s backbone to which the other two proteins bind16. VPS26 is the only member of the core that has been found to have two paralogues, VPS26a and VPS26b17,18, and studies suggest that VPS26b might be differentially expressed in the brain19, 20. Some studies suggest that VPS26a and VPS26b are functionally redundant21, whereas others suggest that they might form distinct cargo-recognition cores20, 22.

The second central module of the retromer assembly is the ‘tubulation’ module, which is made up of proteins that work together in the formation and the stabilization of tubules that extend out of endosomes and that direct the transport of cargo towards its final destination (Fig. 1b). The proteins in this module, which directly binds the cargo-recognition core, are members of the subgroup of the sorting nexin (SNX) family that are characterized by the inclusion of a carboxy-terminal BIN–amphiphysin–RVS (BAR) domain23. These members include SNX1, SNX2, SNX5 and SNX6 (Refs 24,25). As part of the tubulation module, these SNX-BAR proteins exist in different dimeric combinations, but typically SNX1 interacts with SNX5 or SNX6, and SNX2 interacts with SNX5 or SNX6 (Refs 26,27). The EPS15-homology domain 1 (EHD1) protein can be included in this module, as it is involved in stabilizing the tubules formed by the SNX-BAR proteins28.

A third module of the retromer assembly functions to recruit the cargo-recognition core to endosomal membranes and to stabilize the core once it is there (Fig. 1b). Proteins that are part of this ‘membrane-recruiting’ module include SNX3 (Ref. 29), the RAS-related protein RAB7A30, 31,32 and TBC1 domain family member 5 (TBC1D5), which is a member of the TRE2–BUB2–CDC16 (TBC) family of RAB GTPase-activating proteins (GAPs)28. In addition, the lipid phosphatidylinositol-3-phosphate (PtdIns3P), which is found on endosomal membranes, contributes to recruiting most of the retromer-related SNXs through their phox homology domains33. Interestingly, another SNX with a phox homology domain, SNX27, was recently linked to retromer and its function15, 34. SNX27 functions as an adaptor for binding to PDZ ligand-containing cargos that are destined for transport to the cell surface via the recycling pathway. Thus, according to the functional organization of the retromer assembly, SNX27 belongs to the module that engages in cargo recognition and selection.

Recent studies have identified a fourth module of the retromer assembly. The five proteins in this module — WAS protein family homologue 1 (WASH1), FAM21, strumpellin, coiled-coil domain-containing protein 53 (CCDC53) and KIAA1033 (also known as WASH complex subunit 7) — form the WASH complex and function as an ‘actin-remodelling’ module28, 35, 36 (Fig. 1b). Specifically, the WASH complex functions in the rapid polymerization of actin to create patches of actin filaments on endosomal membranes. The complex is recruited to endosomal membranes by binding VPS35 (Ref. 28), and together they divert cargo towards retromer transport pathways and away from the degradation pathway.

The cargos that are transported by retromer include the receptors that directly bind the cargo-recognition core and the ligands of these receptors that are co-transported with the receptors. The receptors that are transported by retromer that have so far been identified to be the most relevant to neurological diseases are the family of VPS10 domain-containing receptors (including sortilin-related receptor 1 (SORL1; also known as SORLA), sortilin, and SORCS1, SORCS2 and SORCS3)7; the cation-independent mannose-6-phosphate receptor (CIM6PR)6, 5; glutamate receptors10; and phagocytic receptors that mediate the clearing function of microglia37. The most disease-relevant ligand to be identified that is trafficked as retromer cargo is the β-amyloid precursor protein (APP)7, 38, 39, 40, 41, which binds SORL1 and perhaps other VPS10 domain-containing receptors42 at the endosomal membrane.

Retromer dysfunction

Guided by retromer’s established function, and on the basis of empirical evidence, there are three well-defined pathophysiological consequences of retromer dysfunction that have proven to be relevant to AD and nervous system disorders. First, retromer dysfunction can cause cargos that typically transit rapidly through the endosome to reside in the endosome for longer than normal durations, such that they can be pathogenically processed into neurotoxic fragments (for example, APP, when stalled in the endosome, is more likely to be processed into amyloid-β, which is implicated in AD43 (Fig. 2a)). Second, by reducing endosomal outflow via impairment of the recycling pathway, retromer dysfunction can lead to a reduction in the number of cell surface receptors that are important for brain health (for example, microglia phagocytic receptors37 (Fig. 2b)).

Figure 2: The pathophysiology of retromer dysfunction.
The pathophysiology of retromer dysfunction.

Retromer dysfunction has three established pathophysiological consequences. In the examples shown, the left graphic represents a cell with normal retromer function and the right graphic represents a cell with a deficit in retromer function. a | Retromer dysfunction causes increased levels of cargo to reside in endosomes. For example, in primary neurons, retromer transports the β-amyloid precursor protein (APP) out of endosomes. Accordingly, retromer dysfunction increases APP levels in endosomes, leading to accelerated APP processing, resulting in an accumulation of neurotoxic fragments of APP (namely, β-carboxy-terminal fragment (βCTF) and amyloid-β) that are pathogenic in Alzheimer disease. b | Retromer dysfunction causes decreased cargo levels at the cell surface. For example, in microglia, retromer mediates the transport of phagocytic receptors to the cell surface and retromer dysfunction results in a decrease in the delivery of these receptors. Studies suggest that this cellular phenotype might have a pathogenic role in Alzheimer disease. c | Retromer dysfunction causes decreased delivery of proteases to the endosome. Retromer is required for the normal retrograde transport of the cation-independent mannose-6-phosphate receptor (CIM6PR) from the endosome back to the trans-Golgi network (TGN). It is in the TGN that this receptor binds cathepsin D and other proteases, and transports them to the endosome, to support the normal function of the endosomal–lysosomal system. By impairing the retrograde transport of the receptor, retromer dysfunction ultimately leads to reduced delivery of cathepsin D to this system. Cathepsin D deficiency has been shown to disrupt the endosomal–lysosomal system and to trigger tau pathology either within endosomes or secondarily in the cytosol.

The third consequence (Fig. 2c) is a result of the established role that retromer has in the retrograde transport of receptors, such as CIM6PR5, 6 or sortilin44, after these receptors transport proteases from the TGN to the endosome. Once at the endosome, the proteases disengage from the receptors, are released into endosomes and migrate to lysosomes. These proteases function in the endosomal–lysosomal system to degrade proteins, protein oligomers and aggregates45. Retromer functions to transfer the ‘naked’ receptor from the endosome back to the TGN via the retrograde pathway5, 6, allowing the receptors to continue in additional rounds of protease delivery. Accordingly, by reducing the normal retrograde transport of these receptors, retromer dysfunction has been shown to reduce the proper delivery of proteases to the endosomal–lysosomal system5,6, which, as discussed below, is a pathophysiological state linked to several brain disorders.

Although requiring further validation, recent studies suggest that retromer dysfunction might be involved in two other mechanisms that have a role in neurological disease. One study suggested that retromer might be involved in trafficking the transmembrane protein autophagy-related protein 9A (ATG9A) to recycling endosomes, from where it can then be trafficked to autophagosome precursors — a trafficking step that is crucial in the formation and the function of autophagosomes46. Autophagy is an important mechanism by which neurons clear neurotoxic aggregates that accumulate in numerous neurodegenerative diseases47. A second study has suggested that retromer dysfunction might enhance the seeding and the cell-to-cell spread of intracellular neurotoxic aggregates48, which have emerged as novel pathophysiological mechanisms that are relevant to AD49, PD50 and other neurodegenerative diseases.

Alzheimer disease

Retromer was first implicated in AD in a molecular profiling study that relied on functional imaging observations in patients and animal models to guide its molecular analysis7. Collectively, neuroimaging studies confirmed that the entorhinal cortex is the region of the hippocampal circuit that is affected first in AD, even in preclinical stages, and suggested that this effect was independent of ageing (as reviewed in Ref. 51). At the same time, neuroimaging studies identified a neighbouring hippocampal region, the dentate gyrus, that is relatively unaffected in AD52. Guided by this information, a study was carried out in which the two regions of the brain were harvested post mortem from patients with AD and from healthy individuals, intentionally covering a broad range of ages. A statistical analysis was applied to the determined molecular profiles of the regions that was designed to address the following question: among the thousands of profiled molecules, which are the ones that are differentially affected in the entorhinal cortex versus the dentate gyrus, in patients versus controls, but that are not affected by age? The final results led to the determination that the brains of patients with AD are deficient in two core retromer proteins — VPS26 and VPS35 (Ref. 7).

Little was known about the receptors of the neuronal retromer, so to understand how retromer deficiency might be mechanistically linked to AD, an analysis was carried out on the molecular data set that looked for transmembrane molecules for which expression levels correlated with VPS35 expression. The top ‘hit’ was the transcript encoding the transmembrane protein SORL1 (Ref. 43). As SORL1 belongs to the family of VPS10-containing receptors and as VPS10 is the main retromer receptor in yeast3, it was postulated that SORL1 and the family of other VPS10-containing proteins (sortillin, SORCS1, SORCS2 and SORCS3) might function as retromer receptors in neurons7. In addition, SORL1 had recently been reported to bind APP53, so if SORL1 was assumed to be a receptor that is trafficked by retromer, then APP might be the cargo that is co-trafficked by retromer. This led to a model in which retromer traffics APP out of endosomes7, which are the organelles in which APP is most likely to be cleaved by βAPP-cleaving enzyme 1 (BACE1; also known as β-secretase 1)43; this is the initial enzymatic step in the pathogenic processing of APP.

Subsequent studies were required to further establish the pathogenic link between retromer and AD, and to test the proposed model. The pathogenic link was further supported by human genetic studies. First, a genetic study investigating the association between AD, the genes encoding the components of the retromer cargo-recognition core and the family of VPS10-containing receptors found that variants of SORL1 increase the risk of developing AD38. This finding was confirmed by numerous studies, including a recent large-scale AD genome-wide association study54. Other genetic studies identified AD-associated variants in genes encoding proteins that are linked to nearly all modules of the retromer assembly55, including genes encoding proteins of the retromer tubulation module (SNX1), genes encoding proteins of the retromer membrane-recruiting module (SNX3 and RAB7A) and genes encoding proteins of the retromer actin-remodelling module (KIAA1033). In addition, nearly all of the genes encoding the family of VPS10-containing retromer receptors have been found to have variants that associate with AD56. Finally, a study found that brain regions that are differentially affected in AD are deficient in PtdIns3P, which is the phospholipid required for recruiting many sorting nexins to endosomal membranes57. Thus, together with the observation that the brains of patients with AD are deficient in VPS26a and VPS35 (Refs 7,37), all modules in the retromer assembly are implicated in AD.

Studies in mice39, 58, 59, flies39 and cells in culture34, 40, 41, 60, 61 have investigated how retromer dysfunction leads to the pathogenic processing of APP. Although rare discrepancies have been observed among these studies62, when viewed in total, the most consistent findings are that retromer dysfunction causes increased pathogenic processing of APP by increasing the time that APP resides in endosomes. Moreover, these studies have confirmed that SORL1 and other VPS10-containing proteins function as APP receptors that mediate APP trafficking out of endosomes.

Retromer has unexpectedly been linked to microglial abnormalities37 — another core feature of AD — which, on the basis of recent genetic findings, seem to have an upstream role in disease pathogenesis54, 63. A recent study found that microglia harvested from the brains of individuals with AD are deficient in VPS35 and provided evidence suggesting that retromer’s recycling pathway regulates the normal delivery of various phagocytic receptors to the cell surface of microglia37, including the phagocytic receptor triggering receptor expressed on myeloid cells 2 (TREM2) (Fig. 2b). Mutations in TREM2 have been linked to AD63, and a recent study indicates that these mutations cause a reduction in its cell surface delivery and accelerate TREM2 degradation, which suggests that the mutations are linked to a recycling defect64. While they are located at the microglial cell surface, these phagocytic receptors function in the clearance of extracellular proteins and other molecules from the extracellular space65. Taken together, these recent studies suggest that defects in the retromer’s recycling pathway can, at least in part, account for the microglial defects observed in the disease.

The microtubule-associated protein tau is the key element of neurofibrillary tangles, which are the other hallmark histological features of AD. Although a firm link between retromer dysfunction and tau toxicity remains to be established, recent insight into tau biology suggests several plausible mechanisms that are worth considering. Tau is a cytosolic protein, but nonetheless, through mechanisms that are still undetermined, it is released into the extracellular space from where it gains access to neuronal endosomes via endocytosis66, 67. In fact, recent studies suggest that the pathogenic processing of tau is triggered after it is endocytosed into neurons and while it resides in endosomes67. Of note, it still remains unknown which specific tau processing step — its phosphorylation, cleavage or aggregation — is an obligate step towards tau-related neurotoxicity. Accordingly, if defects in microglia or in other phagocytic cells reduce their capacity to clear extracellular tau, this would accelerate tau endocytosis in neurons and its pathogenic processing.

A second possibility comes from the established role retromer has in the proper delivery of cathepsin D and other proteases to the endosomal–lysosomal system via CIM6PR or sortilin (Fig. 2c). Studies in sheep, mice and flies68 have shown that cathepsin D deficiency can enhance tau toxicity and that this is mediated by a defective endosomal–lysosomal system68. Whether this mechanism leads to abnormal processing of tau within endosomes or in the cytosol via caspase activation68 remains unclear. As discussed above, retromer dysfunction will lead to a decrease in the normal delivery of cathepsin D to the endosome and will result in endosomal–lysosomal system defects. Retromer dysfunction can therefore be considered as a functional phenocopy of cathepsin D deficiency, which suggests a plausible link between retromer dysfunction and tau toxicity. Nevertheless, although these recent insights establish plausibility and support further investigation into the link between retromer and tau toxicity, whether this link exists and how it may be mediated remain open and outstanding questions.

Parkinson disease

The pathogenic link between retromer and PD is singular and straightforward: exome sequencing has identified autosomal-dominant mutations in VPS35 that cause late-onset PD69, 70, one of a handful of genetic causes of late-onset disease. However, the precise mechanism by which these mutations cause the disease is less clear.

Among a group of recent studies, all46, 48, 71, 72, 73, 74, 75, 76 but one77 strongly suggest that these mutations cause a loss of retromer function. At the molecular level, the mutations do not seem to disrupt mutant VPS35 from interacting normally with VPS26 and VPS29, and from forming the cargo-recognition core. Rather, two studies suggest that the mutations have a restricted effect on the retromer assembly but reduce the ability of VPS35 to associate with the WASH complex46, 75. Studies disagree about the pathophysiological consequences of the mutations. Four studies suggest that the mutations affect the normal retrograde transport of CIM6PR71, 73, 75, 76 from the endosome back to the TGN (Fig. 2c). In this scenario, the normal delivery of cathepsin D to the endosomal–lysosomal system should be reduced and this has been empirically shown73. Cathepsin D has been shown to be the dominant endosomal–lysosomal protease for the normal processing of α-synuclein76, and mutations could therefore lead to abnormal α-synuclein processing and to the formation of α-synuclein aggregates, which are thought to have a key pathogenic role in PD.

A separate study suggested that the mutation might cause a mistrafficking of ATG9, and thereby, as discussed above, reduce the formation and the function of autophagosomes46. Autophagosomes have also been implicated as an intracellular site in which α-synuclein aggregates are cleared. Thus, although future studies are needed to resolve these discrepant findings (which may in fact not be mutually exclusive), these studies are generally in agreement that retromer defects will probably increase the neurotoxic levels of α-synuclein aggregates48.

Several studies in flies71, 74 and in rat neuronal cultures71 provide strong evidence that increasing retromer function by overexpressing VPS35 rescues the neurotoxic effects of the most common PD-causing mutations in leucine-rich repeat kinase 2 (LRRK2). Moreover, a separate study has shown that increasing retromer levels rescues the neurotoxic effect of α-synuclein aggregates in a mouse model48. These findings have immediate therapeutic implications for drugs that increase VPS35 and retromer function, as discussed in the next section, but they also offer mechanistic insight. LRRK2 mutations were found to phenocopy the transport defects caused either by theVPS35 mutations or by knocking down VPS35 (Ref. 71). Together, this and other studies78suggest that LRRK2 might have a role in retromer-dependent transport, but future studies are required to clarify this role.

Other neurological disorders

Besides AD and PD, in which a convergence of findings has established a strong pathogenic link, retromer is being implicated in an increasing number of other neurological disorders. Below, we briefly review three disorders for which the evidence of the involvement of retromer in their pathophysiology is currently the most compelling.

The first of these disorders is Down syndrome (DS), which is caused by an additional copy of chromosome 21. Given the hundreds of genes that are duplicated in DS, it has been difficult to identify which ones drive the intellectual impairments that characterize this condition. A recent elegant study provides strong evidence that a deficiency in the retromer cargo-selection protein SNX27 might be a primary driver for some of these impairments79. This study found that the brains of individuals with DS were deficient in SNX27 and that this deficiency may be caused by an extra copy of a microRNA (miRNA) encoded by human chromosome 21 (the miRNA is produced at elevated levels and thereby decreases SNX27 expression). Consistent with the known role of SNX27 in retromer function, decreased expression of this protein in mice disrupted glutamate receptor recycling in the hippocampus and led to dendritic dysfunction. Importantly, overexpression of SNX27 rescued cognitive and other defects in animal models79, which not only strengthens the causal link between retromer dysfunction and cognitive impairment in DS but also has important therapeutic implications.

Hereditary spastic paraplegia (HSP) is another disorder linked to retromer. HSP is caused by genetic mutations that affect upper motor neurons and is characterized by progressive lower limb spasticity and weakness. Although there are numerous mutations that cause HSP, most are unified by their effects on intracellular transport80. One HSP-associated gene in particular encodes strumpellin81, which is a member of the WASH complex.

The third disorder linked to retromer is neuronal ceroid lipofuscinosis (NCL). NCL is a young-onset neurodegenerative disorder that is part of a larger family of lysosomal storage diseases and is caused by mutations in one of ten identified genes — nine neuronal ceroid lipofuscinosis (CLN) genes and the gene encoding cathepsin D82. Besides cathepsin D, for which the link to retromer has been discussed above, CLN3 seems to function in the normal trafficking of CIM6PR83. However, the most direct link to retromer has been recently described for CLN5, which seems to function, at least in part, as a retromer membrane-recruiting protein84.

