Archive for the ‘Signaling & Cell Circuits’ Category

Effect of mitochondrial stress on epigenetic modifiers

Larry H. Bernstein, MD, FCAP, Curator



Early Mitochondrial Stress Alters Epigenetics, Secures Lifelong Health Benefits

GEN 5/3/2016

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

  • 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             Publication stage: In Press Corrected Proof
  • 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.”


“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

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


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.


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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Embryonic Stem Cell differentiation

Larry H. Bernstein, MD, FCAP, Curator



Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1

Yan Tang1, Ya-Zhen Hong1, Hua-Jun Bai1, Qiang Wu1, Charlie Degui Chen2, Jing-Yu Lang1, Kenneth R. Boheler3 and Huang-Tian Yang1,4,*


Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8-/Y) model, we found that deletion ofphf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8+/Y) and ph8-/Y ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown ofpmaip1 mimicked the phenotype of ph8-/Y by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8-/Y ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells2016


Significance Statement

Embryonic stem cells (ESCs) have the unique ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Thus, ESCs provide a unique model for the study of early embryonic development. We report here previously unrecognized effects of histone demethylase plant homeo domain finger protein 8 (PHF8) on mesodermal and early cardiac differentiation. This effect is resulted from the regulation of PHF8 on apoptosis through activating the transcription of pro-apoptotic gene pmaip1. These findings extend the knowledge in understanding of the epigenetic modification in apoptosis during ESC differentiation and of the link between apoptosis and cell lineage decision as well as cardiogenesis.


Embryonic stem cells (ESCs) have the unique ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Due to this plasticity, mechanisms controlling cell autonomous and regulatory events critical to in vivo mammalian development have benefitted from the in vitro study of differentiating ESCs [1, 2]. Early embryogenesis and cavity formation as well as early ESC differentiation, for example, are accompanied by a reduction in proliferation and increased apoptosis [3-5]. Withdrawal of leukemia inhibitory factor (LIF) from mouse embryonic stem cells (mESCs) cultivated in vitro causes approximately 20%-30% of the cells to die by spontaneous (constitutive) apoptosis [4, 5]. This occurs secondary to the induction of cleaved caspase 3 [3] and apoptosis-inducing factor (AIF)-complex proteins [6]. Blockade of spontaneous apoptosis in vitro by a p38 mitogen-activated protein kinase (MAPK) inhibitor alters the differentiation markers and increases the abundance of both antiapoptotic proteins (Bcl-2, Bcl-XL) and Ca2+-binding proteins [4, 7]. In addition, Ca2+ released from type 3 inositol 1, 4, 5-trisphosphate receptors (IP3R3) negatively regulates this apoptotic response, which in turn modulates the mesodermal lineage commitment of early differentiating mESCs [5]. These findings explain, in part, how apoptosis contributes to specific lineage commitment during early development. However, in contrast to the relatively advanced knowledge of signaling pathways [8], little is known about the contribution of epigenetic regulators, especially, histone lysine demethylases (KDMs), in the regulation of apoptosis during ESC differentiation and how the affected programmed cell death by KDMs contributes to the lineage commitment.

Epigenetic regulators and dynamic histone modifications by KDMs are emerging as important players in ESC fate decisions [9]. Histone modifications coordinate transient changes in gene transcription and help maintaining differential patterns of gene expression during differentiation [10-13]. The molecular and biological functions of many KDMs, however, remain enigmatic during ESC differentiation. PHF8, an X-linked gene encoding an evolutionarily conserved histone demethylase harboring an N-terminal plant homeo domain (PHD) and an active jumonji-C domain (JmjC), is able to catalyze demethylation from histones [14, 15]. It is actively recruited to and enriched in the promoters of transcriptionally active genes [14], and it functions to maintain the active state of rRNA through the removal of the repressive H3K9me2 methylation mark at the active rRNA promoters. Mutation of PHF8 is associated with X-linked mental retardation with cleft lip/cleft palate in human [16-18]. Knockdown of phf8 in mouse embryonic carcinoma P19 cells impairs neuronal differentiation [19] and leads to brain defects in zebrafish by directly regulating the expression of the homeo domain transcription factor MSX1/MSXB [20]. However, the precise function of PHF8 in the regulation of lineage differentiation derived from other germ layers remains to be identified.

Here, we report previously unrecognized effects of the PHF8 histone demethylase on germ layer commitment and differentiation of mESCs. The results are based on an assessment of early steps of differentiation to mesodermal lineages and cardiomyocytes using phf8 knockout (phf8-/Y) and wild-type (phf8+/Y) mESCs. The data show that PHF8 regulates gene transcription of a proapoptotic gene pmaip1 (also named Noxa) [21]. Activation or repression of pmaip1 controlled by PHF8 ultimately determines mESC lineage commitment through the regulation of caspase 3-independent apoptosis during mesodermal and cardiac differentiation. Our data reveal that PHF8-mediated the demethylation of histone proteins coordinates ESC lineage commitment through the regulation of apoptosis in early differentiating ESCs.