Retromer as a therapeutic target

As suggested by the first study implicating retromer in AD7, and in several subsequent studies71,85, increasing the levels of retromer’s cargo-recognition core enhances retromer’s transport function. Motivated by this observation and after a decade-long search86, we identified a novel class of ‘retromer pharmacological chaperones’ that can bind and stabilize retromer’s cargo-recognition core and increase retromer levels in neurons61.

Validating the motivating hypothesis, the chaperones were found to enhance retromer function, as shown by the increased transport of APP out of endosomes and a reduction in the accumulation of APP-derived neurotoxic fragments61. Although there are numerous other pharmacological approaches for enhancing retromer function, this success provides the proof-of-principle that retromer is a tractable therapeutic target.

As retromer functions in all cells, a general concern is whether enhancing its function will have toxic adverse effects. However, studies have found that in stark contrast to even mild retromer deficiencies, increasing retromer levels has no obvious negative consequences in yeast, neuronal cultures, flies or mice40, 48, 61, 71. This might make sense because unlike drugs that, for example, function as inhibitors, simply increasing the normal flow of transport through the endosome might not be cytotoxic.

If retromer drugs are safe and can effectively enhance retromer function in the nervous system — which are still outstanding issues — there are two general indications for considering their clinical application. One rests on the idea that these agents will only be efficacious in patients who have predetermined evidence of retromer dysfunction. The most immediate example is that of individuals with PD that is caused by LRRK2 mutations. As discussed above, several ‘preclinical’ studies in flies and neuronal cultures have already established that increasing retromer levels71, 74can reverse the neurotoxic effects of such mutations and, thus, if this approach is proven to be safe, LRRK2-linked PD might be an appropriate indication for clinical trials.

Alternatively, the pathophysiology of a disease might be such that retromer-enhancing drugs would be efficacious regardless of whether there is documented evidence of retromer dysfunction. AD illustrates this point. As reviewed above, current evidence suggests that retromer-enhancing drugs will, at the very least, decrease pathogenic processing of APP in neurons and enhance microglial function, even if there are no pre-existing defects in retromer.

More generally, histological studies comparing the entorhinal cortex of patients with sporadic AD to age-matched controls have documented that enlarged endosomes are a defining cellular abnormality in AD87, 88. Importantly, enlarged endosomes are uniformly observed in a broad range of patients with sporadic AD, which suggests that enlarged endosomes reflect an intracellular site at which molecular aetiologies converge87. In addition, because they are observed in early stages of the disease in regions of the brain without evidence of amyloid pathology87, enlarged endosomes are thought to be an upstream event. Mechanistically, the most likely cause of enlarged endosomes is either too much cargo flowing into endosomes — as occurs, for example, with apolipoprotein E4 (APOE4), which has been shown to accelerate endocytosis89, 90 — or too little cargo flowing out, as observed in retromer dysfunction40, 61 and related transport defects57. By any mechanism, retromer-enhancing drugs might correct this unifying cellular defect and might be expected to be beneficial regardless of the specific aetiology.

Conclusions

The fact that retromer defects, including those derived from bona fide genetic mutations, seem to differentially target the nervous system suggests that the nervous system is differentially dependent on retromer for its normal function. We think that this reflects the unique cellular properties of neurons and how synaptic biology heavily depends on endosomal transport and trafficking. Although plausible, future studies are required to confirm and to test the details of this hypothesis.

However, currently, it is the clinical rather than the basic neuroscience of retromer that is much better understood, with the established pathophysiological consequences of retromer dysfunction providing a mechanistic link to the disorders in which retromer has been implicated. Nevertheless, many questions remain. The two most interesting questions, which are in fact inversions of each other, relate to regional vulnerability in the nervous system. First, why does retromer dysfunction target specific neuronal populations? Second, how can retromer dysfunction cause diseases that target different regions of the nervous system? Recent evidence hints at answers to both questions, which must somehow be rooted in the functional and molecular diversity of retromer.

The type and the extent of retromer defects linked to different disorders might provide pathophysiological clues as well as reasons for differential vulnerability. As discussed, in AD there seem to be across-the-board defects in retromer, such that each module of the retromer assembly as well as multiple retromer cargos have been pathogenically implicated. By contrast, the profile of retromer defects in PD seems to be more circumscribed, involving selective disruption of the interaction between VPS35 and the WASH complex. These insights might agree with histological87, 88 and large-scale genetic studies54 that suggest that endosomal dysfunction is a unifying focal point in the cellular pathogenesis of AD. In contrast, genetics and other studies91suggest that the cellular pathobiology of PD is more distributed, implicating the endosome but other organelles as well, in particular the mitochondria.

Interestingly, studies suggest that the entorhinal cortex — a region that is differentially vulnerable to AD — has unique dendritic structure and function92, which are highly dependent on endosomal transport. We speculate that it is the unique synaptic biology of the entorhinal cortex that can account for why it might be particularly sensitive to defects in endosomal transport in general and retromer dysfunction in particular, and for why this region is the early site of disease. Future studies are required to investigate this hypothesis, as well as to understand why the substantia nigra or other regions that are differentially vulnerable to PD would be particularly sensitive to the more circumscribed defect in retromer.

Perhaps the most important observation for clinical neuroscience is the now well-established fact that increasing levels of retromer proteins enhances retromer function and has already proved capable of reversing defects associated with AD, PD and DS in either cell culture or in animal models. The relationships between protein levels and function are not always simple, but emerging pharmaceutical technologies that selectively and safely increase protein levels are now a tractable goal in drug discovery93. With the evidence mounting that retromer has a pathogenic role in two of the most common neurodegenerative diseases, we think that targeting retromer to increase its functional activity is an important goal that has strong therapeutic promise.

References

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  • Hussain, N. K., Diering, G. H., Sole, J., Anggono, V. & Huganir, R. L. Sorting nexin 27 regulates basal and activity-dependent trafficking of AMPARs. Proc. Natl Acad. Sci. USA111, 1184011845 (2014).
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……. 93

Affiliations   

Taub Institute for Research on Alzheimer’s Disease and the Ageing Brain, Departments of Neurology, Radiology, and Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

Scott A. Small

Helen and Robert Appel Alzheimer’s Disease Research Institute, Department of Neurology and Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, New York 10065, USA.

Gregory A. Petsko

 

See also:

Neurobiol Aging. 2011 Nov;32(11):2109.e1-14. doi: 10.1016/j.neurobiolaging.2011.05.025.
Altered intrinsic neuronal excitability and reduced Na+ currents in a mouse model of Alzheimer’s disease.
Brown JT, Chin J, Leiser SC, Pangalos MN, Randall AD.

Trends Neurosci. 2013 Jun;36(6):325-35. doi: 10.1016/j.tins.2013.03.002.
Why size matters – balancing mitochondrial dynamics in Alzheimer’s disease.
DuBoff B, Feany M, Götz J.

Neuron. 2014 Dec 3;84(5):1023-33. doi: 10.1016/j.neuron.2014.10.024.
Dendritic structural degeneration is functionally linked to cellular hyperexcitability in a mouse model of Alzheimer’s disease.
Šišková Z, Justus D, Kaneko H, Friedrichs D, Henneberg N, Beutel T, Pitsch J, Schoch S, Becker A, von der Kammer H, Remy S.

 

 

Video: How can we tease out the role of other toxic insults in AD pathogenesis?

https://neuroalzheimerscommunity.nature.com/videos/3896-other-toxic-insults/download.mp4

 

 

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3-D videogames boost memory

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Playing 3-D video games can boost memory formation

http://www.kurzweilai.net/playing-3-d-video-games-can-boost-memory-formation

Video games used in the experiment : screenshot of 2-D Angry Birds (left) and Super Mario 3D World (right) (credit: Gregory D. Clemenson and Craig E.L. Stark/The Journal of Neuroscience)

http://www.kurzweilai.net/images/2D-vs-3D-video-games.jpg

 

Playing three-dimensional video games can boost the formation of memories, especially for people who lose memory as they age or suffer from dementia, according to University of California, Irvine (UCI) neurobiologists.

Craig Stark and Dane Clemenson of UCI’s Center for the Neurobiology of Learning & Memory recruited non-gamer college students to play either a video game with a passive, two-dimensional environment (“Angry Birds”) or one with an intricate, 3-D setting (“Super Mario 3D World”) for 30 minutes per day over two weeks.

Before and after the two-week period, the students took memory tests that engaged the brain’s hippocampus, the region associated with complex learning and memory. They were given a series of pictures of everyday objects to study. Then they were shown images of the same objects, new ones, and others that differed slightly from the original items and asked to categorize them.

Students playing the 3-D video game improved their scores on the memory test by about 12 percent, the same amount it normally decreases between the ages of 45 and 70, while the 2-D gamers did not improve.

 

https://youtu.be/t1YfgMVhhdA

UC Irvine | 3D Video Games and Memory – UC Irvine

 

Role of the hippocampus

Recognition of the slightly altered images requires the hippocampus, Stark said, and his earlier research had demonstrated that the ability to do this clearly declines with age. This is a large part of why it’s so difficult to learn new names or remember where you put your keys as you get older.

In previous studies on rodents, postdoctoral scholar Clemenson and others showed that exploring the environment resulted in the growth of new neurons that became entrenched in the hippocampus’ memory circuit and increased neuronal signaling networks. Stark noted some commonalities between the 3-D game the humans played and the environment the rodents explored — qualities lacking in the 2-D game. “First, the 3-D games have … a lot more spatial information in there to explore. Second, they’re much more complex, with a lot more information to learn,” Stark noted.

Stark added that it’s unclear whether the overall amount of information and complexity in the 3-D game or the spatial relationships and exploration is stimulating the hippocampus. “This is one question we’re following up on,” he said.


Myths of “brain training”

“Results from this study add to the existing literature that playing video games may provide meaningful stimulation to the brain. However, it is important to be cautious when generalizing these results to other instances. Recently, 70 neuroscientists from universities and institutions around the world published a letter discussing the myths of “brain training” (Max Planck Institute for Human Development/Stanford Center on Longevity, 2014. A consensus on the brain training industry from the scientific community. Stanford, CA: Stanford Center on Longevity).

“In contrast to typical brain training, typical video games are not created with specific cognitive processes in mind but rather designed to captivate and immerse the user into charactersand adventure. Rather than isolate single brain processes, modern video games can naturally draw on or require many cognitive processes, including visual, spatial, emotional, motivational, attentional, critical thinking, problem solving, and working memory. It’s quite possible that by explicitly avoiding a narrow focus on a single … cognitive domain and by more closely paralleling natural experience, immersive video games may be better suited to provide enriching experiences that translate into functional gains.”

— Gregory D. Clemenson and Craig E.L. Stark. Virtual Environmental Enrichment through Video Games Improves Hippocampal-Associated Memory. The Journal of Neuroscience.

 

The next step is to determine if environmental enrichment — either through 3-D video games or real-world exploration experiences — can reverse the hippocampal-dependent cognitive deficits present in older populations.

“Can we use this video game approach to help improve hippocampus functioning?” Stark asked. “It’s often suggested that an active, engaged lifestyle can be a real factor in stemming cognitive aging. While we can’t all travel the world on vacation, we can do many other things to keep us cognitively engaged and active. Video games may be a nice, viable route.”

The research is described in a paper published today (Dec. 9) in The Journal of Neuroscience and is funded by a $300,000 Dana Foundation grant.

 

Virtual Environmental Enrichment through Video Games Improves Hippocampal-Associated Memory

Gregory D. Clemenson and Craig E.L. Stark
The Journal of Neuroscience, 9 Dec 2015, 35(49):16116-16125;      http://dx.doi.org:/10.1523/JNEUROSCI.2580-15.2015

 

The positive effects of environmental enrichment and their neural bases have been studied extensively in the rodent (van Praag et al., 2000). For example, simply modifying an animal’s living environment to promote sensory stimulation can lead to (but is not limited to) enhancements in hippocampal cognition and neuroplasticity and can alleviate hippocampal cognitive deficits associated with neurodegenerative diseases and aging. We are interested in whether these manipulations that successfully enhance cognition (or mitigate cognitive decline) have similar influences on humans. Although there are many “enriching” aspects to daily life, we are constantly adapting to new experiences and situations within our own environment on a daily basis. Here, we hypothesize that the exploration of the vast and visually stimulating virtual environments within video games is a human correlate of environmental enrichment. We show that video gamers who specifically favor complex 3D video games performed better on a demanding recognition memory task that assesses participants’ ability to discriminate highly similar lure items from repeated items. In addition, after 2 weeks of training on the 3D video game Super Mario 3D World, naive video gamers showed improved mnemonic discrimination ability and improvements on a virtual water maze task. Two control conditions (passive and training in a 2D game, Angry Birds), showed no such improvements. Furthermore, individual performance in both hippocampal-associated behaviors correlated with performance in Super Mario but not Angry Birds, suggesting that how individuals explored the virtual environment may influence hippocampal behavior.

SIGNIFICANCE STATEMENT The hippocampus has long been associated with episodic memory and is commonly thought to rely on neuroplasticity to adapt to the ever-changing environment. In animals, it is well understood that exposing animals to a more stimulating environment, known as environmental enrichment, can stimulate neuroplasticity and improve hippocampal function and performance on hippocampally mediated memory tasks. Here, we suggest that the exploration of vast and visually stimulating environments within modern-day video games can act as a human correlate of environmental enrichment. Training naive video gamers in a rich 3D, but not 2D, video game, resulted in a significant improvement in hippocampus-associated cognition using several behavioral measures. Our results suggest that modern day video games may provide meaningful stimulation to the human hippocampus.

 

I enjoyed reading the Stanford “consensus on brain training” letter.
http://longevity3.stanford.edu/blog/2014/10/15/the-consensus-on-the-brain-training-industry-from-the-scientific-community-2/

One quote stood out right away about “brain games”: “… In fact, the notion that performance on a single task cannot stand in for an entire ability is a cornerstone of scientific psychology …”

But playing a challenging 3-D video game or exploring a virtual reality like Second Life is far more “educational” and experiential, involving all sorts of cognitive (and, for that matter, psycho-motor and affective) skill domains. Good for the brain and often good for the spirit too.

 

A Consensus on the Brain Training Industry from the Scientific Community

As the baby boomers enter their golden years with mounting concerns about the potential loss of cognitive abilities, markets are responding with products promising to allay anxieties about potential decline. Computer-based cognitive-training software –popularly known as brain games– claim a growing share of the marketplace. The promotion of these products reassures and entices a worried public.

Consumers are told that playing brain games will make them smarter, more alert, and able to learn faster and better. In other words, the promise is that if you adhere to a prescribed regimen of cognitive exercise, you will reduce cognitive slowing and forgetfulness, and will fundamentally improve your mind and brain.

brain training

http://longevity3.stanford.edu/wp-content/uploads/2014/10/iStock_000018505398XSmall-347×320-copy-300×276.jpg

 

It is customary for advertising to highlight the benefits and overstate potential advantages of their products. In the brain-game market, advertisements also reassure consumers that claims and promises are based on solid scientific evidence, as the games are “designed by neuroscientists” at top universities and research centers. Some companies present lists of credentialed scientific consultants and keep registries of scientific studies pertinent to cognitive training. Often, however, the cited research is only tangentially related to the scientific claims of the company, and to the games they sell. In addition, even published peer-reviewed studies merit critical evaluation. A prudent approach calls for integrating findings over a body of research rather than relying on single studies that often include only a small number of participants.

The Stanford Center on Longevity and the Berlin Max Planck Institute for Human Development gathered many of the world’s leading cognitive psychologists and neuroscientists –people who have dedicated their careers to studying the aging mind and brain– to share their views about brain games and offer a consensus report to the public. What do expert scientists think about these claims and promises? Do they have specific recommendations for effective ways to boost cognition in healthy, older adults? Are there merits to the claimed benefits of the brain games and if so, do older adults benefit from brain-game learning in the same ways younger people do? How large are the gains associated with computer-based cognitive exercises? Are the gains restricted to specific skills or does general cognitive aptitude improve? How does playing games compare with other proposed means of mitigating age-related declines, such as physical activity and exercise, meditation, or social engagement?

The search for effective means of mitigating or postponing age-related cognitive declines has taught most of us to recognize the enormous complexity of the subject matter. Like many challenging scientific topics, this is a devil of many details. The consensus of the group is that claims promoting brain games are frequently exaggerated and at times misleading. Cognitive training produces statistically significant improvement in practiced skills that sometimes extends to improvement on other cognitive tasks administered in the lab. In some studies, such gains endure, while other reports document dissipation over time. In commercial promotion, these small, narrow, and fleeting advances are often billed as general and lasting improvements of mind and brain. The aggressive advertising entices consumers to spend money on products and to take up new behaviors, such as gaming, based on these exaggerated claims. As frequently happens, initial findings, based on small samples, generate understandable excitement by suggesting that some brain games may enhance specific aspects of behavior and even alter related brain structures and functions. However, as the findings accumulate, compelling evidence of general and enduring positive effects on the way people’s minds and brains age has remained elusive.

Mind_circle copyThese conclusions do not mean that the brain does not remain malleable, even in old age. Any mentally effortful new experience, such as learning a language, acquiring a motor skill, navigating in a new environment, and, yes, playing commercially available computer games, will produce changes in those neural systems that support acquisition of the new skill. For example, there may be an increase in the number of synapses, the number of neurons and supporting cells, or a strengthening of the connections among them. This type of brain plasticity is possible throughout the life span, though younger brains seem to have an advantage over the older ones. It would be appropriate to conclude from such work that the potential to learn new skills remains intact throughout the life span. However at this point it is not appropriate to conclude that training-induced changes go significantly beyond the learned skills, that they affect broad abilities with real-world relevance, or that they generally promote “brain health”.

As we take a closer look at the evidence on brain games, one issue needs to be kept in mind: It is not sufficient to test the hypothesis of training-induced benefits against the assumption that training brings no performance increases at all. Rather, we need to establish that observed benefits are not easily and more parsimoniously explained by factors that are long known to benefit performance, such as the acquisition of new strategies or changes in motivation. It is well established, for example, that improvements on a particular memory task often result from subtle changes instrategy thatreflect improvement in managing the demands of that particular task. Such improvement is rewarding for players (the fun factor) but does not imply a general improvement in memory. In fact, the notion that performance on a single task cannot stand in for an entire ability is a cornerstone of scientific psychology. Claims about brain games often ignore this tenet. In psychology, it is good scientific practice to combine information provided by many tasks to generate an overall index representing a given ability. According to the American Psychological Association, newly developed psychological tests must meet specific psychometric standards, including reliability and validity. The same standards should be extended into the brain game industry, but this is not the state of affairs today.

To date, there is little evidence that playing brain games improves underlying broad cognitive abilities, or that it enables one to better navigate a complex realm of everyday life. Some intriguing isolated reports do inspire additional research, however. For instance, some studies suggest that both non-computerized reasoning and computerized speed-of-processing training are associated with improved driving in older adults and a reduction in the number of accidents. Another study revealed, for a sample of younger adults, that 100 days of practicing 12 different computerized cognitive tasks resulted in small general improvements in the cognitive abilities of reasoning and episodic memory, some of which were maintained over a period of two years. In other studies, older adults have reported that they felt better about everyday functioning after cognitive training, but no objective measures supported that impression. Additional systematic research is needed to replicate, clarify, consolidate, and expand such results. To be fully credible, an empirical test of the usefulness of brain games needs to address the following questions. Does the improvement encompass a broad array of tasks that constitute a particular ability, or does it just reflect the acquisition of specific skills? Do the gains persist for a reasonable amount of time? Are the positive changes noticed in real life indices of cognitive health? What role do motivation and expectations play in bringing about improvements in cognition when they are observed?

iStock_000000218284XSmall-180x180 copyIn a balanced evaluation of brain games, we also need to keep in mind opportunity costs. Time spent playing the games is time not spent reading, socializing, gardening, exercising, or engaging in many other activities that may benefit cognitive and physical health of older adults. Given that the effects of playing the games tend to be task-specific, it may be advisable to train an activity that by itself comes with benefits for everyday life. Another drawback of publicizing computer games as a fix to deteriorating cognitive performance is that it diverts attention and resources from prevention efforts. The promise of a magic bullet detracts from the message that cognitive vigor in old age, to the extent that it can be influenced by the lives we live, reflects the long-term effects of a healthy and active lifestyle.

We also must keep in mind that studies reporting positive effects of brain games on cognition are more likely to be published than studies with null results –the so-called “file drawer effect”– such that even the available evidence is likely to draw an overly positive picture of the true state of affairs. Statistical methods such meta-analysis, which integrates the results of many studies in a given field of inquiry, allow estimation of effect magnitude as well as the likelihood of the file-drawer effect. While some meta-analyses report small positive effects of training on cognition, others note substantial disparities in methodological rigor among the studies that cast doubt on any firm conclusion. Further, the problems that haunt individual studies do not simply disappear when results from such studies are summarized in a meta-analysis. In particular, the practice of assessing specific tests rather than broader assays of ability is just as problematic on the level of meta-analytic integration as it is on the level of individual studies.

In summary, research on aging has shown that the human mind is malleable throughout life span. In developed countries around the world, later-born cohorts live longer and reach old age with higher levels of cognitive functioning than those who were born in earlier times. When researchers follow people across their adult lives, they find that those who live cognitively active, socially connected lives and maintain healthy lifestyles are less likely to suffer debilitating illness and early cognitive decline in their golden years than their sedentary, cognitively and socially disengaged counterparts. The goal of research on the effectiveness of computer-based cognitive exercise is to provide experimental evidence to support or qualify these observations. Some of the initial results are promising and make further research highly desirable. However, at present, these findings do not provide a sound basis for the claims made by commercial companies selling brain games. Many scientists cringe at exuberant advertisements claiming improvements in the speed and efficiency of cognitive processing and dramatic gains in “intelligence”, in particular when these appear in otherwise trusted news sources. In the judgment of the signatories below, exaggerated and misleading claims exploit the anxiety of adults facing old age for commercial purposes. Perhaps the most pernicious claim, devoid of any scientifically credible evidence, is that brain games prevent or reverse Alzheimer’s disease.

In closing, we offer five recommendations. Some of these recommendations reflect experimental findings in human populations, whereas others are based on a synthesis of correlational evidence in humans and mechanistic knowledge about risks and protective factors.

  • Much more research needs to be done before we understand whether and what types of challenges and engagements benefit cognitive functioning in everyday life. In the absence of clear evidence, the recommendation of the group, based largely on correlational findings, is that individuals lead physically active, intellectually challenging, and socially engaged lives, in ways that work for them. Before investing time and money on brain games, consider what economists call opportunity costs: If an hour spent doing solo software drills is an hour not spent hiking, learning Italian, making a new recipe, or playing with your grandchildren, it may not be worth it. But if it replaces time spent in a sedentary state, like watching television, the choice may make more sense for you.
  • Physical exercise is a moderately effective way to improve general health, including brain fitness. Scientists have found that regular aerobic exercise increases blood flow to the brain, and helps to support formation of new neural and vascular connections. Physical exercise has been shown to improve attention, reasoning, and components of memory. All said, one can expect small but noticeable gains in cognitive performance, or attenuation of loss, from taking up aerobic exercise training.
  • A single study, conducted by researchers with financial interests in the product, or one quote from a scientist advocating the product, is not enough to assume that a game has been rigorously examined. Findings need to be replicated at multiple sites, based on studies conducted by independent researchers who are funded by independent sources. Moreover, participants of training programs should show evidence of significant advantage over a comparison group that does not receive the treatment but is otherwise treated exactly the same as the trained group.
  • No studies have demonstrated that playing brain games cures or prevents Alzheimer’s disease or other forms of dementia.
  • Do not expect that cognitively challenging activities will work like one-shot treatments or vaccines; there is little evidence that you can do something once (or even for a concentrated period) and be inoculated against the effects of aging in an enduring way. In all likelihood, gains won’t last long after you stop the challenge.

In summary: We object to the claim that brain games offer consumers a scientifically grounded avenue to reduce or reverse cognitive decline when there is no compelling scientific evidence to date that they do. The promise of a magic bullet detracts from the best evidence to date, which is that cognitive health in old age reflects the long-term effects of healthy, engaged lifestyles. In the judgment of the signatories, exaggerated and misleading claims exploit the anxiety of older adults about impending cognitive decline. We encourage continued careful research and validation in this field.

 

 

 

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Human Genetics and Childhood Diseases

Curator: Larry H. Bernstein, MD, FCAP

 

 

 

Publication Roundup: HGMD

HGMD®, the Human Gene Mutation Database is used by scientists around the world to find information on reported genetic mutations. The papers below use the database to advance our understanding of disease, DNA dynamics, and more.

https://www.qiagenbioinformatics.com/blog/translational/publication-roundup-hgmd

Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes
First author: Albino Bacolla

Scientists in the US and UK published results in Nucleic Acids Research of a detailed analysis of single-base substitutions and indels in the human genome. Their findings show that certain base positions are more susceptible to mutagenesis than others. They used HGMD Professional to find mutations in specific genomic regions for analysis; the paper includes charts showing mutation patterns, germline SNPs, and more from HGMD data.

High prevalence of CDH23 mutations in patients with congenital high-frequency sporadic or recessively inherited hearing loss
First author: Kunio Mizutari

This Orphanet Journal of Rare Diseases paper from scientists in Japan sequenced 72 patients with unexplained hearing loss, finding several CDH23 mutations, some of which were novel. Mutations in the gene have been linked to Usher syndrome and other forms of hereditary hearing loss. The scientists used HGMD to find all known CDH23 mutations within nearly 70 coding regions.

Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation
First author: Q.L. Bai

In Genetics and Molecular Research, scientists report the utility of mutation analysis of the interleukin-2 receptor gamma gene to assess carrier status and perform prenatal diagnosis for X-linked severe combined immunodeficiency. They studied two high-risk families, along with 100 controls, to evaluate the approach. Sequence variation was determined using HGMD Professional and an X-SCID database, and a new mutation was discovered in the project.

Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease
First author: Tomoko Oeda

Researchers from three hospitals in Japan published this Neurobiology of Aging report that may help stratify Parkinson’s disease patients by prognosis. They sequenced mutations in the GBA gene in 215 patients, finding that those who had mutations associated with Gaucher disease suffered dementia and psychosis much earlier than those who didn’t. The team found previously reported GBA mutations using HGMD Professional.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort
First author: Elisabet Selga

In this PLoS One publication, scientists from a number of institutions in Spain examined genetic variation among patients with Brugada syndrome, a rare genetic cardiac arrhythmia. They sequenced 14 genes in 55 patients, identifying 61 variants and finding the subset that appear pathogenic. Variants were filtered against a number of databases, including HGMD.

 

 

Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes

Albino Bacolla1Xiao Zhu2Hanning Chen3Katy Howells4David N. Cooper4 and Karen M. Vasquez1

Nucl. Acids Res. (26 May 2015) 43(10): 5065-5080.   http://dx.doi.org:/10.1093/nar/gkv364

Single base substitutions (SBSs) and insertions/deletions are critical for generating population diversity and can lead both to inherited disease and cancer. Whereas on a genome-wide scale SBSs are influenced by cellular factors, on a fine scale SBSs are influenced by the local DNA sequence-context, although the role of flanking sequence is often unclear. Herein, we used bioinformatics, molecular dynamics and hybrid quantum mechanics/molecular mechanics to analyze sequence context-dependent mutagenesis at mononucleotide repeats (A-tracts and G-tracts) in human population variation and in cancer genomes. SBSs and insertions/deletions occur predominantly at the first and last base-pairs of A-tracts, whereas they are concentrated at the second and third base-pairs in G-tracts. These positions correspond to the most flexible sites along A-tracts, and to sites where a ‘hole’, generated by the loss of an electron through oxidation, is most likely to be localized in G-tracts. For A-tracts, most SBSs occur in the direction of the base-pair flanking the tracts. We conclude that intrinsic features of local DNA structure, i.e. base-pair flexibility and charge transfer, render specific nucleotides along mononucleotide runs susceptible to base modification, which then yields mutations. Thus, local DNA dynamics contributes to phenotypic variation and disease in the human population.

INTRODUCTION

Changes in human genomic DNA in the form of base substitutions and insertions/deletions (indels) are essential to ensure population diversity, adaptation to the environment, defense from pathogens and self-recognition; they are also a critical source of human inherited disease and cancer. On a genome-wide scale, base substitutions result from the combined action of several factors, including replication fidelity, lagging versus leading strand DNA synthesis, repair, recombination, replication timing, transcription, nucleosome occupancy, etc., both in the germline and in cancer (14). On a much finer scale [(over a few base pairs (bp)], rates of base substitutions may be strongly influenced by interrelationships between base–protein and base–base interactions. For example, the mutator role of activation-induced deaminase (AID) in B-cells during class-switch recombination and somatic hypermutation (5) targets preferentially cytosines within WRC (W: A|T; R: A|G) sequences (6), whereas apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) overexpression displays a preference for base substitutions at cytosines in TCW contexts (7). Other examples, such as the induction of C→T transitions at CG:CG dinucleotides by cytosine-5-methylation and the role of UV light in promoting base substitutions at pyrimidine dimers have been well documented (reviewed in (4,8)). More recently, complex patterns of base substitution at guanosines in cancer genomes have been found to correlate with changes in guanosine ionization potentials as a result of electronic interactions with flanking bases (9), suggesting a role for electron transfer and oxidation reactions in sequence-dependent mutagenesis. However, despite these advances, the increasing number of sequence-dependent patterns of mutation noted in genome-wide sequencing studies has met with a lack of understanding of most of the underlying mechanisms (10). Thus, a picture is emerging in which mutations are often heavily dependent on sequence-context, but for which our comprehension is limited.

Mononucleotide repeats comprise blocks of identical base pairs (A|T or C|G; hereafter referred to as A-tracts and G-tracts) and display distinct features: they are abundant in vertebrate genomes; mutations within the tracts occur more frequently than the genome-wide average; mutations generally increase with increasing tract length; length instability is a hallmark of mismatch repair-deficiency in cancers; and sequence polymorphism within the general population has been linked to phenotypic diversity (1115). Thus, mononucleotide repeats appear ideal for addressing the question of sequence-dependent mutagenesis since base pairs within the tracts are flanked by identical neighbors. Both historic and recent investigations concur with the conclusion that a major source of mononucleotide repeat polymorphism is the occurrence of slippage (i.e. repeat misalignment) during semiconservative DNA replication, which gives rise to the addition or deletion of repeat units (11,12). An additional and equally important source of mutation has recently been suggested to arise from errors in DNA replication by translesion synthesis DNA polymerases, such as pol η and pol κ (13), also on slipped intermediates, leading to single base substitutions.

A key question that remains unanswered in these studies and which is relevant to the issue of sequence context-dependent mutagenesis is whether all base pairs within mononucleotide repeats display identical susceptibility to single base changes and whether indels (which are consequent to DNA breakage) occur randomly within the tracts.

Herein, we combine bioinformatics analyses on mononucleotide repeat variants from the 1000 Genomes Project and cancer genomes with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations to address the question of sequence-dependent mutagenesis within these tracts. We show that mutations along both A-tracts and G-tracts are highly non-uniform. Specifically, both base substitutions and indels occur preferentially at the first and last bp of A-tracts, whereas they are concentrated between the second and third G:C base pairs in G-tracts. These positions coincide with the most flexible base pairs for A-tracts and with the preferential localization of a ‘hole’ that results when one electron is lost due to an oxidation reaction anywhere along G-tracts. Thus, despite the uniformity of sequence composition, mutations occur in a sequence-dependent context at homopolymeric runs according to a hierarchy that is imposed by both local DNA structural features and long-range base–base interactions. We also show that the repair processes leading to base substitution must differ between A- and G-tracts, since in the former, but not in the latter, base substitutions occur predominantly in the direction of the base immediately flanking the tracts. Additional sequence-dependent patterns of mutation are likely to arise from studies of more heterogeneous sequence combinations, possibly involving other aspects intrinsic to the structure of DNA.

 

RESULTS

Mononucleotide repeat variation is defined by tract length and flanking base composition

We define mononucleotide repeats in the GRCh37/hg19 (hg19) human genome assembly as uninterrupted runs of A:T and G:C base pairs (hereafter referred to as A-tracts and G-tracts, respectively) from 4 to 13 base pairs in length (Figure 1A). We retrieved a total of 48,767,945 A-tracts and 13,633,781 G-tracts, both of which displayed a biphasic distribution with an inflection point between tract lengths of 8 and 9 (bp) and with the number of runs declining with length more dramatically for G-tracts than for A-tracts (Figure 1B), as noted previously (29). Both the number of short tracts and the extent of decline varied with flanking base composition, TA[n]T runs being two- to three-fold more abundant than CA[n]Cs (Supplementary Figure S1A) and AG[n]As declining the most rapidly (Supplementary Figure S1B). Thus, mononucleotide runs exist as a collection of separate pools of sequences in extant human genomes, each maintained at distinctive rates of sequence stability, as determined by factors such as bp composition (A:T versus G:C), tract length and flanking sequence composition.

Figure 1.

Mononucleotide repeat variation, evolutionary conservation and association with transcription. (A) The search algorithm was designed to retrieve runs of As or Ts (A-tracts) and Gs or Cs (G-tracts) length n (n = 4 to 13), along with their 5′ (n = 0) and 3′ (n = n + 1) nearest neighbors from hg19. Tract bases were numbered 5′ to 3′ with respect to the purine-rich sequence. The panel exemplifies the nomenclature for A- and G-tracts of length 4. (B) Logarithmic plot of the number of A-tracts (closed circles) and G-tracts (open circles) in hg19 as a function of length. (C) Normalized fractions of polymorphic tracts (F SNV) (number of SNVs divided by both hg19 number of tracts and n) from the 1KGP for A-tracts (closed circles) and G-tracts (open circles). (D) Radial plot of SNVs in the 1KGP at the 5′ and 3′ nearest neighbors of A-tracts. Periphery, tract length; horizontal axis, scale for the fraction of SNVs (F SNV). (E) Radial plot of SNVs in the 1KGP at the 5′ and 3′ nearest neighbors of G-tracts. (F) Percent difference in the numbers of A-tracts (closed circles) and G-tracts (open circles) between syntenic regions of hg19 and HN genomes. (G) The exponents of Benjamini-corrected P-values for A-tract-containing genes enriched in transcription-factor binding sites plotted as a function of A-tract length (triangles); each value represents the median of the top 11 USCS_TFBS terms. The percent A-tracts (closed circles) and G-tracts (open circles) intersecting genomic regions pulled-down by chromatin immunoprecipitation using antibodies against transcription factors are plotted as a function of tract length. (H) List of gene enrichment terms with a Benjamini-corrected P-value of <0.05 in common between genes containing A- and G-tracts of lengths 4–13, excluding the UCSC_TFBS terms.

 We examined the extent of sequence variation in the human population by mapping 38,878,546 single nucleotide variants (SNVs) from 1092 haplotype-resolved genomes (the 1000 Genomes Project, 1KGP) (30) to the hg19 A- and G-tracts. The normalized fractions of polymorphic tracts (F SNV) were greater for G-tracts than A-tracts and both displayed Gaussian-type distributions, with maxima of 0.067 for G-tracts of length 8 and 0.017 for A-tracts of length 9 (Figure 1C). CA[n]C and AG[n]A runs displayed the highest F SNV values for A- and G-tracts, respectively (Supplementary Figure S1C and D), with F SNV values for AG[n]As attaining ∼0.10 at length 8. We conclude that flanking base composition influences the rates of SNV within mononucleotide runs and, as a consequence, their representation in the reference human genome.