Deletion of phf8 Promotes Mesodermal and Cardiac Lineage Commitment

The PHF8 protein was detectable in undifferentiated ESCs, but its abundance significantly increased within one day of LIF withdrawal. Then it gradually decreased to a level at day 5 lower than that observed in the undifferentiated ESCs (Fig. 1A).


Figure 1. Plant homeo domain finger protein 8 (PHF8) regulates the mesodermal and early cardiac differentiation of mouse embryonic stem cells (mESCs). (A): Western blot analysis of PHF8 expression in undifferentiated and differentiating ESCs. n = 3. (B): quantitative RT-PCR (qRT-PCR) analysis of pluripotency markers nanog, rex1, sox2, and oct4. n = 8. (C): qRT-PCR analysis of gene expression of pluripotency marker oct4; early mesodermal markers brachyury (T), gsc, eomes, and mesp1; cardiovascular progenitor markers flk-1 and nrp1; and the cardiac transcription factors hand1, tbx5, and mef2c during ESC differentiation. n = 5. (D): qRT-PCR analysis of the early ectodermal markersnestin and fgf5 during ESC differentiation. n = 3. (E): qRT-PCR analysis of early endodermal markers afp, foxa2, sox17, and gata4 during ESC differentiation. n = 3. Data are presented as mean ± SEM *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value.

To determine the significance of phf8 gene expression on ESC fate decision, we knocked out the X-chromosome-encoded phf8 gene in one allele of male SCR012 ESCs by deletion of exons 7 and 8 through Cre-mediated recombination (Supporting Information Fig. S1A). Gene inactivation was confirmed by the lack of Phf8 mRNA and PHF8 protein expression in these targeted ESCs (Supporting Information Fig. S1B). Transcripts for pluripotency marker genes nanog, rex1 (zfp42), sox2, and oct4 (pou5f1) were not significantly different between phf8+/Y and phf8-/Y ESCs (Fig. 1B). No significant difference was observed in cell morphology (Supporting Information Fig. S1C) of undifferentiated phf8+/Y and phf8-/Y ESCs or in alkaline phosphatase activity (Supporting Information Fig. S1D). Immunofluorescence staining confirmed that the expression of pluripotency marker SOX2 and SSEA-1 did not differ between the phf8+/Y and phf8-/Y ESCs (Supporting Information Fig. S1E). These results indicate that phf8 may be dispensable for normal growth and maintenance of mESCs.

We then analyzed the role of PHF8 in the mesodermal and cardiac lineage commitment. By microarray analysis of differentiating phf8+/Y and phf8-/Y cells from days 0, 1, to 3.5, we found a significant decrease in transcripts for pluripotency markers, accompanied by a significant increase in transcripts for ectoderm, mesoderm and endoderm, while in phf8-/Y cells some transcripts for mesodermal and cardiac lineage commitment were significantly enhanced compared with those in phf8+/Y cells (Supporting Information Fig. S2A). These differentiation-dependent changes in transcript abundance were confirmed by qRT-PCR for early mesodermal markers brachyury (T) [28], goosecoid (gsc),eomes[29], and mesp1[30], cardiovascular progenitor marker flk-1[31, 32] and neuropilin 1 (nrp1) [33]. Early cardiac transcription factors, including myocyte enhancer factor 2C (mef2c) [34], hand1[35], and tbx5[36, 37] were also up-regulated in phf8-/Y cells at differentiation day 5, while no difference in the expression levels of pluripotent markersoct4 (Fig. 1C), rex1, and nanog (Supporting Information Fig. S2B) were detected between phf8+/Y and phf8-/Y cells at the time points examined.

Because mESCs can differentiate into all three germ layers, we also examined whether phf8 affected ectodermal and endodermal differentiation. qRT-PCR analysis did not show any significantly difference in the expression of early ectodermal markers nestin and fgf5 between the phf8+/Y and phf8-/Y cells (Fig. 1D). Moreover, in the induced early ectodermal differentiation system [23], the expression of ectodermal markers nestin, fgf5, and pax6 were comparable between the phf8+/Y and phf8-/Y cells (Supporting Information Fig. S3A). Besides, the expression of endodermal markers afp, foxa2, sox17, and gata4 were not significantly different between the phf8+/Y and phf8-/Y cells (Fig.1E). Consistently, the expression of endodermal markers foxa2, sox17, and gata4 were comparable during induced endodermal differentiation [24] between the phf8+/Y andphf8-/Y cells (Supporting Information Fig. S3B). Thus, phf8 appears not to affect early ectodermal and endodermal differentiation.

The increased mesodermal and cardiac marker expressions were associated with a significant increase in the total number of cardiac progenitors and cardiomyocytes in differentiating phf8-/Y cells. By flow cytometry analysis, marked increases in the population of FLK-1 positive (FLK-1+) cells were detected in phf8-/Y cells at differentiation day 3 and day 4 (Fig. 2A). Consistently, the percentage of contracting EBs was higher in phf8-/Y cells than in phf8+/Y cells (Fig. 2B). The transcripts for progenitor marker nrp1, early cardiac transcription factor tbx5, and cardiac specific genes tnnt2, myh6, myl2, and gja1 were higher in phf8-/Y EBs than those in phf8+/Y ones (Fig. 2C). The areas of immunostained EBs positive for the cardiac cytoskeletal and myofilamental proteins α-actinin and TNNT2 were also greater in phf8-/Y than in phf8+/Y EBs (Fig. 2D). Flow cytometry analysis of MYH6+ (Fig. 2E) and TNNT2+ (Fig. 2F) cells at differentiation day 9 further confirmed the increase of cardiomyocytes in phf8-/Y cells. Taken together, these data indicate that the phf8 deletion facilitates the differentiation of mesodermal and cardiac linage commitment.