F SNV values at the flanking 5′ and 3′ bp were similar between A- and G-tracts, except for minor differences for the least represented (i.e. longest) tracts and did not exceed 0.02 (Supplementary Figure S1E). These fractions are expected to be greater than at more distant positions from the tracts, based on previous data (29). SNVs at G-tracts, but not at A-tracts, were more frequent than at flanking base pairs. F SNVs for base pairs flanking short (≤8 bp) tracts were at least twice as high as those flanking long tracts; F SNVs also displayed distinct sequence preference with most (∼0.1) variants occurring at Ts 3′ of G-tracts (Figure 1D and E). In summary, SNVs at mononucleotide runs do not increase monotonically with length but peak at 8–9 bp. This behavior mirrors the genomic distributions, both with respect to the total number of tracts (Figure 1B) and the subsets flanked by specific-sequence combinations (Supplementary Figure S1A–D). Variation at flanking base pairs also displayed a biphasic pattern centered at a length of 8–9 bp, with a greater chance of variation adjacent to G- than A-tracts and with characteristic sequence preferences.

Long tracts are evolutionarily conserved and associated with high transcription

To assess whether more variable monosatellite runs (Figure 1C) might have undergone a greater reduction in number in extant humans relative to extinct hominids, we compared the number of A- and G-tracts between syntenic regions of five individuals comprising hg19 and three Neanderthal (HN) specimens (31). The difference between hg19 and HN was very small (<±2%) for the short tracts, but it displayed more negative values in hg19 with increasing tract length, which reached a maximum of −11.8 and −32.7% for A- and G-tracts, respectively, of length 9. Beyond this threshold, the numbers of tracts converged for A-tracts, whereas they were more abundant in hg19 for G-tracts >11 bp (Figure 1F). In summary, the largest difference in the number of mononucleotide runs between hg19 and HN sequences was centered at 9 bp for both A- and G-tracts, suggesting that the length distributions (Figure 1A and Supplementary Figure S1A and B) reflect distinct rates of evolutionary gains and losses due to differential sequence mutability (Figure 1C) as a function of length and flanking sequence composition (12).

The fact that long (>9 bp) mononucleotide runs display low variability in the human population (Figure 1C) and sequence conservation during evolutionary divergence (Figure 1F) raises the possibility that they might serve functional roles. Through gene enrichment analyses, we found that genes containing A- and G-tracts were enriched for genes associated with the term ‘UCSC_TFBS’, which pertains to transcripts harboring frequent transcription factor binding sites (32,33). For A-tract-containing genes, the median P-values for the top 11 UCSC_TFBS terms decreased from 2.95E-26 for tracts of length 4 to 5.22E-241 for tracts of length 13 (Figure 1G). The percent of A-tracts intersecting genomic fragments amplified from chromatin immunoprecipitation using transcription-factor binding antibodies (32,33) also increased from 8.7 to 9.9 from length 6 to 13, whereas it was constant (mean ± SD, 22.4 ± 1.1) for G-tracts (Figure1G). For gene classes excluding ‘UCSC_TFBS’, a search for categories enriched at P < 0.05 and common to all A- and G-tract-containing genes returned a set of 25 terms, 22 of which were associated with high levels of tissue-specific gene expression (Figure 1H). In summary, these analyses extend prior work (14) supporting a role for mononucleotide tracts in enhancing gene expression, a function that for A-tracts appears to increase with increasing tract length.

Repeat variability is highly skewed

Next we addressed whether bp along A- and G-tracts display equal probability and type of variation. In the 1KGP dataset, the number of SNVs at each position along both A- and G-tracts of length 4 was within a two-fold difference (144,000–240,000); for both types of sequence, transitions (i.e. A→G and G→A) were the predominant (51–78%) type of base substitution (Supplementary Figure S2A and B). However, with increasing length, the number of SNVs decreased up to 30-fold more drastically for G-tracts than for A-tracts, with increasing numbers of transversions (A→T and G→C|T) being predominant. Normalizing the data for the number of tracts genome-wide revealed that the extent of SNV varied by up to 10-fold, depending upon tract length and bp position. Specifically, the highest degree of variation was observed at the first and last A within the A-tracts (i.e. A1 and An), which underwent up to 61% A→T and 43% A→C transversions, respectively, at length 9 (Figure 2A). Likewise, for G-tracts, the most polymorphic sites were G3, followed by G2, for mid-size tracts of 8–10 bp, with 44% G→C transversions at G3 for tracts of length 8 (Figure2B). Thus, the extent of SNV at mononucleotide runs is grossly skewed in human genomes, both along the sequence itself and across tract length, which must account for the bell-shape behavior in F SNV for the tracts as a whole (Figure 1C).

Figure 2.

Population variation spectra. (A) Variation spectra of A-tracts. Percent (number of SNVs at each position divided by the number of tracts in hg19 × 100) of A→T (black), A→C (red) and A→G (green) SNVs in the 1KGP dataset (left). Percent SNVs at A1 as a function of tract length (right). (B) Variation spectra of G-tracts. As in panel A with G→T (black), G→C (red) and G→A (cyan) (left). Percent SNVs at G3 as a function of tract length (right). (C) Percent A→T, A→C and A→G transitions at each position along A-tracts (stars) preceded and followed by a T (TA[n]T, left), C (CA[n]C), center) and G (GA[n]G, right) as a function of tract length. (D) Percent G→T, G→C and G→A transitions at each position along G-tracts (stars) preceded and followed by a T (TG[n]T, left), C (CG[n]C), center) and A (AG[n]A, right) as a function of tract length. (E) Percent transitions at base pairs (stars) preceding or following A-tracts (left) and G-tracts (right) as a function of tract length (n). *, mutated position.

We assessed whether SNV hypervariability was associated with specific combinations of nearest neighbors. For A-tracts flanked 5′ by a T, C or G, the highest percentage of SNVs was observed at A1 when preceded by a T, which reached 7.9% for TA[n] tracts of length 9 (Supplementary Figure S2C). By contrast, for 3′ T, C or G, the greatest effect was elicited by a C, with the highest percentage (7.1%) of SNVs at An for A[n]C tracts of length 9 (Supplementary Figure S2D). Therefore, flanking base pairs play a critical role both in the spectra and frequencies of SNVs at A-tracts. More detailed plots along A-tracts either preceded (Supplementary Figure S2E), followed (Supplementary Figure S2F) or preceded and followed (Figure 2C) by a T, C or G revealed the dramatic and long-range (up to 9–10 bp for the longest tracts, higher than the value of 4 bp predicted by mathematical models of slippage (11)) influence of flanking base pairs on variation spectra, in which up to 95% of the changes were in the direction of the base flanking the tract. Because the number of A-tracts preceded or followed by a specific base varies by up to three-fold (Supplementary Figure S2G), we conclude that for A-tracts, the overall mutation fractions and spectra are the result of at least three variables; length, position along the tract, and base composition of the 5′ and 3′ nearest-neighbors.

For G-tracts flanked 5′ by a T, C or A, high percentages (10–12%) of SNVs were observed at G1 for tracts preceded by a C, an effect that decreased with increasing tract length (Supplementary Figure S3A). This result, together with an exceedingly low number of G→A transitions at G1 for tracts not preceded by a C (Supplementary Figure S3C) relative to all tracts (Supplementary Figure S2B), is consistent with the known high mutability of CG:CG dinucleotides as a result of cytosine-5 methylation (9). The hypermutability at G2 was observed preferentially for tracts preceded by an A, and to a lesser extent T, whereas that at G3 was insensitive to flanking sequence composition. Likewise, G-tracts flanked 3′ by a T, C or A did not display marked sequence-dependent effects (Supplementary Figure S3B). Detailed plots of the SNV spectra along G-tracts either preceded (Supplementary Figure S3D), followed (Supplementary Figure S3E), or preceded and followed (Figure 2D) by a T, C or A revealed a noticeable effect only for 5′ T in association with G→T substitutions at G1for tracts of length ≥8. Thus, despite a consistent over-representation of G-tracts flanked 5′ by a T (Supplementary Figures S3F and S1B), which must account for the high absolute number of SNVs at G1 for TG[n] relative to AG[n] and CG[n] (Supplementary Figure S3G), nearest-neighbor base composition seems to play a lesser role in SNV spectra at G-tracts than at A-tracts.

With respect to SNVs at the flanking 5′ and 3′ nearest positions, no B→A or H→G substitutions (Figure 1A) were found above a length threshold of 9 for A-tracts and 8 for G-tracts (Figure 2E, gray shading) out of 5969 SNVs, implying that tract expansion by recruiting flanking base pairs is disfavored at these lengths. In summary, base substitution along mononucleotide repeats is strongly skewed towards the edges of A-tracts and within the 5′ half of G-tracts, with frequencies that peak at midsize lengths (8–9 bp). For A-tracts ≥7 bp, base substitution occurred almost exclusively in the direction of the flanking nearest-neighbors. Finally, base substitution at flanking bases did not contribute to tract expansion for mononucleotide runs longer than 8–9 bp.

Insertions and deletions display length and positional preference

In addition to SNVs, mononucleotide runs are polymorphic in length as a result of indels. Herein, we consider separately two types of indels: one in which tract length changes by ±1 and flanking bp composition is not altered (slippage); the other comprising all other cases involving the addition or removal of 1–200 bp (indels). Slippage is a widely accepted mutational mechanism (1112,34), whereby DNA replication errors at reiterated DNA motifs cause changes in the number of motifs (most often +/−1). The normalized fractions of slippage in the 1KGP dataset peaked at lengths of 8 bp for A-tracts and 9 bp for G-tracts (Figure 3A), generating bell-shaped curves similar to those observed for SNVs (Figure1C) and with no differences in the highest fraction of ‘slipped’ tracts, which peaked at ∼0.02. By contrast, +1 slippage occurred more frequently than −1 slippage at A-tracts (Figure 3B). These results support recent studies on microsatellite repeats (12) and contrast with previous conclusions that slippage increases monotonically with tract length, and that the extent of slippage differs between A- and G-tracts (35,36).

Figure 3.

Population insertions and deletions. (A) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage in the 1KGP dataset as a function of tract length. Data were obtained by dividing the number of events by both the number of hg19 tracts and tract length (n). (B) Ratio of the number of +1 to −1 slippage for A-tracts (closed circles) and G-tracts (open circles). (C) Indels at A-tracts. For positions along the tracts (‘Tract’), ‘F Indel’ is the ratio between the number of indels and the number of tracts in hg19 multiplied by tract length. For the positions immediately flanking the tracts genomic coordinates (‘Before tract’ and ‘After tract’), ‘F Indel’ is the ratio between the number of indels and the number of tracts in hg19. (D) Indels at G-tracts, calculated as described in panel C. (E) Heatmap representation of insertions along A-tracts. The percent insertions (i.e. the number of insertions at each position divided by the number of tracts in hg19) (y-axis) plotted as a function of location (x-axis) from position 0 (insertion between the bp 5′ to the tract and the first bp of the tract) to position n + 1 (insertion between the bp 3′ to the last bp of the tract and the following bp) (see Figure 1A) and as a function of tract length (z-axis). (F) Heatmap representation of insertions along G-tracts.

With respect to indels, the normalized fractions were low (<1 × 10−3) along short (4–6 bp) A- and G-tracts, but rose to a plateau for longer tracts as reported earlier (11); this plateau was 10-fold higher for G-tracts (∼0.03) than for A-tracts (∼0.003) (Figure 3C and D). Indels also occurred more frequently (up to six-fold for A-tracts of length 11) at nearest-neighboring base pairs (‘Before tract’ and ‘After tract’ in Figure 3C and D) than along the tracts. Thus, contrary to SNVs and slippage, indels increased to a plateau with mononucleotide tract length.

We analyzed in detail the locations of insertions along the tracts and the flanking positions with respect to the 5′ to 3′ orientation of the tracts (Figure 1A). The normalized fractions demonstrated that insertions peaked at the 3′, and to a lesser extent 5′, ends of the longest A-tracts (Figure 3E), but remained low. For G-tracts, insertions occurred most efficiently at two locations (G2–3 and G5) (Figure 3F), they increased with tract length (up to ∼0.04), and attained ∼10-fold higher values than for A-tracts. In conclusion, insertion sites at A- and G-tracts followed the patterns observed for SNVs (Figure 2A and B), suggesting that factors associated with local DNA dynamics sensitize specific bases along the tracts to genetic alteration, inducing both SBS and indels.

Base pair flexibility and charge localization map to sites of sequence changes

To elucidate elements of intrinsic DNA dynamics that may be responsible for the biases in SNV and insertion sites, we performed molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations on model A[6], A[9], G[6] and G[9] duplex DNA fragments. We focused on water bridge coordination (Figure 4A), bp step flexibility, and for the G[6] and G[9], charge localization, as these properties are known to impact the susceptibility of DNA to base damage, repair and mutation. The fractions of one water coordination increased along the A[9] and A[6] structures in a 5′ to 3′ direction, irrespective of flanking sequence composition, in concert with a decrease in minor groove width (Figure 4B and Supplementary Figure S4A) as predicted (37). Vstep, a measure of bp structural fluctuation, displayed a prominent peak of ∼40 Å3deg3 at the 5′-TA-3′ step for both structures (Figure 4C and Supplementary Figure S4B), which together with low water occupancy points to 5′-TA-3′ being a preferred location for base modification and mutation. In the G[9] and G[6] structures water coordination involved mostly two-water bridges due to wide (∼14 Å) minor grooves (Figure 4Dand Supplementary Figure S4C), whereas flexibility was modest (∼20–22 Å3deg3, Figure 4E and Supplementary Figure S4D). Thus, bp dynamics are likely to impact mutations at A-tracts to a greater extent than at G-tracts. Guanine has the lowest ionization potential (IP) of all four bases and IP further decreases at guanine runs, rendering them targets for electron loss, charge localization, oxidation and eventually mutation (4,38). Because after electron loss the ensuing charge (hole) can migrate along the DNA double-helix and relocalize at specific guanines, we addressed whether the preferred sites of mutation along G-tracts, i.e. G2–3 and G5, would also be preferred sites for charge localization. The QM/MM determinations indicated that whereas for the short G[6] fragment the difference in the density-derived atomic partial charges (DDAPC) (i.e. the hole) localized most often (∼50%) to the first position (Figure 4F), for the long G[9] fragment charge localization shifted downstream (mostly to the second, but also to positions 6–7, Figure 4G). Importantly, the charge was found exclusively around the guanine rings (Figure 4H). Thus, the two main sites of sequence change along G-tracts, i.e. G2–3 and G5, coincide with positions where charge localization and hence one-electron oxidation reactions is predicted to occur most frequently. In summary, bp flexibility at A-tracts and charge transfer at G-tracts likely represent intrinsic DNA features underlying the bias in SNV and insertions at mononucleotide runs in human genomes.

Figure 4.

MD and QM/MM simulations. (A) Molecular modeling of one (left) and two (right) minor groove water bridge coordination. (B) Fraction of one-water bridge occupancy (left axis) at A[9] DNA sequences flanked 5′ and 3′ by a T (black circles), C (red circles) or G (green circles). Minor groove widths (right axis), as determined from intrastrand phosphate-to-phosphate distances. (C) Vstep for A[9] DNA sequences, determined as the product of the square root of the eigenvalues (λi) described by the six bp step parameters shift, slide, rise, tilt, roll and twist; i.e. Vstep=6i=1λi−−√. (D) Fraction of one- (black circles) and two-water (red circles) bridge occupancy (left axis) at G[9] DNA sequences. Minor groove widths (right axis), as assessed from intrastrand phosphate-to-phosphate distances. (E) Vstep for G9 DNA sequences. (F) Average charge redistribution (open circles and right axis) for G[6] DNA structures upon vertical ionization, examined by calculating the difference on the density-derived atomic partial charges (DDAPC) for the neutral and negatively charged states. Histogram of the number of instances (left axis) in which the largest charge redistribution occurred at a specific position along the G[6] structures. (G) DDAPC for G[9] DNA structures (open circles and right axis) and histogram of the number of instances (left axis) in which the largest charge redistribution occurred at a specific position. (H) VMD rendering of a G[9] DNA structure displaying hole localization at G2. Capped base pairs were removed for clarity.

Position and orientation along nucleosome core particles modulate sequence variation

DNA wrapped around histones in nucleosomes is subject to local deformation (39), which may impact mutation. Thus, we analyzed the 1KGP SNVs at A- and G-tracts predicted to overlap with well-positioned nucleosome core particles (NCPs) (16). In hg19, the percentage of tracts that overlap with NCPs decreased moderately from ∼90% at length of 4 to 81% and 71% for A- and G-tracts of length 13, respectively (Figure 5A), suggesting that mononucleotide runs are not depleted in NCPs in human genomes as previously proposed (40). A-tracts of lengths 4–8 base pairs displayed distinctive peaks along the NCP surface in phase with the helical repeat of DNA (10.5 bp) and with minor grooves facing toward the inner protein core (lengths 4–5) (16) (Figure 5B and Supplementary Figure S5A). A-tracts of length of 9–13 bp exhibited only half (six) the peaks evident for the shorter tracts. For the G-tracts, only small peaks with no clear minor groove-inward-facing regions were detected (Supplementary Figure S5B).

Figure 5.

Positioning along nucleosome core particles. (A) Percent of A-tract (open circles) and G-tract (closed circles) base pairs in hg19 overlapping with well-positioned NCP genomic coordinates as a function of tract length. (B) Counts of base pairs in hg19 A-tracts of length 5 overlapping with NCPs genomic regions as a function of distance from the histone octamer dyad axis. Minor groove-inward-facing regions (gray) were derived from the X-ray crystal structure of NCP147 (41). (C) Percent SNVs in the 1KGP dataset (left axis) at every bp along A-tracts of length 5 for tracts centered at maxima (black) and minima (gray) along NCPs (Figure 5B). Percent increase (right axis) of SNVs at minima relative to maxima (green). P-values for paired t-tests: 0.013 (*), 0.002 (**) and 4.7 × 10−6 (***). (D) Whisker plots of%SNVs (left axis) at A1 for A-tracts of length 5 centered at maxima and minima (black) along NCPs (Figure 5B). Percent difference (right axis) in the number of A-tracts of length 5 in hg19 preceded by C, T or G (red) between those centered at minima and those centered at maxima (Figure5B). (E) C-containing/G-containing ratios (see text) for G-tracts of length 5 in hg19 as a function of distance from the NCP dyad axis (black) and location of core histones (maroon and green). Peaks correspond to negative iSAT (i.e. tilt parameters multiplied by the corresponding sin θ) values (gray) (39). Ratios of%SNV at G1 (upshifted by 0.5 for clarity) between C-containing (5′-CCCCCG-3′ sequences on the hg19 forward strand) and G-containing (5′-CGGGGG-3′ sequences on the hg19 forward strand) (Figure 1A) CG[5] tracts mapping NCP Chip-seq genomic intervals (red) fitted by a non-parametric local regression (loess; sampling proportion, 0.100; polynomial degree, 3). (F) VMD rendering (top) of TATTT residues 34–38 (yellow) and the complementary AAATA residues 672–753 (pink) from the 1EQZ pdb nucleosomal crystal structure, corresponding to peak area from −40 to −36 in Figure 5E. The switch in G-tract (lengths of 5 and 7) orientation along NCPs (bottom) serves to position the C-containing strand on the outside (yellow) and, correspondingly, the G-containing strand on the inside (pink).