Figure 2. phf8 deletion promotes cardiac differentiation of mouse embryonic stem cells (mESCs). (A): Left, representative flow cytometry plots showing FLK-1 expression at differentiation day 3 (n = 6), day 4 and day 5 (n = 3 each). Right, the quantification of flow cytometry data. (B): Differentiation profile of cardiomyocytes during embryoid bodies (EB) outgrowth. n = 6. (C): qRT-PCR analysis of ESCs for the expression of cardiac markers at differentiation day 14. n = 3. (D): Immunofluorescence analysis of TNNT2 and α-actinin in day 14 EBs. Scale bars = 400 μm. (E) Flow cytometry analysis of MYH6 positive cells and (F) TNNT2 positive cells in day 9 EBs. n = 3 each. Data are presented as mean ± SEM *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value.

PHF8 Inactivation Increases Cell Viability but not Proliferation of the Differentiating ESCs

Differentiation of both phf8+/Y and phf8-/Y ESCs via EB formation produced normal round shaped EBs but, by day 3, phf8-/Y EBs were larger than those generated from phf8+/YESCs, and the size differences were visibly obvious at differentiation days 5 and 7 (Fig. 3A). Although no significant differences in cell viability could be demonstrated between undifferentiated phf8+/Y and phf8-/Y ESCs (Fig. 3B), the viability of phf8-/Y cells was significantly greater than that in phf8+/Y cells at differentiation days 3 to 7 (Fig. 3C). However, no significant change in BrdU staining was detected by flow cytometry between phf8+/Y and phf8-/Y ESCs at differentiation days 0, 3, or 5 (Fig. 3D). Moreover, no significant difference in the cell cycle distribution between the differentiating Phf8+/Y and Phf8-/Y ESCs was detected, although the percentage of cells in S phase gradually decreased while those in G1 phase increased upon differentiation (Fig. 3E). Knockout of phf8 thus increases cell numbers in the early differentiating ESCs through the improvement of cell viability without changes in cell proliferation or cell cycle progression.

Figure 3. phf8 deletion increases cell viability in differentiating mouse embryonic stem cells (mESCs) without affecting cell proliferation. (A): Left, phase-contrast images of embryoid bodies (EB) morphology during EB formation from ESCs. Scale bar = 200 μm. Right, the diameter of EB formed from ESCs. (B): Cell viability of undifferentiated and (C): differentiating ESCs analyzed by MTT assay for seven consecutive days. n = 3. (D): Flow cytometry analysis of BrdU positive proportion of undifferentiated (n = 4) and differentiating ESCs at day 3 and day 5.n = 5 each. (E): Flow cytometry analysis of cell cycle distribution by propidium iodide (PI) staining at differentiation day 3 (n = 6) and day 5 (n = 3). Data are presented as mean ± SEM *, p < .05; ***, p < .001 compared with the corresponding phf8+/Y value.

PHF8 Regulates Apoptosis During the Early Stage of Cardiac Lineage Commitment

We then examined whether cell death might account for the differences in the cell viability observed between the differentiating phf8+/Y and phf8-/Y ESCs. In undifferentiated ESCs, no significant difference was demonstrated with Annexin V (an early apoptosis marker) staining, TUNEL assay, total DNA fragmentation or caspase 3 protein cleavage between phf8+/Y and phf8-/Y cells (Fig. 4A–4C, 4E). In contrast, Annexin V staining (Fig. 4A) and TUNEL assay (Fig. 4B) showed significant decreases in the number of apoptotic cells in phf8-/Y ESCs at differentiation days 3 and 5 compared with those in phf8+/Y cells. Genomic DNA fragmentation with a pattern typical for apoptosis was detected in phf8+/Y cells at differentiation days 3 and 5, but it was reduced in phf8-/Y cells at the same time points (Fig. 4C). Moreover, approximately 35% of Annexin V+ cells were present in FLK-1+/phf8+/Y cells at differentiation day 4, whereas only 9% of the cells were Annexin V+ in FLK-1+/phf8-/Y cells (Fig. 4D). The ratio of TUNEL+ to either NESTIN+ (ectoderm) or SOX17+ (endoderm) cells did not differ between the phf8+/Y and phf8-/Y cells (Supporting Information Fig. S3C, S3D). In addition, phf8+/Y ESCs at differentiation days 3 and 5 increased the caspase 3 cleavage (Fig. 4E, upper and lower left panels) and the ratio of cleaved caspase 3 to total caspase 3 protein (Fig. 4E, lower right panel). Unexpectedly, the ratio of cleaved caspase 3 to total caspase 3 in phf8-/Y ESCs did not significantly differ from that observed in phf8+/Y ones. Consistently, a significant enhancement of the downstream target PARP1 cleavage [38, 39] was observed at differentiation days 3 and 5, but it was comparable between the phf8+/Y andphf8-/Y cells (Fig. 4F). These data suggest that the cell death associated with phf8 function does not operate through the conventional caspase 3-mediated apoptosis.

Figure 4. Plant homeo domain finger protein 8 (PHF8) regulates apoptosis during the early mouse embryonic stem cells (mESC) differentiation. (A): Left, representative flow cytometry plots showing Annexin V (x-axis), and PI (y-axis) staining in ECSs at differentiation day 0 (n = 4), day 3 (n = 3) and day 5 (n = 7). Right, the quantification of flow cytometry data. (B): Flow cytometry detection of apoptotic responses of TUNEL positive cells at differentiation day 0 (n = 3), day 3 (n = 4), and day 5 (n = 4). (C): DNA laddering analysis at differentiation days 0, 3, and 5. n = 6 each. (D): Cells double stained with FLK-1 and Annexin V were analyzed by flow cytometry at differentiation day 4. n = 3. (E): Western blot analysis of caspase 3 during the mESC differentiation. β-actin was used as a loading control. n = 4. (F): Western blot analysis of PARP1 expression during the differentiation. β-actin was used as a loading control. n = 4. Data are presented as mean ± SEM *, p < .05; ***, p < .001 compared with the corresponding phf8+/Yvalue; #, p < .05; ##, p < .01 compared with the corresponding d0 value.

pmaip1 is a Direct Target Gene of PHF8 in the Early Differentiating ESCs

To understand how PHF8 might regulate apoptosis during early ESC differentiation, we compared the profiles of apoptosis-related gene transcripts in undifferentiated and early differentiating phf8+/Y and phf8-/Y ESCs using gene expression microarrays. Among the apoptosis-related genes, the transcript to pmaip1, a proapoptotic Bcl-2 family member crucial in fine-turning the cell death decision [21, 40-42], was markedly increased during early differentiation of phf8+/Y cells but it was reduced in phf8-/Y cells at differentiation days 1 and 3.5 (Fig. 5A). These expression patterns were confirmed by qRT-PCR during cardiac differentiation (Fig. 5B), and the results were consistent with the apoptotic pattern observed during the early ESC differentiation (Fig. 4A–4C). In addition, qRT-PCR analysis showed that the expression of pmaip1 did not show any significant difference between the phf8+/Y and phf8-/Y cells during the induced ectodermal (Supporting Information Fig. S3E) or endodermal (Supporting Information Fig. S3F) differentiation.

Figure 5. pmaip1 is a direct target gene of plant homeo domain finger protein 8 (PHF8) in mouse embryonic stem cells (mESCs). (A): Microarray gene expression heat map depicting the expression of apoptosis-related genes at differentiation days 0, 1 and 3.5 in phf8+/Y and phf8-/Y ESCs. The expression values in log2 scale were calculated and presented on the heat map with red representing highly abundant transcripts and green representing poorly abundant transcripts. n = 3. (B): qRT-PCR analysis of pmaip1 during the ESC differentiation. (C): ChIP assay of PHF8 around the TSS of pmaip1 in phf8+/Y andphf8-/Y ESCs at differentiation days 0 and 3. n = 4 each. (D): Western blot analysis of H3K9me2 and H3 in phf8+/Y and phf8-/Y ESCs during the differentiation. H3 was used as a loading control. n = 9. (E): H3K9me2 staining inphf8+/Y and phf8-/Y embryoid bodies (EBs) at differentiation day 1. Scale bars = 25 μm. (F): ChIP assay of H3K9me2 around the TSS of pmaip1 in phf8+/Y andphf8-/Y ESCs at differentiation day 3. n = 4. Data are presented as mean ± SEM. *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value or d0.

A direct link between the PHF8 and pmaip1 was then confirmed by ChIP analysis. We detected the endogenous binding of PHF8 at the transcription start site (TSS, from −45 bp to 104 bp) of pmaip1 in phf8+/Y ESCs and determined that binding was enhanced at differentiation day 3. The binding of PHF8 was not detectable above the IgG control levels in phf8-/Y cells (Fig. 5C). Global methylation (H3K9me2 normalized to H3) was unchanged at differentiation days 3 and 5, but it was significantly enhanced at differentiation day 1 in phf8-/Y cells (Fig. 5D). The augmentation of H3K9me2 methylation in phf8-/Y ESCs was then confirmed by immunostaining at differentiation day 1 (Fig.5E). An increase in the repressive mark of H3K9me2 was also observed at the TSS of pmaip1 in the early differentiating phf8-/Y ESCs (Fig. 5F), indicating that the PHF8 demethylase activity is actively involved in the regulation of pmaip1 gene.