 To assess if tract-positioning along NCPs influences SNVs, we selected A-tracts of lengths 5, 7 and 9 bp and G-tracts of lengths 5 and 7 bp whose central positions coincided with either the maxima or minima (41) (Figure 5B and Supplementary Figure S5A and B) and conducted pair-wiset-tests (330 total) between permutations of ‘categories’, including ‘tracts centered at maxima versus minima’, ‘position along the tracts’, ‘flanking sequence composition’, ‘specific NCP locations’ and ‘tract orientation’. For A-tracts, 79/207 (38%) significant pairs were found, 68 (86%) of which were related to differences between tracts centered at maxima versus minima, with a preponderance (63%) of tests displaying increased %SNVs at minima (Supplementary Figure S5C and E). For example, %SNVs at length 5 bp were greater at minima than at maxima at each position along the A-tracts (Figure 5C). A→C substitutions at A1 were more abundant at maxima than at minima (mean ± SD, 18.7 ± 0.7% at max and 17.6 ± 0.8% at min; P-value 0.001), whereas A→T substitutions at the same position displayed the opposite trend (mean ± SD, 18.4 ± 0.5% at max and 19.8 ± 1.1% at min; P-value 0.0005) (Figure 5D). A-tracts of length 7 also exhibited a similar pattern at A7 (Supplementary Figure S5H). The percentages of CA[5] and A[7]C tracts in hg19 centered at maxima were greater than at minima and the reverse was observed for the TA[5] and A[7T] tracts (Figure 5D and Supplementary Figure S5H). Thus, we conclude that positioning along the NCP surface of both the double-helical grooves and junctions with flanking base pairs influence SNVs along A-tracts. However, this influence is complex and for the most part, difficult to predict.

For G-tracts, most pairwise comparisons (18/34, 53%) indicated SNV variation according to sequence orientation (Supplementary Figure S5F and G). In hg19, the ratio of the numbers of G-tracts of lengths 5 and 7 for which the C-containing strand coincided with the forward sequence (downstream example sequence in Figure 1A) to the numbers of G-tracts for which the G-containing strand coincided with the forward sequence (upstream example sequence in Figure 1A) (C-containing/G-containing ratios) displayed a prominent 10.5-bp oscillation in phase with iSAT (Figure 5E), a measure of ‘inside’ and ‘outside’ bases, according to the bp step tilt parameter (39). Analysis of the helical path of a 146-bp DNA fragment wrapped around histones showed that the oscillation in the C-containing/G-containing ratios corresponds to a preference for guanine bases to face the protein core (Figure 5F). We analyzed the subset of G-tracts preceded by a 5′ C (i.e. CG[5]) to assess whether SNVs at G1, the position known to be mutable due to CpG methylation also oscillated with the C-containing/G-containing ratios. Oscillation in SNV-C-containing/SNV-G-containing values was evident, with peaks aligning to the hg19 troughs (Figure 5E) implying that the cytosines facing the protein surface harbor more variants than those facing away. We conclude that A- and G-tracts display preferential positioning (the former) and orientation (the latter) along NCPs, which in turn modulate the rate of sequence variation.

Mutations associated with human disease

Knowing that the first and last As of long A-tracts and G2–3 in G-tracts are the major sites of SNV in the human population, we addressed whether these features are also discernible in mutated mononucleotide tracts associated with human genetic disease. We collected 9,450,456 unique SBSs (both SBSs and SNVs refer to single base changes) from sequenced cancer genomes and normalized the percent mutations along A- and G-tracts to enable a direct comparison with the 1KGP dataset. For A-tracts (Figure 6A and Supplementary Figure S6A), SBSs displayed the same trend as the 1KGP data (Figure 2A) with respect to the bell-shape increase in mutations at A1 and An and the mutation spectra, although the susceptibility to mutation as a function of tract length attained greater values (6.36% for length 11 in cancer versus 4.15% for length 9 in the 1KGP datasets at A1). The first and last 3 bp also harbored more SBSs than in the 1KGP dataset for tracts >7 bp, a feature that we found to be due exclusively to a large cancer dataset (42) containing high-level microsatellite instability (MSI) samples (Supplementary Figure S6B and C), which are known to result from mismatch-repair deficiency (15). Thus, A-tracts display similar patterns of base substitution between the germline and somatic cancer tissues. For G-tracts, mutation spectra were characterized by G→T transversions at tract lengths >7, particularly at G1, the most frequently mutated position for tracts lengths up to 11 bp (Figure 6B and Supplementary Figure S6D). This trend persisted even when the high rates of methylation-mediated deamination mutations at the CG dinucleotide were removed (Supplementary Figure S6E). Thus, mutation patterns in cancer genomes contrast with those observed in the germline, both with respect to the most mutable position (G1 versus G2–3) and the types of base substitution (G→T in cancer genomes versus G→T and G→C in the germline).

Figure 6.

Mutation patterns in cancer genomes. (A) Mutation spectra for SBSs at A-tracts. Percent values were obtained by dividing the total number of SBSs at each position by the number of tracts in hg19 and then multiplying by 3.2516 to equalize the percentage of A-tracts of length 4 between the cancer genomes and the 1KGP datasets. (B) Mutation spectra for SBSs at G-tracts in cancer genomes. Percent values were obtained as in (A) using a multiplication factor of 3.7419. (C) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage, obtained by dividing the number of events by both the number of tracts in hg19 and tract length. (D) Indels at A-tracts, calculated as described in Figure 3C. (E) Indels at G-tracts, calculated as described in Figure3C. (F) Heatmap representation of insertions along G-tracts, as described in Figure 3E.

 With respect to slippage, the fractions for A-tracts elicited an excess at lengths 9 and 10 bp relative to the 1KGP dataset, which was also due to the MSI-containing dataset. For G-tracts, the fractions peaked at length 8, as for the 1KGP dataset (Figures 3A and 6C), implying that the propensity to undergo slippage is indistinguishable between the germline and soma. Indels were also more abundant at flanking base pairs than along the tracts (Figure 6D and E), particularly for G-tracts of length >7, similar to the 1KGP dataset (Figure 3C and D). Detailed analyses of insertions revealed that both G1 and the preceding position were the most significant sites of mutation (F-values up to 0.08 at G1 for tracts of length 8) (Figure 6F). Thus, the 5′ end of long G-tracts is the most susceptible site for both SBSs and insertions in cancer genomes, in contrast to the germline where these occur within the runs, typically at G2–3.

We also extracted the mutated A- and G-tracts from the Human Gene Mutation Database (HGMD), a collection of >150,000 germline gene mutations associated with human inherited disease. A total of 1519 genes were mutated at A- or G-tracts out of a total of 3972 (38%); 3480 SBSs and 2866 slippage events were noted within these tracts, 85 and 46% of which were predicted to be disease-causing, respectively (Figure 7A and Supplementary Table S1). Ranking genes by the number of literature reports indicated that among the top 10 entries three were associated with cancer (BRCA1, BRCA2 and APC), two with hemophilia (F8 and F9), four with debilitating lesions of the skin (COL71A), muscle (DMD), lung (CFTR) and kidney (PKD1), with one causing hypercholesterolemia (LDLR) (Figure 7B). Thus, mutations within A- and G-tracts carry a high social burden by contributing to some of the most common human pathological conditions.

Figure 7.

Mutation patterns in HGMD and model for sequence context-dependent changes. (A) Number of germline SBSs and slippage events (Slip.) at A- and G-tracts in HGMD. Gene alterations were classified as disease-causing mutation (DM), likely disease-causing mutation (DM?), disease-associated and putatively functional polymorphism (DFP), disease-associated polymorphism with additional supporting functional evidence (DP) and invitro/laboratory orinvivo functional polymorphism (FP). Codon changes (SIFT predictor) were classified as damaging (d), null (n), tolerated (t) and low-confidence prediction (l). (B) The 10 most commonly reported genes in HGMD with mutations at A- and G-tracts. Various mutated tracts were generally reported for the same gene in different reports. (C) Mutation spectra for SBSs at A- (left) and G-tracts (right) in HGMD. Percent values were obtained by dividing the total number of SBSs at each position by the number of tracts in hg19 exons. A|G→T (black), A|G→C (red), A→G (green), G→A (cyan). (D) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage, obtained by dividing the total number of events by the number of tracts in hg19 exons and by tract length. (E) Model for sequence context-dependent changes at A-tracts (left) and G-tracts (right). *, site of base modification.

 For both A- and G-tracts, SBSs occurred mostly at tract lengths of 4–7, with patterns more similar to those in the 1KGP than in the cancer datasets, both with respect to the location of the most mutable positions (first and last As and first/second Gs) and the types of base substitution (A→T and G→H) (Figure 7C and Supplementary Figure S6F). Likewise, slippage events peaked at tract lengths of 7–9 as observed in the 1KGP dataset (Figure 7D). In summary, the patterns of both SBSs and slippage in the HGMD dataset followed the trend observed in the 1KGP dataset, suggesting that germline variants at mononucleotide repeats leading to either population variation or human inherited disease may have arisen through similar mechanisms.
DISCUSSION

Why are specific A:T and G:C base pairs within A- and G-tracts more susceptible to sequence changes than their identical neighbors? For A-tracts, bp flexibility may play a role. Chemical damage to DNA, such as by hydroxyl radicals has been shown to be proportional to the geometrical solvent-accessible surface of the atomic groups, which increases with DNA flexibility (43). Along A-tracts flexibility is restricted, but it is high at both the 5′ and 3′ junctions. Thus, the fact that the highest rates of mutation coincide with the highest degree of flexibility at the 5′-TA-3′ bp step is consistent with the view that this position may be susceptible to DNA damage as a result of flexibility. Other sources of DNA dynamics are also likely to be relevant, such as sugar flexibility at the junctions, which increases with tract length (44). Chemical modification at these junctions may then lead to base substitution and indels, the latter as a result of strand breaks.

With respect to SNV mutation spectra, these were found mostly in the direction of flanking base composition above a length of 7–8 bp. We interpret this behavior in terms of DNA slippage along A-tracts when attempts are made during translesion synthesis (TLS) to bypass a damaged site (Figure 7Ei). Two scenarios may be considered to account for A→T transitions at A1. In the first, the last tract-template base would loop out into the polymerase active site permitting base-pairing and strand elongation (Figure 7Eii) using the tract-flanking base as a template (34,4546). In the second (Figure 7Eiii), slippage would occur behind the polymerase, prompting extension past the newly created A*:T mispair generated by primer/template misalignment. Either pathway would yield a common intermediate (Figure 7Eiv) that contains the base complementary to the junction across from the damaged site upon slippage resolution (34). Following DNA synthesis (S) and/or repair (R) (Figure 7Ev and vi), this mispair will generate a base change that is always identical to the tract-flanking base.

For G-tracts, the high rates of G→T transversions at G1 in cancer genomes are also consistent with preferred chemical attack at this site due to high flexibility (Figure 7F top). Direct chemical attack at a guanine is known to result in stable products, such as 8-oxo-G and Fapy-G, both of which are known to yield G→T transversions (4750). Thus, G1 may be the most susceptible site for such reactions for G-tracts of lengths ≥7 (Figure 7Fright), which in cancer genomes would become a mutation hotspot. In the germline, SNVs peaked inside G-tract base pairs, while mutational spectra were insensitive to flanking base composition; these events are inconsistent with a role for template misalignment and slippage as noted for A-tracts. Rather, the correspondence between hotspot mutations at G2–3 and G5 and the QM/MM simulations suggest a role for charge transfer. A large body of work during the past 20 years using computational, theoretical chemistry and biophysical techniques on short oligonucleotides, has shown that guanine is the most easily oxidizable base in DNA and that indeed a guanine radical cation can be generated through long-range hole transfer from an oxidant via one-electron oxidation mechanisms (5155). GGG triplets were found to act as the most effective traps in hole transfer by both experimental and theoretical work (5659), demonstrating that the resulting guanine radical cation (or its neutral deprotonated form) became rather delocalized, but it preferentially centered at the first and second G. These well-established patterns of chemical reactivity are consistent with our experimental observation of high mutation frequencies at G1 for short G-tracts and the results from QM/MM simulations on G6. For longer tracts, the downstream shift in mutation hotspots, i.e., G2–3 and G5, also correlate well with the charge localization predicted from QM/MM simulations, which explicitly included solvent effects and structural fluctuations. Thus, in conjunction with the constrained density functional theory (60), both the neutral and oxidized forms of a guanine nucleobase can be reliably constructed to infer the accurate determination of mutational patterns of mononucleotide repeats in human genomic DNA.

The compact organization of the sperm genome (61), and presumably low levels of oxidative stress in the germline, may enable guanine oxidization through one-electron oxidation reactions rather than by direct chemical attack, thereby favoring the formation of radical cations. A charge injected at G1 by electron loss would then migrate to neighboring guanines and localize at sites of low IP, such as G2 (Figure 7F left). Guanine radical cations are known to readily undergo further chemical modification leading to products such as 8-oxo-G, oxazolone, imidazolone, guanidinohydantoin, and spiroiminodyhydantoin (62) (M in Figure 7F), to yield G→T, G→C and G→A substitutions (4,63). Our model is in line with recent observations in which mutations at guanines within short G-runs (1–4 bp) correlate with sequence-dependent IPs at the target guanine in cancer genomes (9). Interestingly, these correlations were not observed in the germline (9). We interpret these composite observations as follows. The IP values for G-runs have been shown to decrease asymptotically with tract length, although the absolute values vary according to the methods and assumptions used (we obtained a value of 5.43 eV for both G[6] and G[9]) (64,65). We suggest that short G-runs with high IPs undergo one-electron oxidation reactions in the oxidative environment of cancer cells but would be refractory to such a mechanism in the germline (Figure 7Fright yellow and left white sectors). As length increases and IP values fall, G-runs would be attacked directly by oxidants abundant in tumor cells (Figure 7F orange sector), whereas oxidation will be limited to electron loss in the germline environment (Figure 7F left yellow sector).

These models (template misalignment for A-tracts and charge transfer for G-tracts) suggest a more complex scenario for mechanisms underlying mononucleotide repeat polymorphism in the human population than recently proposed (13), in which nucleotide misincorporation by error-prone polymerases is proposed as a primary source of mutations at both A- and G-tracts. As already stated, the directionality of SNVs toward tract-flanking bases in A-tracts and the hotspot mutations at G2–3, supports multiple and distinct mechanisms of base substitution at mononucleotide repeats.

Our analyses highlight additional information, including the lack of mutations in the direction of tract-base composition for base pairs flanking long tracts, the association with gene expression and the preference of guanines for the inner NCP surface, and extend prior observations (12) such as the bell-shape character of base substitution and slippage, whose mechanisms remain to be fully clarified. Finally, we document the contribution of mononucleotide mutagenesis to key aspects of human pathology beyond the well-established MSI instability in cancer (15), including hemophilia and tissue degeneration. Our collective work supports the conclusion that as the human genome undergoes evolutionary diversification and along the way suffers disease-associated mutations, oxidation reactions including charge transfer may play a prominent role.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

 

 

Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation

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Genet. Mol. Res. 14 (2): 6164 – 6172   DOI: 10.4238/2015.June.9.2
Severe combined immunodeficiency diseases (SCIDs) are a group of primary immunodeficiency diseases characterized by a severe lack of T cells (or T cell dysfunction) caused by various gene abnormalities and accompanied by B cell dysfunction (WHO, 1992; Buckley et al., 1997). The incidence rates in infants were 1/75,000-1/10,0000 (WHO, 1992), but no morbidity statistics are available in China. The 2 genetic modes of SCID include X-linked recessive and autosomal recessive genetic inheritance. X-linked severe combined immunodeficiency (X-SCID) is the most common form, accounting for 50-60% of SCID cases (Noguchi et al., 1993). Immune system abnormalities in patients with X-SCID include T-B+NK-, in which T cells (CD3+) and natural killer (NK) cells (CD16+/CD56+) are absent or significantly reduced, and the number of B cells (CD19+) is normal or increased, causing reduced immunoglobulin production and class switching disorder (Buckley, 2004; Fischer et al., 2005). The IL- 2Rg gene mutation has been confirmed to be a major cause of X-SCID (Noguchi et al., 1993). In recent years, great progress has been made in understanding the pathogenesis of primary immunodeficiency disease and its application in clinical treatment, particularly regarding the development of critical care medicine and immune reconstruction technology. With timely control of infection and early bone marrow or stem cell transplantation, X-SCID patients can be treated, prolonging survival time. Therefore, early diagnosis of X-SCID is very important for patient treatment. Gene diagnosis has become a better early diagnosis or differential diagnosis method. In addition, familial X-SCID brings a great psychological burden to the relatives of patients. Ordinary chromosome analysis and immunological evaluation cannot be used for female carrier identification and fetal diagnosis, and gene diagnosis is the most effective method of carrier detection and prenatal diagnosis. In this study, we detected mutations in 2 families with X-SCID and identified 2 novel mutations, confirming the X-SCID pedigrees. Prenatal diagnosis was performed for the pregnant fetus in the mother of one of the probands based on gene diagnosis. Female individuals in this family were subjected to carrier detection.
IL2Rg gene mutation test Direct sequencing of 1-8 exons and the flanking region of the IL2Rg gene by PCR in family 1 showed that the 3rd exon of the proband contained the c.361-363delGAG heterozygous deletion mutation, which led to deletion of the 121st amino acid glutamate (p.E121del) in its coding product. There were no sequence variations in other coding regions or in the shear zone. The proband’s mother carried the same heterozygous mutation, while his father did not carry the mutation site (Figure 2a, b, c). This mutation was not observed in any cases of the control group, and this family was identified as an X-SCID family. The c.510-511insGAACT insertion heterozygous mutation was present in the 4th exon of the proband’s mother in family 2. This mutation was a 5-base repeat of GAACT, resulting in a change in amino acid 173 from tryptophan into a stop codon (p.W173X). While there were no sequence variations in other coding regions or in the shear zone, the patient’s father did not carry the mutation (see Figure 2d, e). We did not find this mutation in the healthy control group. We presumed that the 4th exon of the deceased child in family 2 contained the c.510-511insGAACT insertion mutation, leading to X-SCID symptoms, and thus we speculated that this family was an X-SCID pedigree. Prenatal diagnosis We verified the chorionic villus status of the fetus in family 1 using the PowerPlex 16 HS System kit. The results of prenatal diagnosis showed that the fetal tissue contained no maternal contamination and that this fetus was female. The results of prenatal diagnosis showed that there was no c.361-363delGAG (p.E121del) heterozygous mutation in the female fetus of family 1.
Figure 2. Sequencing graph of IL2Rg gene in 2 pedigrees with X-chain severe combined immunodeficiency. a.-c. Family 1. a. Normal control (rectangle indicates 3 edentulous bases of this patient). b. Proband carrying the c.361- 363delGAG (p.E121del) mutation (arrow indicates deletion of fragment connection sites). c. The proband’s mother contained a c.361-363delGAG (p.E121del) heterozygous mutation (arrow). d.-e. Family 2. d. The proband’s mother carried the c.510-511insGAACT (p.W173X) heterozygous mutation (arrow indicates that the reverse sequencing graph was positive). e. Normal control (rectangular box indicates 2 normal copies of GAACT (the mutation fragment was 3 copies). Carrier detection results For the c.361-363delGAG (p.E121del) site, the gene analysis results of the female individual in family 1 showed that I2 (proband’s grandmother) was a heterozygous carrier and that II3 (proband’s aunt) was a non-carrier and had no mutations.
IL-2 can combine with the IL-2 receptor (IL-2R) of the immune cell membrane. IL-2R is composed of 3 subunits, including the IL-2Ra chain (CD25), IL-2Rb chain (CD122), and IL- 2Rg chain (CD132). IL-2Rg functional units in common with IL-4, IL-7, IL-9, IL-15, IL-21, and other cytokine receptors, and these regions are referred to as the total chain (Li et al., 2000). The IL-2Rg chain can maintain the integrity of the IL-2R complex and is required for the internalization of the IL-2/IL-2R complex; it is also the link that contacts the cell membrane surface factor region and downstream cell signal transduction molecules. Therefore, the integrity of the IL-2Rg chain is vital for the immune function of an organism (Malka et al., 2008; Shi et al., 2009).
Mutations in the IL2Rg gene, which encodes IL-2Rg, were identified to be a major cause of X-SCID in 1993 (Noguchi et al., 1993). The IL2Rg gene is located on chromosome X q21.3-22, is 37.5 kb length, and contains 8 exons, which encode 369 IL-2Rg amino acids. The IL2Rg chain exhibits varying structural regions, such as the signal peptide [amino acids (AA) 1-22], extracellular domain (AA 23-262), transmembrane region (AA 263-283), and intracellular region (AA 284-369). The WSXWS motif is located in the extracellular region (AA 237-241), while Box 1 is located in the intracellular region (AA 286-294).
By the end of 2013, the Human Gene Mutation Database contained a total of 200 mutations in the IL2Rg gene (HGMD Professional 2013.4). The most common mutation types in the IL2Rg gene were the missense or nonsense mutations, which result from single base changes. A total of 100 missense or nonsense mutations have been identified, followed by insertion or deletion mutations in a total of 50 species. The 3rd most common type of mutations includes shear mutations in approximately 30 species. Eight exons contained mutations, and mutations in 3rd or 4th exons were the highest, accounting for a total mutation rate of 43% (86/200). According to the X-SCID gene database (IL2RGbase) (http://research.nhgri. nih.gov/scid/), the gene mutations in IL2Rg mainly occurred in the extracellular region of the IL2Rg chain (Fugmann et al., 1998). Zhang et al. (2013) reported that the IL2Rg gene mutations in 10 patients with X-SCID in China were located in the extracellular region. Two mutations reported in our study were also located in the extracellular region. The mutation of IL2Rg gene in family 1 was a codon mutation in the 3rd exon, resulting in a 3-base deletion. The c.361-363delGAG (p.E121del) mutation was located in the extracellular area of the IL- 2Rg subunit, and we inferred that the 121 glutamate deletion caused by the mutation would lead to changes in the structure of the peptide chain, affecting signal transmission and resulting in serious symptoms. The mutation of family 2 was a GAACT repeat of ILR2g gene; this repeat of 5 bases resulted in 173 codon changes from tryptophan into a stop codon. Generation of the peptide chain with the mutation lacked 196 amino acids compared to the normal chain, including the intracellular, transmembrane, and some extracellular regions, directly affecting the structure and function of receptors and causing disease. No studies have been reported regarding these 2 mutations. We combined with the mutation characteristics and clinical manifestations and diagnosed family 1 as X-SCID pedigrees. Although the patient in family 2 was deceased, it can be speculated that the 2 deceased patients in family 2 were X-SCID pedigrees caused by c.510-511insGAACT (W173X).
Prenatal diagnosis can accurately identify fetal situations and be used to avoid birth defects, which can also ease the anxiety of the pregnant mother. Gene diagnosis for pedigrees of patients based on DNA samples has advanced recently, particularly with the application of high-throughput sequencing technology (Alsina et al., 2013). We can now perform gene analysis for varied clinical infectious diseases for differential diagnosis. However, the effectiveness of prenatal diagnosis for pedigrees in which the proband is dead remains unclear. Because the gene mutations in the proband is unknown in these cases, the patient’s situation was only inferred by his mother’s genotypes. However, we considered that for the deceased, if we can define the mother was a pathogenic gene carrier, even if the proband is not X-SCID, the woman also has a risk of having X-SCID children and this pedigree may be X-linked recessive inheritance. Prenatal diagnosis may provide a choice for preventing the birth of patients in these families in the premise of informed consent.
Gene diagnosis of IL2Rg can also be used for carrier detection of suspected females in the family.
In the present study, we performed carrier detection of the patient’s grandmother and aunt in family 1 and determined that the patient’s pathogenic mutations were from his grandmother. His aunt did not inherit the pathogenic gene, and thus she was a non-carrier and her fertility will not be affected. In this study, we used direct sequencing of PCR products and identified IL2Rg gene mutations in 2 pedigrees with X-SCID. We found 2 unreported mutations in the IL2Rg gene, and prenatal diagnosis and carrier detection were conducted in 1 X-SCID family. Because the incidence rate of X-SCID is extremely low, it is difficult to promote the widespread use and application of genetic diagnosis. However, this study may provide some implications for the diagnosis of infants with immunodeficiency, and gene diagnosis techniques such as conventional or high-throughput sequencing should be used as soon as possible during pregnancy, which can be used to guide treatment. This method can also provide reliable prenatal diagnosis and carrier detection service for these families.
MEF2A gene mutations and susceptibility to coronary artery disease in the Chinese population
J. Li1 , H.-X. Chen2 , J.-G. Yang3 , W. Li3 , R. Du3 and L. Tian3       DOI http://dx.doi.org/10.4238/2014.October.20.15
Coronary artery disease (CAD) has high morbidity and mortality rates worldwide. Thus, the pathogenesis of CAD has long been the focus of medical studies. Myocyte enhancer factor 2A (MEF2A) was first discovered as a CAD-related gene by Wang (2005) and Wang et al. (2003, 2005). Three mutation points in exon 7 of MEF2A were subsequently identified by Bhagavatula et al. (2004); however, Altshuler and Hirschhorn (2005) and Weng et al. (2005) predicted that the MEF2A gene lacked mutations. Zhou et al. (2006a,b) analyzed the mutations and polymorphisms in exons 7 and 11 of the MEF2A gene in the Han population in Beijing, and various rare mutations were found in exon 11 rather than in exon 7. The clinical significance of specific 21-bp deletions in MEF2A was also explored, and previous studies have shown mixed results. In this study, polymerase chain reaction-singlestrand conformation polymorphism (PCR-SSCP) and DNA sequencing were used to detect exon 11 of the MEF2A gene in samples collected from 210 CAD patients and 190 healthy controls and to investigate the function of the MEF2A gene in CAD pathogenesis and their correlation.
CAD, a common disease in China, is induced by multiple factors, such as genetics, the environment, and lifestyle. Thus, a multi-faceted approach is necessary in the study of CAD pathogenesis, particularly in molecular biology research, which is important for developing comprehensive treatment of CAD based on gene therapy. The MEF2A gene was first identified as a CAD-related gene through linkage analysis of a large family with CAD (9 of 13 patients developed MI) in 2003.
In this study, we found the following mutations: 1) codon 451G/T (147191) heterozygous or homozygous mutation; 2) loss of 1 (Q), 2 (QQ), 3 (QQP), 6 (425QQQQQQ430), and 7 (424QQQQQQQ430) amino acids (147108-147131); and 3) codon 435G/A (147143) heterozygous mutation. Among these mutations, the synonymous mutation at locus 147191 was confirmed by reference to the National Center for Biotechnology Information (NCBI) database to be a single nucleotide polymorphism, which was also demonstrated in our study by the extensive presence of this polymorphism in healthy controls. However, the heterozygous mutation at locus 147143 was only found in the genomes of CAD patients, and was therefore identified as a mutation.
Given that MEF2A is a CAD-related gene, the results of various studies are controversial among several countries. Weng et al. (2005) screened gene mutations in exon 11 of the MEF2A gene from 300 CAD patients and 1500 healthy controls. They hypothesized that the changes in 5-12 CAG repeats are genetic polymorphisms and that the 21-base deletion in exon 11 of the MEF2A gene did not induce autosomal dominant genetic CAD. Gonzalez et al. (2006) suggested that the CAG repeat polymorphism was independent of MI susceptibility in Spanish patients. Kajimoto et al. (2005) reported that the CAG repeat sequence was not correlated with MI susceptibility in Japanese patients. Horan et al. (2006) also found that the CAG repeat sequence was not associated with the susceptibility to early-onset familial CAD in an Irish population. Hsu et al. (2010) identified no correlation between the CAG repeat sequence and CAD susceptibility in the Taiwanese population. Dai et al. (2010) found that the structural change in exon 11 was not related to CAD in the Chinese Han population. Lieb et al. (2008) and Guella et al. (2009) hypothesized that MEF2A was independent of CAD. However, Yuan et al. (2006) and Han et al. (2007) suggested that the CAG repeat sequence was correlated with CAD because 9 CAG repeats was an independent predictor of CAD. Elhawari et al. (2010) and Maiolino et al. (2011) suggested that MEF2A is a susceptibility gene for CAD. Dai et al. (2013) showed that mutations in exon 12 are associated with the early onset of CAD in the Chinese population. Liu et al. (2012) failed to demonstrate a correlation between the CAG repeat sequence and CAD through case-control analysis, systematic review, and meta-analysis, but found that the 21- base deletion in exon 11 was strongly associated with CAD, and that genetic variations in MEF2A may be a relatively rare, but specific, pathogenic gene for CAD/MI. Kajimoto et al. (2005) reported 4-15 CAG repeats. However, only 4-11 CAG repeats were observed in our study, possibly because of genetic differences in patients in this study. Eleven CAG repeats were observed in most samples from the control group, and the proportion of 10, 9, and 8 repeats exceeded 1%. The heterozygous mutation at 147143, as well as the 4 and 5 CAG repeats, was only observed in CAD patients. Thus, we speculated that the CAG repeat sequence is correlated with CAD susceptibility, and the presence of 4 or 5 repeats may be a risk factor for CAD, which was inconsistent with the results obtained by Han et al. (2007). The inconsistency in these results may be explained by the differences in subjects and sample sizes among studies.
Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease

Homozygous and compound heterozygous mutations in GBA encoding glucocerebrosidase lead to Gaucher disease (GD). A link between heterozygous GBAmutations and Parkinson’s disease (PD) has been suggested ( Bembi et al., 2003,Goker-Alpan et al., 2004, Halperin et al., 2006, Machaczka et al., 1999, Neudorfer et al., 1996, Tayebi et al., 2001 and Tayebi et al., 2003). In 2009, a 16-center worldwide analysis of GBA revealed that heterozygous GBA mutation carriers have a strong risk of PD ( Sidransky et al., 2009).

In addition, heterozygote GBA mutations not only carry a risk for PD development but also the possibility of some risk burden on the progression of PD clinical course. In cross-sectional analyses of GBA mutations in PD patients, earlier disease onset, increased cognitive impairment, a greater family history of PD, and more frequent pain were reported in patients with mutations, compared with no mutations ( Chahine et al., 2013,Clark et al., 2007, Gan-Or et al., 2008, Kresojevic et al., 2015, Lwin et al., 2004, Malec-Litwinowicz et al., 2014, Mitsui et al., 2009, Neumann et al., 2009, Nichols et al., 2009,Seto-Salvia et al., 2012, Sidransky et al., 2009, Swan and Saunders-Pullman, 2013 and Wang et al., 2012). Recently, a few prospective studies have investigated clinical features of PD with GBA and showed a more rapid progression of motor impairment and cognitive decline in GBA mutation cases than in PD controls ( Beavan et al., 2015, Brockmann et al., 2015 and Winder-Rhodes et al., 2013). However, in terms of motor complications such as wearing-off and dyskinesia, no studies exist in the longitudinal course of PD with GBA mutations.

Here, we conducted a multicenter retrospective cohort analysis, and the data were investigated by survival time analysis to show the impact of GBA mutations on PD clinical course. We also investigated regional cerebral blood flow (rCBF) and cardiac sympathetic nerve degeneration of subjects with GBA mutations, compared with matched PD controls.

3.1. Subjects

Among the 224 eligible PD patients (the subjects were not related to each other), 9 subjects were excluded from the analysis (4 due to multiple system atrophy findings on subsequent brain MRI and 5 because of insufficient clinical information). Therefore, 215 PD patients [female, 52.1%; age, 66.7 ± 10.8 (mean ± standard deviation)] were analyzed. For non-PD healthy controls, 126 patients’ spouses (female, 58.7%; age, 67.3 ± 10.3) without a family history of PD or GD were enrolled.

3.2. GBA mutations and risk ratios for PD

In the PD subjects, we identified 10 nonsynonymous and 2 synonymous GBA variants. Within the nonsynonymous variants, 7 mutations were previously reported in GD [R120W, L444P-A456P-V460 (RecNciI), L444P, D409H, A384D, D380N, and444L(1447-1466 del 20, insTG)] as GD-associated mutations. Three nonsynonymous mutations have never been reported in GD patients [I(-20)V, I489V, and there was one novel mutation (Y11H)].

GD-associated GBA mutations were found in 19 of the 215 (8.8%) PD patients but none in the healthy controls. The risk of PD development relative to these GD-associated mutations was estimated as an OR of 25.1 [95% confidence interval (CI), 1.50–420,p = 0.0001] with 0-cell correction. The nonsynonymous mutations that were not reported in GD patients had no association with PD development (p = 0.506; OR, 1.3; 95% CI, 0.7–2.6) ( Table 1). Four subjects had double mutations. For subsequent analyses, 2 subjects with double mutations of I (-20)V and K466K were adopted to the group of mutations unreported in GD, and 2 subjects with double mutations of R120W and I(-20)V, and of R120W and L336L were adopted to the group of GD-associated mutations.

Table 1.Frequency of glucocerebrosidase gene allele in Parkinson’s disease patients and controls

Allele name PD (n = 215) Controls (n = 126) p Odds ratio (95% CI)
GD-associated mutations
 R120W 7a 0 0.050 9.1 (0.5–160.8)
 RecNciI (L444P-A456P-V460) 4 0
 L444P 4 0
 D409H 1 0
 A384D 1 0
 D380N 1 0
444L(1447-1466 del 20, insTG) 1 0
 Subtotal, n (%) 19 (8.8%) 0 (0%) <0.001 25.1 (1.5–419.8)b
Nonsynonymous mutations not reported in GD
 I(-20)V 27a 13 0.603 1.3 (0.6–2.5)
 I489V 3 0
 Y11Hc 0 1
 Subtotal, n (%) 30 (14.0%) 14 (11.1%) 0.506 1.3 (0.7–2.6)
Synonymous, n
 K466K 2a 1
 L336L 1a 0
Allele names refer to the processed protein (excluding the 39-residue signal peptide).

Key: CI, confidence interval; GD, Gaucher disease; PD, Parkinson’s disease.

a Four subjects had double mutations; 2 of I(-20)V and K466K, 1 of I(-20)V and R120W, and 1 of R120W and L336L.
b Odds ratio was calculated by adding 0.5 to each value.
c Novel mutation.
3.3. Clinical features of PD patients by GBA mutation groups

The clinical features of PD patients with GD-associated mutations, those with mutations unreported in GD, and those without mutations are shown in Table 2. In the GD-associated mutation group, females, those with a family history and those with dementia (DSM IV) were significantly more frequent than those in the no-mutation group (p = 0.047, 0.012, and 0.020, respectively). The age of PD onset was lower in patients with GD-associated mutations (55.2 ± 9.9 years ± standard deviation), compared with those without mutations (59.3 ± 11.5), although the statistical difference was not significant. There were no differences in clinical manifestations between subjects with mutations unreported in GD and those without mutations, except for dopamine agonist dosage (p = 0.026) ( Table 2).