Transient Knockdown of pmaip1 Decreases Apoptosis and Promotes Mesodermal and Cardiac Differentiation

To clarify the role of pmaip1 in mESC differentiation, we transfected specific siRNAs against pmaip1 (si-Pmaip1) into phf8+/Y ESCs followed by EB formation. The negative control siRNA (si-NC) did not alter pmaip1 transcript levels compared with untreated (NT) cells, while si-Pmaip1 significantly inhibited pmaip1 transcripts by 74%-76% at differentiation days 0 and 1 (Fig. 6A-a). si-Pmaip1 cells had fewer TUNEL+ cells compared with the NT and si-NC cells at differentiation day 3 in both phf8+/Y and phf8-/Y cells (Fig. 6A-b). We then examined whether the pmaip1 knockdown influences mesodermal and early cardiac differentiation. As shown in Figure 6B, the apoptosis of FLK-1+ cells was significantly decreased in si-Pmaip1 mESCs (Fig. 6B). The expression of T and gsc as well as nrp1 and flk-1 were increased in si-Pmaip1 cells compared with those in NT and si-NC cells at differentiation day 3. In addition, the expression of cardiac transcript factors mef2c and tbx5 was up-regulated at differentiation day 5, and myh6 andtnnt2 were up-regulated at differentiation day 9 (Fig. 6C). We also transfected si-Pmaip1 into phf8-/Y ESCs. The expression of pmaip1 was downregulated at differentiation day 0 and day 1 in phf8-/Y ESCs with si-Pmaip1 (Supporting Information as Fig. S4A-a), accompanied by a decrease in TUNEL+ cells compared with NT and si-NC (Fig. 6A-b), while Annexin V remained unchanged (Supporting Information Fig. S4A-b). The expression of nrp1 and flk1 did not significantly change in phf8-/Y ESCs with si-Pmaip1 at differentiation day 3, while mef2c was upregulated at differentiation day 5, and myh6 was upregulated at differentiation day 9 (Supporting Information as Fig. S4B). These results suggest that downregulation of pmaip1 in phf8-/Y ESCs may not lead to as robust of a phenotype as it did in phf8+/Y ESCs. This difference is likely due to the level ofpmaip1 during early differentiation of phf8-/Y ESCs was already decreased to a low level similar to that observed in the undifferentiated cells (Fig. 5B). Taken together, these data demonstrate that the decreased apoptosis via down-regulation of pmaip1 contributes, at least partially, to the phf8-/Y-facilitated mesodermal and cardiomyocyte commitment.

Figure 6. Plant homeo domain finger protein 8 (PHF8) regulates the mesodermal and cardiac differentiation through pmaip1. (A-a): qRT-PCR analysis of thepmaip1 expression in phf8+/Y ESCs after being transiently transfected with si-NC or si-Pmaip1. n = 4. (A-b): Apoptosis cells were quantified by flow cytometry analysis of TUNEL assay at differentiation day 3 in phf8+/Y and phf8-/Y ESCs after transient transfection with si-NC or si-Pmaip1. n = 3. (B): Cells double stained with FLK-1 and Annexin V were analyzed by flow cytometry at differentiation day 4. n = 4. (C): qRT-PCR analysis of the expression of T, gsc, flk-1, nrp1, tbx5, mef2c, myh6, and tnnt2 in phf8+/Y ESCs after transient transfection with si-NC or si-Pmaip1. n = 5. (D): Flow cytometry detection of TUNEL positive cells at differentiation day 3, Annexin V positive cells and double stained FLK-1 and Annexin V at differentiation day 4 in phf8-/Y, phf8-NC-/+, phf8-pmaip1-/+, and phf8-hPHF8-/+ mouse embryonic stem cells (mESCs). n = 4. (E): qRT-PCR analysis of the expression of T, gsc, flk-1, nrp1, tbx5, mef2c, myh6, and tnnt2 in phf8-/Y, phf8-NC-/+, phf8-pmaip1-/+, and phf8-hPHF8-/+ mESCs. n = 3. Data are presented as mean ± SEM. *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y or phf8-/Y value.

Overexpression of pmaip1 or hPHF8 in phf8-/Y ESCs Increases Apoptosis and Weakens Mesodermal and Cardiac Differentiation

To further determine whether PHF8 contributes to mesoderm and cardiac cell commitment through the regulation of apoptosis via targeting pmaip1, we rescued the expression of pmaip1 and phf8 in phf8-/Y ESCs by generating pmaip1-ovexpressing phf8-/Y mESCs (phf8-pmaip1-/+ mESCs) and hPHF8-overexpressing phf8-/Y mESCs (phf8-hPHF8-/+ mESCs). The qRT-PCR analysis confirmed that the expression of hPHF8 or pmaip1 was significantly upregulated in the respective overexpressing cell lines (Supporting Information Fig. S4C). The expression of pmaip1 in undifferentiated phf8-/Y mESCs was not affected by hPHF8 overexpression. However, Pmaip1 transcripts increased by differentiation day 3 in overexpressing cells (Supporting Information Fig. S4D), indicating that PHF8 does regulate the expression of pmaip1 during differentiation. Both TUNEL and Annexin V analysis revealed significant increases of apoptosis in phf8-pmaip1-/+ and phf8-hPHF8-/+ mESCs compared with the phf8-/Y andphf8-NC-/+ mESCs at differentiation day 3 or day 4, accompanied by a higher apoptosis ratio in FLK-1+ cells (Fig. 6D). Moreover, the expression of T and gsc as well as nrp1and flk-1 were significantly decreased in phf8-pmaip1-/+ and phf8-hPHF8-/+ mESCs at differentiation day 3, followed by a down-regulation of mef2c and tbx5 at differentiation day 5, and myh6 and tnnt2 at differentiation day 9 (Fig. 6E). In addition, TUNEL analysis showed no changes in the apoptotic responses either through knockout or overexpression of phf8 compared with the corresponding wild-type cells or phf8+/Y cells during induced ectodermal differentiation (Supporting information Fig. S4E). These data are consistent with a regulatory role of phf8 on mesodermal and cardiac differentiation through targeting of pmaip1


This is the first study to unravel a regulatory role of histone demethylase in the differentiation of ESCs through the control of apoptosis and subsequent effects on cell lineage commitment. The role of PHF8 in the regulation of ESC differentiation to the mesodermal lineage and cardiac differentiation is supported by selective changes in RNA markers for mesodermal lineages, and an increase in cardiomyocyte progenitors and cardiomyocytes (Figs. 1C, 2C). Moreover, deletion of phf8 specifically inhibits apoptosis of Flk-1+ mesodermal cells with a concomitant reduction in Annexin V+ staining (Fig. 4D) and cardiac differentiation (Fig. 2B–E), while the ratio of TUNEL+ to either NESTIN+(ectodermal cells) or SOX17+ (endodermal cells) cells does not differ between the phf8+/Y and phf8-/Y lines (Supporting Information Fig. S3C, S3D). Consistently, the proportion of early apoptotic cells (Annexin V+) in pmaip1-knockdown (Fig. 6B) is also decreased, while pmaip1-overexpression or hPHF8-overexpression in phf8-/Y cells increase the proportion of TUNEL+ and Annexin V+ cells simultaneously with a reduction in mesodermal and cardiac differentiation (Fig. 6D, 6E). These findings indicate that the PHF8 functions, at least partially, through regulation of apoptosis.

It is well known that the regulation of apoptosis is of critical importance for proper ESC differentiation and embryo development [8, 43]. ESC differentiation is regulated by apoptosis induced by MAPK activation [7] and IP3R3-regulated Ca2+ release [5]. Previously only histone 3 lysine 4 methyltransferase MLL2 had been shown to activate the antiapoptotic gene bcl2 to inhibit apoptosis during ESC differentiation [44]. The data presented, here, extends and reveals the importance of epigenetic controls in the activation of proapoptotic gene associated with ESC differentiation.

Mesodermal and cardiac differentiation have been shown to be regulated by the histone demethylase ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX)[13, 45] and jumonji domain–containing protein 3 (JMJD3) [12] through transcriptional activation of mesodermal and cardiac genes. These findings together with those presented in this paper support the critical role of histone demethylases in lineage commitment through regulatory mechanisms that control the expression of core lineage specific transcription factors and apoptotic genes. The decrease in apoptosis through deletion of phf8 can be attributed to the maintenance of repressive H3K9me2 mark on the TSS of pmaip1 after phf8 deletion, resulting in a ∼70% downregulation of pmaip1 at differentiation day 3 in the phf8-/Y cells (Fig. 5B). The pro-apoptotic gene pmaip1 is, therefore, epigenetically regulated by the histone demethylase, which subsequently affects the mesodermal and cardiac differentiation.

PMAIP1 is a Bcl2 homology domain 3 (BH3)-only protein that acts as an important mediator of apoptosis [46]. Its expression is regulated transcriptionally by various transcription factors and, when present, it acts to promote cell death in a variety of ways [21] including caspase 3 dependent [47] and independent apoptosis [48] and autophagy [40]. Here, we find that PHF8 and its regulation on the pmaip1 promote DNA fragmentation and cell death most likely through a caspase 3-independent pathway. This conclusion is based on the observation that neither the ratio of cleaved caspase 3 to total caspase 3 [49, 50] nor PARP1, a downstream target of caspase 3, is significantly affected. While this may be explained as the inhibitor of apoptosis proteins can counteract the function of caspase 3 [51, 52], the exact mechanisms we observed here need to be further explored.

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Chemotherapy Benefit in Early Breast Cancer Patients

Larry H Bernstein, MD, FCAP, Curator



Agendia’s MammaPrint® First and Only Genomic Assay to Receive Level 1A Clinical Utility Evidence for Chemotherapy Benefit in Early Breast Cancer Patients

  • Clinical high-risk patients with a low-risk MammaPrint® result, including 48 percent node-positive, had five-year distant metastasis-free survival rate in excess of 94 percent, whether randomized to receive adjuvant chemotherapy or not
  • MammaPrint could change clinical practice by substantially de-escalating the use of adjuvant chemotherapy and sparing many patients an aggressive treatment they will not benefit from
  • Forty-six percent overall reduction in chemotherapy prescription among clinically high-risk patients

April 19, 2016 / B3C newswire / Agendia, Inc., together with the European Organisation for Research and Treatment of Cancer (EORTC) and Breast International Group (BIG), announced results from the initial analysis of the primary objective of the Microarray In Node-negative (and 1 to 3 positive lymph node) Disease may Avoid ChemoTherapy (MINDACT) study at the American Association for Cancer Research Annual Meeting 2016 in New Orleans, LA.