Table 2.Epidemiological and clinical features of PD patients with Gaucher disease–associated GBA mutations, those with mutations previously unreported in GD and those without mutations

Variables Total n = 215 Mutation (-) GD-associated mutations


Mutations unreported in GD


167 19a pb 29c pd
Sex Female, n (%) 83 (49.7) 14 (73.7) 0.047 15 (51.7) ns
Age Mean (SD) 67.0 (10.8) 62.2 (10.7) 0.063e 67.5 (11.2) nsf
Disease duration (y) Mean (SD) 7.7 (5.5) 6.9 (4.6) nsf 7.2 (4.9) nsf
Onset age Mean (SD) 59.3 (11.5) 55.2 (9.9) ns 60.3 (11.8) ns
Family history Yes, n (%) 17 (11.0)g 6 (31.6) 0.012 0 (0.0) ns
Dementia (DSM-IV) Yes, n (%) 29 (17.4) 9 (47.4) 0.020 5 (17.2) ns
MMSE Mean (SD) 25.8 (5.4)h 23.3 (7.7) nsf 27.0 (3.4)i nsf
Onset symptom (tremor vs. others) Tremor, n (%) 78 (46.8) 9 (47.4) ns 15 (51.7) ns
Modified H-Y on (<3 vs. ≥3) ≥3, n (%) 82 (49.1) 14 (73.7) 0.042 16 (55.2) ns
UPDRS part 3 Mean (SD) 23.6 (12.2)j 28.5 (13.8) nsf 21.9 (8.7) nsf
Wearing off Yes, n (%) 70 (41.9) 9 (47.4) ns 13 (44.8) ns
Dyskinesia Yes, n (%) 49 (29.3) 8 (42.1) ns 8 (27.6) ns
Mood disorder Yes, n (%) 43 (25.7) 8 (42.1) ns 7 (24.1) ns
Orthostatic hypotension symptom Yes, n (%) 21 (12.6) 5 (26.3) ns 7 (24.1) ns
Psychosis history Yes, n (%) 59 (35.3) 10 (52.6) ns 7 (24.1) ns
ICD history Yes, n (%) 8 (4.8) 1 (5.3) ns 1 (3.4) ns
Stereotactic brain surgery for PD Yes, n (%) 4 (2.4) 0 (0.0) ns 0 (0.0) ns
Agonist LED mg/d Mean (SD) 92.8 (114.2) 72.1 (137.7) nse 163.7 (155.6) 0.026e
Levodopa LED mg/d Mean (SD) 400.7 (184.2) 456.7 (206.9) nsf 369.2 (230.3) nse
Total LED mg/d Mean (SD) 496.4 (233.7) 537.9 (258.9) nsf 525.7 (287.4) nsf
Categorical data were examined by Fisher’s exact test.

Key: DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; GBA, glucocerebrosidase gene; GD, Gaucher disease; H-Y, Hoehn and Yahr; ICD, impulse control disorder; LED, levodopa equivalent dose; ns, not significant; MMSE, Mini-Mental State Examination; PD, Parkinson’s disease; SD, standard deviation; UPDRS, Unified Parkinson’s Disease Rating Scale.

a Including a double-mutation subject (with a mutation unreported in GD).
b GD-associated mutations versus mutation (-).
c Two subjects with double mutation, including GD-associated mutations, were assigned to GD-associated mutation group.
d Other mutations versus mutation (-).
e Examined by Student t test after Levene’s test for equality of variances.
f Examined by Mann-Whitney U-test because of non-Gaussian distribution.
g    n = 155 due to 10 missing data.
h    n = 164 due to 3 missing data.
i     n = 28 due to 1 missing datum.
j     n = 165 due to 2 missing data.

3.4. Survival time analyses to develop dementia, psychosis, dyskinesia, and wearing-off

Time to develop clinical outcomes (dementia, psychosis, dyskinesia, and wearing-off) was compared in 19 subjects with GD-associated mutations, 29 with mutations unreported in GD, and 167 without mutation. The median observation time was 6.0 years. The subjects with GD-associated mutations showed a significantly earlier development of dementia and psychosis, compared with subjects without mutation (p < 0.001 and p = 0.017) ( Supplementary Table e-1, Fig. 1A and B). We rereviewed the clinical record of the subject who showed early dementia (defined by DSM IV) ( Fig. 1A) and made sure it did not satisfy the criteria of DLB ( McKeith et al., 2005).

Kaplan–Meier curves of dementia and psychosis in Parkinson's disease (PD) ...

Fig. 1.

Kaplan–Meier curves of dementia and psychosis in Parkinson’s disease (PD) patients with Gaucher disease (GD)-associated glucocerebrosidase gene (GBA) mutations and those without mutations. PD patients with GD-associated GBA mutations and those without GBA mutations were compared to investigate the time taken to develop dementia (A) and psychosis (B). Because of insufficient information in several patients, the numbers in each analysis were different. The patients with and without mutations were 17 and 165 (A), 18 and 165 (B) against a total of 19 and 167. DSM IV, Diagnostic and Statistical Manual of Mental Disorders, revised fourth edition. p-Values were calculated by log-rank tests.

The associations of GBA mutations and these symptoms were estimated as HRs, adjusting for sex and age at PD onset. HRs were 8.3 for dementia (95% CI, 3.3–20.9; p < 0.001) and 3.1 for psychosis (95% CI, 1.5–6.4; p = 0.002). The time until development of wearing-off and dyskinesia complications was not statistically significant, with HRs of 1.5 (95% CI, 0.8–3.1; p = 0.219) and 1.9 (95% CI, 0.9–4.1; p = 0.086) ( Table 3).

Table 3.Hazard ratios of GBA pathogenic mutations for clinical symptoms

Model Clinical feature Hazard ratio 95% CI p
1 Dementia (DSM-IV) 8.3 3.3–20.9 <0.001
2 Psychosis 3.1 1.5–6.4 0.002
3 Wearing-off 1.5 0.8–3.1 0.219
4 Dyskinesia 1.9 0.9–4.1 0.086
Each model was adjusted for sex and age at onset.

Key: CI, confidence interval; DSM-IV; The Diagnostic and Statistical Manual of Mental Disorders part 1IV; GBA, glucocerebrosidase.

Subjects with mutations unreported in GD did not show significant differences in time to develop all 4 outcomes, compared with no mutation subjects. Therefore, subjects with GD-unreported mutations were regarded as subjects without GBA mutations in further analyses.

3.5. rCBF on SPECT in patients with GD-associated GBA mutations

We conducted pixel-by-pixel comparisons of rCBF on SPECT between PD subjects with mutations (cases) and sex-, age-, and disease duration-matched PD subjects without any mutations in GBA (controls). Four controls were adopted for each case (except for a 34-year-old female case who was matched to a control), and in total 12 cases (female 50%, age at SPECT mean ± standard error (SE); 58.9 ± 3.3 years, disease duration at SPECT 7.3 ± 1.5 years) and 45 controls (female 64.4%, age at SPECT mean ± SE; 61.0 ± 1.3 years, disease duration at SPECT 7.1 ± 0.7 years) were analyzed. As a result, a significantly lower rCBF was seen in the cases compared to the controls in the bilateral parietal cortex, including the precuneus ( Fig. 2).

Regional cerebral blood flow in the group with GD-associated mutations compared ...

Fig. 2.

Regional cerebral blood flow in the group with GD-associated mutations compared with the matched Parkinson’s disease group without mutations. Regions with lower regional cerebral blood flow in the group with GD-associated mutations displayed on an anatomic reference map. Abbreviation: GD, Gaucher disease.

3.6. H/M ratios on MIBG scintigraphy in patients with GD-associated GBA mutations

Cardiac MIBG scintigraphy visualizes catecholaminergic terminals in vivo that are reduced as well as brain dopaminergic neurons in PD patients. We also investigated MIBG scintigraphy between 16 cases (female 68.8%, age at examination mean ± SE; 60.2 ± 2.6 years, disease duration at examination 6.2 ± 1.2 years) and sex-, age- and disease duration-matched 61 controls [(63.8 %, age 62.0 ± 1.1 years, disease duration 5.5 ± 0.6 years) (1:4 except for 1 young 34-year-old female case who was matched to a control)]. In the results, both early and late H/M ratios declined in both groups and did not show any significant differences (p = 0.309 and 0.244) ( Supplementary Table e-2).

4. Discussion

4.1. Contributions of GD-associated GBA mutations to the development of PD

In the analysis of 215 PD patients and 126 non-PD controls, we identified 10 nonsynonymous heterozygous GBA mutations, including 1 novel mutation. Among these mutations, 7 were GD-associated, and the patients carrying these mutations represented 8.8% of the PD cohort. No significant association was found between the GD-unreported mutations and PD development, which suggests that only the GD-associated mutations are a genetic risk for PD. According to a worldwide multicenter analysis of 1883 fully sequenced PD patients, 7% of the GD-associated mutations are found in non-Ashkenazi Jewish PD patients ( Sidransky et al., 2009). Although the mutation frequency in the present study was similar to previous results, the OR of GD-associated heterozygous mutations (25.1) was significantly greater than the OR (5.43) of other ethnic cohorts (Sidransky et al., 2009) and was consistent with an OR of 28.0 from a previous Japanese report ( Mitsui et al., 2009). These results, taken together, suggest the possibility thatGBA mutations are at a distinct risk for PD in the Japanese population. However, a larger Japanese cohort study is required to confirm this.

4.2. Cross-sectional clinical figures of PD with GBA mutations

Before the survival time analyses, we investigated clinical features at enrollment between mutation groups. The lower onset age, more frequent family history and dementia, and worse disease severity of PD in patients with GBA mutations, compared with those without mutations, were consistent with previous cross-sectional case-control reports ( Anheim et al., 2012, Brockmann et al., 2011, Chahine et al., 2013, Lesage et al., 2011, Li et al., 2013, Mitsui et al., 2009, Neumann et al., 2009, Seto-Salvia et al., 2012 and Sidransky et al., 2009). In contrast, female-predominance (73.7%, p = 0.047) in patients with mutations observed in the present study is inconsistent ( Neumann et al., 2009 and Seto-Salvia et al., 2012).

4.3. Impact of GBA mutations on the clinical course of PD

To investigate the impact of GBA mutations on the clinical course of PD, a prospective-designed study over a long period is preferred. Although there has been a few longitudinally designed study to date, follow-up clinical data for a median of 6 years of 121 PD cases from a community-based incident cohort was recently reanalyzed; results demonstrate that progression to dementia defined by DSM IV (HR 5.7) and Hoehn and Yahr stage 3 (HR 3.2) are significantly earlier in 4 GBA mutation-carrier patients compared with 117 patients with wild-type GBA ( Winder-Rhodes et al., 2013). A 2-year follow-up clinical report of 28 heterozygous GBA carriers who were recruited from relatives of GD-patients shows slight but significant deterioration of cognition and smelling, compared to healthy controls ( Beavan et al., 2015). Brockmann et al. (2015)assessed motor and nonmotor symptoms including cognitive and mood disturbances for 3 years in 20 PD patients with GBA mutations and showed a more rapid disease progression of motor impairment and cognitive decline in GBA mutation cases comparing to sporadic PD controls. The current long-term retrospective cohort study up to 12 years reinforced these results. It revealed that dementia and psychosis developed significantly earlier in subjects with GD-associated mutations compared with those without mutation, and the HRs of GBA mutations were estimated at 8.3 for dementia and 23.1 for psychosis, with adjustments for sex and PD onset age. In contrast, the results showed no significant difference in developing wearing-off and dyskinesia.

In this study, we also investigated whether GD-unreported mutations affected the clinical course of PD. In both cross-sectional and survival time analyses, the mutations unreported in GD carried no increased burden on clinical symptoms such as dementia, psychosis, wearing-off, and dyskinesia.

4.4. Reduced rCBF in PD with GBA mutations compared with matched PD controls

We found a significantly decreased rCBF, reflecting decreased synaptic activity, in the bilateral parietal cortex including the precuneus, in subjects with GD-associated mutations compared with matched subjects without mutations. The pattern of reduced rCBF was very similar to the pattern of H215O positron-emission tomography that Goker-Alpan et.al. (2012) reported, showing decreased resting rCBF in the lateral parietal association cortex and the precuneus bilaterally in GD subjects with parkinsonism (7 subjects with homozygous or compound heterozygous GBA mutations), compared with 11 PD without GBA mutations. Results suggest that PD with heterozygous GBAmutations and GD patients presenting parkinsonism had a common reduced pattern of rCBF. Interestingly, in their study, rCBF in the precuneus—but not in the lateral parietal cortex—correlated with IQ, suggesting that the involvement of the precuneus is critical for defining GBA-associated patterns.

4.5. Reduced cardiac MIBG H/M ratios as well as matched PD controls

We also showed that cardiac MIBG H/M ratios in subjects with GD-associated mutations were lower than the cutoff point for PD discrimination (Sawada et al., 2009), suggesting that postganglionic sympathetic nerve terminals to the epicardium were denervated, as well as in PD without mutations.

4.6. Mechanisms of impact on PD clinical course by GD-associated GBA mutations

Experimental studies suggesting a bidirectional pathogenic loop between α-synuclein and glucocerebrosidase have been accumulated (Fishbein et al., 2014, Gegg et al., 2012, Mazzulli et al., 2011, Noelker et al., 2015, Schondorf et al., 2014 and Uemura et al., 2015). Loss of glucocerebrosidase function compromises α-synuclein degradation in lysosome, whereas aggregated α-synuclein inhibits normal lysosomal function of glucocerebrosidase. The pathogenic loop may facilitate neurodegeneration in GD-associated PD brain, resulting in early development of dementia or psychosis as shown in the present study. Several recent researches propose the possibility that the similar mechanism as in PD with GBA mutations exists even in idiopathic PD brain ( Alcalay et al., 2015, Chiasserini et al., 2015, Gegg et al., 2012 and Murphy et al., 2014). On the other hand, the impacts of GD-associated GBA mutations for the development of motor complications such as wearing-off and dyskinesia were not statistically significant, suggesting other pathophysiological mechanisms in the striatal circuit brought out after long-term therapy especially by l-dopa.

4.7. Limitations

Our study has several limitations. In the design of the study, we assumed that the sample size was 215 (PD patients) for survival time analyses and investigated 224 PD patients. We assumed that the mutation prevalence would be 9.4%, and in fact, we found 19 patients with mutations (8.5%) of the 224 patients. Based on these figures, we estimated the risk ratios of heterozygous GBA mutations for the risk of PD development and PD clinical symptoms as ORs in the cross-sectional multivariate analyses, although the 95% CIs were broad. More of subject numbers will be needed to determine robust risk ratios.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort

PLOS   Published: July 14, 2015   DOI: http://dx.doi.org:/10.1371/journal.pone.0132888

Brugada syndrome (BrS) was identified as a new clinical entity in 1992 [1]. Six years later, the first genetic basis for the disease was identified, with the discovery of genetic variations inSCN5A [2]. Nowadays, more than 300 pathogenic variations in this first gene are known to be associated with BrS [3]. SCN5A encodes for the α subunit of the cardiac voltage-dependent sodium channel (Nav1.5), which is responsible for inward sodium current (INa), and thus plays an essential role in phase 0 of the cardiac action potential (AP). Genetic variations in this gene can explain around 20–25% of BrS cases [3].

Since BrS was classified as a genetic disease, several other genes have been described to confer BrS-susceptibility [47]. Pathogenic variations have been mainly described in: 1) genes encoding proteins that modulate Nav1.5 function, and 2) other calcium and potassium channels and their regulatory subunits. All these proteins participate, either directly or indirectly, in the development of the cardiac AP. Although the incidence of pathogenic variations in these BrS-associated genes is low [6], it is considered that, among all of them, they could provide a genetic diagnosis for up to an extra 5–10% of BrS cases. Hence, altogether, a genetic diagnosis can be achieved approximately in 35% of clinically diagnosed BrS patients.
Other types of genetic abnormalities have been suggested to explain the remaining percentage of undiagnosed patients. Indeed, multiplex ligation-dependent probe amplification (MLPA) has allowed the detection of large-scale gene rearrangements involving one or several exons ofSCN5A in BrS cases. However, the low proportion of BrS patients carrying large genetic imbalances identified to date suggests that this type of rearrangements will provide a genetic diagnosis for a modest percentage of BrS cases [810].
BrS has been associated with an increased risk of sudden cardiac death (SCD), despite the reported variability in disease penetrance and expressivity [11]. The prevalence of BrS is estimated at about 1.34 cases per 100 000 individuals per year, with a higher incidence in Asia than in the United States and Europe [12]. However, the dynamic nature of the typical electrocardiogram (ECG) and the fact that it is often concealed, hinder the diagnosis of BrS. Therefore, an exhaustive genetic testing and subsequent family screening may prove to be crucial in identifying silent carriers. A large percentage of these pathogenic variation carriers are clinically asymptomatic, and may be at risk of SCD, which is, sometimes, the first manifestation of the disease [13].
In the present work, we aimed to determine the spectrum and prevalence of genetic variations in BrS-susceptibility genes in a Spanish cohort diagnosed with BrS, and to identify variation carriers among relatives, which would enable the adoption of preventive measures to avoid SCD in their families.

Results  
Study population 

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Table 1. Demographics of the 55 Spanish BrS patients included in the study.

The table shows the demographic characteristics of all the patients included in the study. Numbers in parentheses represent the relative percentages for each condition. T1 ECG refers to Type 1 BrS diagnostic electrocardiogram (ECG), obtained either spontaneously, or after drug challenge. The information regarding both the electrophysiological studies (EPS) and the treatment was not available for all the patients. Two of the patients that didn’t receive any treatment died, and were not taken into account for the calculations of percentages (+2 dead). ICD, intracardiac cardioverter defibrillator.

http://dx.doi.org:/10.1371/journal.pone.0132888.t001

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Table 2. Characteristics of the Spanish BrS patients carrying rare genetic variations.

The table shows the clinical characteristics of the probands who carried rare genetic variations in SCN5A, SCN2B, or RANGRF. All of them are potentially pathogenic except that found in RANGRF, which is of unknown significance (see discussion). All the potentially pathogenic variations (PPVs) that had been previously reported, except p.P1725L and p.R1898C, had been identified in BrS patients. p.P1725L had been associated with Long QT Syndrome and p.R1898C was found in Exome Variant Server with a MAF of 0.0079%. No rare variations were identified in the control population. Patient’s age is expressed in years. Bold identifies the patients carrying variations that had not been described previously. M, male; F, female; S, syncope; ICD, intracardiac cardioverter defibrillator; UK, unknown; EPS, electrophysiological studies (+, positive response;-, negative response; N/P, not performed). The two patients who carried two PPVs each are identified by a and b, respectively.

http://dx.doi.org:/10.1371/journal.pone.0132888.t002

Sequencing of genes associated with BrS

We performed a genetic screening of 14 genes (SCN5A, CACNA1C, CACNB2, GPD1L,SCN1B, SCN2B, SCN3B, SCN4B, KCNE3, RANGRF, HCN4, KCNJ8, KCND3, and KCNE1L), which allowed the identification of 61 genetic variations in our cohort. Of these, 20 were classified as potentially pathogenic variations (PPVs), one variation of unknown significance, and 40 common or synonymous variants considered benign.