Using the company’s MammaPrint® assay, patients with early-stage breast cancer who were considered at high risk for disease recurrence based on clinical and biological criteria had a distant metastasis-free survival at five years in excess of 94 percent.The MammaPrint test—the first and only genomic assay with FDA 510(k) clearance for use in risk assessment for women of all ages with early stage breast cancer—identified a large group of patients for whom five-year distant metastasis–free survival was equally good whether or not they received adjuvant chemotherapy (chemotherapy given post-surgery).

“The MINDACT trial design is the optimal way to prove clinical utility of a genomic assay,” said Prof. Laura van ’t Veer, CRO at Agendia, Leader, Breast Oncology Program, and Director, Applied Genomics at UCSF Helen Diller Family Comprehensive Cancer Center. “It gives the level 1A clinical evidence (prospective, randomized and controlled) that empowers physicians to clearly and confidently know when chemotherapy is part of optimal early-stage breast cancer therapy.  In this trial, MammaPrint (70-gene assay) was compared to the standard of care physicians use today, to decide what is the best treatment option for an early-stage breast cancer patient.”

The MINDACT trial is the first prospective randomized controlled clinical trial of a breast cancer recurrence genomic assay with level 1A clinical evidence and the first prospective translational research study of this magnitude in breast cancer to report the results of its primary objective.

Among the 3,356 patients enrolled in the MINDACT trial, who were categorized as having a high risk of breast cancer recurrence based on common clinical and pathological criteria (C-high), the MammaPrint assay reduced the chemotherapy treatment prescription by 46 percent.Using the 70-gene assay, MammaPrint, 48 percent of lymph-node positive breast cancer patients considered clinically high-risk (Clinical-high) and genomic low-risk (MammaPrint-low) had an excellent distant metastasis-free survival at five years in excess of 94 percent.

“Traditionally, physicians have relied on clinical-pathological factors such as age, tumor size, tumor grade, lymph node involvement, and hormone receptor status to make breast cancer treatment decisions,” said Massimo Cristofanilli, MD, Associate Director of Translational Research and Precision Medicine at the Robert H. Lurie Comprehensive Cancer Center, Northwestern University in Chicago. “These findings provide level 1A clinical utility evidence by demonstrating that the detection of low-risk of distant recurrence reported by the MammaPrint test can be safely used in the management of thousands of women by identifying those who can be spared from a toxic and unnecessary treatment.”

MINDACT is a randomized phase III trial that investigates the clinical utility of MammaPrint, when compared (or – “used in conjunction with”) to the standard clinical pathological criteria, for the selection of patients unlikely to benefit from adjuvant chemotherapy. From 2007 to 2011, 6,693 women who had undergone surgery for early-stage breast cancer enrolled in the trial (111 centers in nine countries). Participants were categorized as low or high risk for tumor recurrence in two ways: first, through analysis of tumor tissue using MammaPrint at a central location in Amsterdam; and second, using Adjuvant! Online, a tool that calculates risk of breast cancer recurrence based on common clinical and biological criteria.

Patients characterized in both clinical and genomic assessments as “low- risk” are spared chemotherapy, while patients characterized as “high- risk” are advised chemotherapy. Those with conflicting results are randomized to use either clinical or genomic risk (MammaPrint) evaluation to decide on chemotherapy treatment.

The MINDACT trial is managed and sponsored by the EORTC as part of an extensive and complex partnership in collaboration with Agendia and BIG, and many other academic and commercial partners, as well as patient advocates.

“These MINDACT trial results are a testament that the science of the MammaPrint test is the most robust in the genomic breast recurrence assay market.  Agendia will continue to collaborate with pharmaceutical companies, leading cancer centers and academic groups on additional clinical research and in the pursuit of bringing more effective, individualized treatments within reach of cancer patients,” said Mark Straley, Chief Executive Officer at Agendia. “We value the partnership with the EORTC and BIG and it’s a great honor to share this critical milestone.”

Breast cancer is the most frequently diagnosed cancer in women worldwide(1). In 2012, there were nearly 1.7 million new breast cancer cases among women worldwide, accounting for 25 percent of all new cancer cases in women(2).

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CRISPR/Cas9, Familial Amyloid Polyneuropathy ( FAP) and Neurodegenerative Disease

CRISPR/Cas9, Familial Amyloid Polyneuropathy ( FAP) and Neurodegenerative Disease

Curator: Larry H. Bernstein, MD, FCAP


CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The Biology of Cas9

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus (10).

Three types of CRISPR mechanisms have been identified, of which type II is the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (11).