The 20 PPVs were found in 18 of the 55 patients (32.7% of the patients, 83.3% males; Table 2). Sixteen patients (88.9%) carried one PPV, and two patients (11.1%) carried two different PPVs each. Nineteen out of the 20 PPVs identified were localized in SCN5A and one in SCN2B.

The vast majority of the PPVs identified were missense (70%). We also detected 2 nonsense variations (10%), 3 insertions or deletions causing frameshifts (15%), and one splicing variation (5%). The three frameshifts (p.R569Pfs*151, p.E625Rfs*95 and p.R1623Efs*7) were identified in SCN5A. These were not found in any of the databases consulted (see Methods), and were thus considered potentially pathogenic (see below). The other 16 rare variations identified inSCN5A had been previously described, and hence were also considered potentially pathogenic. Fourteen of them had been identified in BrS patients. Of these, 6 had also been identified in individuals diagnosed with other cardiac electric diseases (i.e. Sick Sinus Syndrome, Long QT Syndrome, Sudden Unexplained Nocturnal Death Syndrome or Idiopathic Ventricular Fibrillation [2,15,16,20,21,25]). The other 2, p.P1725L and p.R1898C, had only been associated with Long QT Syndrome or found in Exome Variant Server with a MAF of 0.0079%, respectively. Furthermore, we identified a variation in SCN2B (c.632A>G in exon 4 of the gene, resulting in p.D211G) which was considered pathogenic. This patient was included within our cohort, but the functional characterization of channels expressing SCN2B p.D211G was object of a previous study from our group [7]. We also identified a nonsense variation in RANGRFwhich has been formerly reported as rare genetic variation of unknown significance [29].

Additionally, we screened the relatives of those probands carrying a PPV. We analysed a total of 129 relatives, 69 of which (53.5%) were variation carriers. Genotype-phenotype correlations evidenced that 8 of the families displayed complete penetrance (S3 Table). Additionally, no relatives were available for one of the probands carrying a PPV, thus hampering genotype-phenotype correlation assessment. The other 12 families showed incomplete penetrance.

 

MLPA analysis

The 37 patients with negative results after the genetic screening of the 14 BrS-associated genes underwent MLPA analyses of SCN5A. This technique did not reveal any large exon deletion or duplication in this gene for any of the patients.

 

SCN5A p.R569Pfs*151 (c.1705dupC), a novel PPV

A 41-year-old asymptomatic male presented a type 3 BrS ECG which was suggestive of BrS. Flecainide challenge unmasked a type 1 BrS ECG (Fig 1A, left), which was also spontaneously observed sometimes during medical follow up. Sequencing of SCN5A revealed a duplication of a cytosine at position 1705 (c.1705dupC; Fig 1A, right), which originated a frameshift that lead to a truncated Nav1.5 channel (p.R569Pfs*151). The proband’s sister also carried this duplication, but had never presented signs of arrhythmogenesis. The proband’s twin daughters were also variation carriers, displayed normal ECGs and, to date, are asymptomatic (Fig 1A, middle). Thus, p.R569Pfs*151 represents a novel genetic alteration in the Nav1.5 channel that could potentially lead to BrS, but with incomplete penetrance.

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Fig 1. Characteristics of the probands carrying non-reported potentially pathogenic variations (PPVs) in SCN5A and their families.

Left: Electrocardiograms of the probands: (A) patient carrying the p.R569Pfs*151 variation, showing the ST elevation characteristic of BrS in V1 at the time of the flecainide test; (B) patient carrying the p.E625Rfs*95 variation, showing the spontaneous ST elevation characteristic of BrS in V1 and V2; and (C) patient carrying the p.R1623Efs*7 variation, showing the spontaneous ST elevation characteristic of BrS in V1 and V2. Middle: Family pedigrees. Open symbols designate clinically normal subjects, filled symbols mark clinically affected individuals and question marks identify subjects without an available clinical diagnosis. Plus signs indicate the carriers of the PPVs and minus signs, non-carriers. The crosses mark deceased individuals and arrows identify the proband. Right: Detail of the electropherograms obtained after SCN5Asequence analysis of a control subject (left panels) and of the probands (right panels).

http://dx.doi.org:/10.1371/journal.pone.0132888.g001

SCN5A p.E625Rfs*95 (c.1872dupA), a novel PPV

A 51-year-old asymptomatic male was diagnosed with BrS since he presented a spontaneous ST segment elevation in leads V1 and V2 characteristic of type 1 BrS ECG (Fig 1B, left). The sequencing of SCN5A evidenced an adenine duplication at position 1872 (c.1872dupA, Fig 1B, right). This genetic variation results in a truncated Nav1.5 channel (p.E625Rfs*95). The genetic analysis of the proband’s relatives proved that only her mother carried the variation (Fig 1B, middle). She was asymptomatic, but a BrS ECG was unmasked upon ajmaline challenge. The proband’s sister was found dead in her crib at 6 months of age, which suggests that her death might be compatible with BrS. Therefore, the p.E625Rfs*95 variation in the Nav1.5 channel represents a novel genetic alteration potentially causing BrS.

SCN5A p.R1623Efs*7 (c.4867delC), a novel PPV

The proband, a 31-year-old male, was admitted to hospital after suffering a syncope. His baseline 12-lead ECG showed a ST segment elevation in leads V1 and V2 that strongly suggested BrS type 1 (Fig 1C, left). A deletion of the cytosine at position 4867 (c.4867delC) was observed upon SCN5A sequencing (Fig 1C, right). This base deletion leads to a frameshift that originates a truncated Nav1.5 channel (p.R1623Efs*7). Genetic screening of his parents and sisters evidenced that none of them carried this novel variation (Fig 1C, middle). None of them had presented any signs of arrhythmogenicity, nor had a BrS ECG. Nevertheless, in uterogenetic analysis of one of his daughters proved that she had inherited the variation. She died when she was 1 year of age of non-arrhythmogenic causes. Hence, the p.R1623Efs*7 variation in the Nav1.5 channel is a novel genetic alteration originated de novo in the proband that could potentially lead to BrS.

Synonymous and common genetic variations portrayal

In our cohort, we identified 40 single nucleotide variations which were common genetic variants and/or synonymous variants (S2 Table). Twenty-nine had a minor allele frequency (MAF) over 1%, and were thus considered common genetic variants.

We also identified 11 variants with MAF less than 1%. Of them, 9 were synonymous variants, what made us assume that they were not disease-causing. Four of these synonymous variants were not found in any of the databases consulted, and thus their MAF was considered to be less than 1%. Each of these synonymous variations was identified in 1 patient of the cohort. A similar proportion of individuals carrying these novel variations was detected upon sequencing of 300 healthy Spanish individuals (600 alleles). The remaining 2 variants were missense, and although they had either a MAF of less than 1% or an unknown MAF according to the Exome Variant Server and dbSNP websites, they were common in our cohort (29.2 and 50%, respectively; S2 Table), and a similar MAF was detected in a Spanish cohort of healthy individuals (26.7% and 48.8%, respectively).

Influence of phenotype and age on PPV discovery

To assess if a connection existed between the probands’ phenotype and the PPV detection yield, we classified the patients in our cohort according to their ECG (spontaneous or induced type 1), the presence of BrS cases within their families, and the presence/absence of symptoms. Even though the overall PPV detection yield was 32.7%, it was even higher for symptomatic patients (Fig 2). Indeed, in this group of patients, having a family history of BrS was identified as a factor for increased PPV discovery yield. In the case of absence of BrS in the family, the variation discovery yield was almost double for those patients having a spontaneous type 1 BrS ECG than for patients with drug-induced type 1 ECG (45.5% vs 25%, respectively). In addition, we identified a PPV in 44.4% of the asymptomatic patients who presented family history of BrS and a spontaneous type 1 BrS ECG. When the patient presented drug-induced type 1 ECG or in the absence of family history of BrS, the PPV discovery yield was of around 15%.

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Fig 2. Influence of the phenotype on PPV discovery yield.

Bar graph comparing the PPV detection yield in 8 different clinical categories (stated below the graph). Each bar shows the total number of patients for each clinical category divided in those with a PPV (black) and those without an identified PPV (white). The number of patients (in brackets) and percentages are given. Pos, positive; Neg, negative; Spont, spontaneous type 1 BrS ECG; Drug, drug-induced type 1 BrS ECG; n, number of patients.

http://dx.doi.org/:10.1371/journal.pone.0132888.g002

We also investigated the role of age on the PPV occurrence. No significant age differences were observed between variation carriers and non-carriers (38.6±10.3 and 43.5±14.4, respectively, p = 0.16). However, the PPV discovery yield was higher for patients with ages between 30 and 50 years: out of the total of patients carrying a PPV, 83.3% of the patients were in this age range, while 11.1% were younger and 5.6% were older patients (Fig 3A, upper panel). The PPV discovery yield was significantly higher for symptomatic than for asymptomatic patients (42.3% vs 24.1%, respectively; Fig 3A, lower panels).

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Fig 3. Influence of the age on PPVs discovery yield.

(A) Pie charts showing the distribution of patients in the overall population as well as in the categories of symptomatic and asymptomatic patients regarding PPV discovery. The percentage and the number of patients (in brackets) are given for each group. The small pie charts correspond to the age distribution of patients with an identified PPV. (B) Bar graphs of the PPV detection yields obtained for each of the age groups (< 30 years, 30–50 years and > 50 years). Numbers inside each bar correspond to the number of patients carrying a PPV for each category and the percentages represent the variation detection yield.

http://dx.doi.org:/10.1371/journal.pone.0132888.g003

Noteworthy, in the 30–50 age range, 52.9% (9/17) of the symptomatic patients and 35.3% (6/17) of asymptomatic patients carried one PPV (Fig 3B, middle). Additionally, 40% (2/5) of the symptomatic young patients (< 30 years) were variation carriers, while no PPVs were identified in asymptomatic patients within this age range.

Overall, 55 unrelated Spanish patients clinically diagnosed with BrS were included in our study.Table 1 shows the demographics of this cohort, and Table 2 and S1 Table show the clinical and genetic characteristics of all the patients included in the study. The mean age at clinical diagnosis was of 41.9±13.3 years. Although the majority of patients were males (74.5%), their age at diagnosis was not different than that of females (41.8±12.1 years and 42.3±16.3 years, respectively; p = 0.92). A type 1 BrS ECG was present spontaneously in 37 patients (67.3%), and drug challenge revealed a type 1 BrS ECG for the remaining 18 patients (32.7%). Almost half of the patients had experienced symptoms, including 2 SCD and 4 aborted SCD. Patients who had not previously experienced any signs of arrhythmogenicity despite having a BrS ECG were considered asymptomatic. Comparison of symptomatic vs asymptomatic patients evidenced a similar percentage of males (73.1% and 75.9%, respectively). However, the mean age at diagnosis was different between the two groups of patients (37.7±14.3 and 45.7±11.4, respectively; p<0.05).

Discussion

To the best of our knowledge, this is the first comprehensive genetic evaluation of 14 BrS-susceptibility genes and MLPA of SCN5A in a Spanish cohort. Well delimited BrS cohorts from Japan, China, Greece and even Spain have been genetically studied [24,3032]. Additionally, an international compendium of BrS genetic variations identified in more than 2100 unrelated patients from different countries was published in 2010 [3]. However, all these studies screenedSCN5A exclusively. In 2012, Crotti et al. reported the spectrum and prevalence of genetic variations in 12 BrS-susceptibility genes in a BrS cohort [5]. However, this study included patients of different ethnicity. Here, we report the analysis of 14 genes which has been conducted on a well-defined BrS cohort of the same ethnicity.

Our results confirm that SCN5A is still the most prevalent gene associated with BrS. Indeed,SCN5A-mediated BrS in our cohort (30.9%) is higher than the proportion described in other European reports [3,23], where a potentially causative variation is identified in only 20–25% of BrS patients. The reason for this discrepancy is unclear but could point towards a higher prevalence of SCN5A PPVs in the Spanish population or to selection bias. Additionally, we identified a genetic variation in SCN2B (c.632A>G, which results in p.D211G). We have formerly published the comprehensive electrophysiological characterization of this variation, and showed that indeed this variation could be responsible of the phenotype of the patient, thus linking SCN2B with BrS for the first time [7]. Also, we identified a variation in RANGRF. This variation (c.181G>T leading to p.E61X) had been previously reported in a Danish atrial fibrillation cohort [33]. Surprisingly, the authors reported an incidence of 0.4% for this variation in the healthy Danish population, which brought into question its pathogenicity. Our finding of this variation in an asymptomatic patient displaying a type 2 BrS ECG also points toward considering it as a rare genetic variation with a potential modifier effect on the phenotype but not clearly responsible for the disease [29].

No PPVs were identified in the other genes tested. Certainly, it is well accepted that the contribution of these genes to the disease is minor, and thus should only be considered under special circumstances [13,34]. In addition, recent studies have questioned the causality of variations identified in some of these minority genes [35].

We also used the MLPA technique for the detection of large exon duplications and/or deletions in SCN5A in patients without PPVs, and no large rearrangements were identified. This is in accordance with previous reports, which revealed that such imbalances are uncommon [810].

Kapplinger et al. [3] reported a predominance of PPVs in transmembrane regions of Nav1.5. Indeed, it has been proposed that most rare genetic variations in interdomain linkers may be considered as non-pathogenic [36]. In contrast, PPVs identified in this study are mainly located in extracellular loops and cytosolic linker regions of Nav1.5 (Fig 4). Additionally, 2 of our non-previously reported frameshifts are located in the DI-DII linker. These 2 genetic variations lead to truncated proteins, which would lack around 75% of the protein sequence, and thus are presupposed to be pathogenic.

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Fig 4. Nav1.5 channel scheme showing the relative position of the SCN5A PPVs identified in our cohort.

Open symbols indicate already described variations and closed symbols locate novel variations reported in this study. DI to DIV designate the 4 domains of the protein, and numbers 1–6 identify the different segments within each domain. Crosses mark the voltage sensor.

http://dx.doi.org:/10.1371/journal.pone.0132888.g004

In our cohort, we have identified 40 synonymous or common genetic variations, 4 of which have not been previously reported. These variations are gradually becoming more and more important in the explanation of certain phenotypes of genetic diseases. Only a few common variations identified here are already published as phenotypic modifiers [37,38]. The effect of these and other common variants identified in our cohort on BrS phenotype should be further studied.

Unexpectedly, almost 40% (7/18) of the PPV carriers did not present signs of arrhythmogenicity. We also performed genotype-phenotype correlations of the PPVs identified in the families (S3 Table). These studies uncovered relatives, most of whom were young individuals, who carried a familial variation but had never exhibited any clinical manifestations of the disease. This is in agreement with Crotti et al. and Priori et al. [5,23], who postulated that a positive genetic testing result is not always associated with the presence of symptoms. Indeed, the existence of asymptomatic patients carrying genetic variations described to cause a severe Nav1.5 channel dysfunction has been reported [39]. The identification of silent carriers is of paramount importance since it allows the adoption of preventive measures before any lethal episode takes place. Unknown environmental factors, medication and modifier genes have been suggested to influence and/or predispose to arrhythmogenesis [11]. Hence, this group of patients has to be cautiously followed in order to avoid fatal events.

Our studies on the connection between patients’ phenotype and the PPV detection yield highlighted the presence of symptoms as a factor for an increased variation discovery yield. Within the group of symptomatic individuals, a PPV was identified in a higher proportion of patients displaying a spontaneous type 1 BrS ECG than for patients showing a drug-induced ECG. Likewise, within the asymptomatic patients with family history of BrS, those who presented spontaneous type 1 BrS ECG carried a PPV more often than those with a drug-induced ECG (Fig 2). Referring to age, the vast majority (17/20, 85%) of the PPVs were identified in patients around their fourth decade of age (30–50 years). This is in accordance with the accepted mean age of disease manifestation. Moreover, in this age range, more than 50% of the patients who presented symptoms carried a variation that could be pathogenic (Fig 3). Importantly, 35.3% of asymptomatic patients of around 40 years of age also carried one of such variations. These data highlight the importance of performing a genetic test even in the absence of clinical manifestations of the disease, and particularly when in the 30–50 years range, which is in accordance with consensus recommendations [13,34].

In conclusion, we have analysed for the first time 14 BrS-susceptibility genes and performed MLPA of SCN5A in a Spanish BrS cohort. Our cohort showed male prevalence with a mean age of disease manifestation around 40 years. BrS in this cohort was almost exclusivelySCN5A-mediated. The mean PPV discovery yield in our Spanish BrS patients is higher than that described for other BrS cohorts (32.7% vs 20–25%, respectively), and is even higher for patients in the 30–50 years age range (up to 53% for symptomatic patients). All these evidences support the genetic testing, at least of SCN5A, in all clinically well diagnosed BrS patients.

 

Study Limitations

First of all, drug challenge tests were not performed for all the relatives who were asymptomatic variation carriers. This fact hampered their clinical diagnosis and represents an impediment to definitely assess the link between PPVs and BrS. These patients are nowadays under follow-up.

New PPVs have been identified in our cohort. The clinical information available for the families suggests that these new variations could be pathogenic. Still, in vitro studies of these variations are required in order to evaluate their functional effects and verify their pathogenic role. Additionally, genotyping in an independent cohort would help reduce the likelihood of type I (false positive) error in genetic variant discovery.

We have to acknowledge that the study set is relatively small. Consequently, the classification of patients according to the different clinical categories rendered rather small sub-groups, which may lead to over-interpretation of the results. Future studies will be directed to the genetic screening of additional Spanish BrS patients, which will probably reinforce the significance of the tendencies observed here.

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