Figure 1. Cas9 in vivo: Bacterial Adaptive Immunity

In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)

One Cas protein, Cas9 (also known as Csn1), has been shown, through knockdown and rescue experiments to be a key player in certain CRISPR mechanisms (specifically type II CRISPR systems). The type II CRISPR mechanism is unique compared to other CRISPR systems, as only one Cas protein (Cas9) is required for gene silencing (12). In type II systems, Cas9 participates in the processing of crRNAs (12), and is responsible for the destruction of the target DNA (11). Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13).

To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA (11). The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9 (12).

During the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double-stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript (11, 14). The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

The double-stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2–5 nts) known as protospacer-associated motif (PAM), follows immediately 3´- of the crRNA complementary sequence (15). In fact, even fully complementary sequences are ignored by Cas9-RNA in the absence of a PAM sequence (16).

Cas9 and CRISPR as a New Tool in Molecular Biology

The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) makes this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier labs (11). Based on the type II CRISPR system described previously, the authors developed a simplified two-component system by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). sgRNAprogrammed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations (Figure 2A).

To date, three different variants of the Cas9 nuclease have been adopted in genome-editing protocols. The first is wild-type Cas9, which can site-specifically cleave double-stranded DNA, resulting in the activation of the doublestrand break (DSB) repair machinery. DSBs can be repaired by the cellular Non-Homologous End Joining (NHEJ) pathway (17), resulting in insertions and/or deletions (indels) which disrupt the targeted locus. Alternatively, if a donor template with homology to the targeted locus is supplied, the DSB may be repaired by the homology-directed repair (HDR) pathway allowing for precise replacement mutations to be made (Figure 2A) (17, 18).

Cong and colleagues (1) took the Cas9 system a step further towards increased precision by developing a mutant form, known as Cas9D10A, with only nickase activity. This means it cleaves only one DNA strand, and does not activate NHEJ. Instead, when provided with a homologous repair template, DNA repairs are conducted via the high-fidelity HDR pathway only, resulting in reduced indel mutations (1, 11, 19). Cas9D10A is even more appealing in terms of target specificity when loci are targeted by paired Cas9 complexes designed to generate adjacent DNA nicks (20) (see further details about “paired nickases” in Figure 2B).

The third variant is a nuclease-deficient Cas9 (dCas9, Figure 2C) (21). Mutations H840A in the HNH domain and D10A in the RuvC domain inactivate cleavage activity, but do not prevent DNA binding (11, 22). Therefore, this variant can be used to sequence-specifically target any region of the genome without cleavage. Instead, by fusing with various effector domains, dCas9 can be used either as a gene silencing or activation tool (21, 23–26). Furthermore, it can be used as a visualization tool. For instance, Chen and colleagues used dCas9 fused to Enhanced Green Fluorescent Protein (EGFP) to visualize repetitive DNA sequences with a single sgRNA or nonrepetitive loci using multiple sgRNAs (27).

Figure 2. CRISPR/Cas9 System Applications

  1. Wild-type Cas9 nuclease site specifically cleaves double-stranded DNA activating double-strand break repair machinery. In the absence of a homologous repair template non-homologous end joining can result in indels disrupting the target sequence. Alternatively, precise mutations and knock-ins can be made by providing a homologous repair template and exploiting the homology directed repair pathway.
    B. Mutated Cas9 makes a site specific single-strand nick. Two sgRNA can be used to introduce a staggered double-stranded break which can then undergo homology directed repair.
    C. Nuclease-deficient Cas9 can be fused with various effector domains allowing specific localization. For example, transcriptional activators, repressors, and fluorescent proteins.

Targeting Efficiency and Off-target Mutations

Targeting efficiency, or the percentage of desired mutation achieved, is one of the most important parameters by which to assess a genome-editing tool. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs or ZFNs (8). For example, in human cells, custom-designed ZFNs and TALENs could only achieve efficiencies ranging from 1% to 50% (29–31). In contrast, the Cas9 system has been reported to have efficiencies up to >70% in zebrafish (32) and plants (33), and ranging from 2–5% in induced pluripotent stem cells (34). In addition, Zhou and colleagues were able to improve genome targeting up to 78% in one-cell mouse embryos, and achieved effective germline transmission through the use of dual sgRNAs to simultaneously target an individual gene (35).

A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (36, 37) (Figure 3). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wildtype DNA strand (37).

Figure 3. T7 Endonuclease I Targeting Efficiency Assay

Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding 3 possible structures. Duplexes containing a mismatch are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to calculate targeting efficiency.

Another important parameter is the incidence of off-target mutations. Such mutations are likely to appear in sites that have differences of only a few nucleotides compared to the original sequence, as long as they are adjacent to a PAM sequence. This occurs as Cas9 can tolerate up to 5 base mismatches within the protospacer region (36) or a single base difference in the PAM sequence (38). Off-target mutations are generally more difficult to detect, requiring whole-genome sequencing to rule them out completely.

Recent improvements to the CRISPR system for reducing off-target mutations have been made through the use of truncated gRNA (truncated within the crRNA-derived sequence) or by adding two extra guanine (G) nucleotides to the 5´ end (28, 37). Another way researchers have attempted to minimize off-target effects is with the use of “paired nickases” (20). This strategy uses D10A Cas9 and two sgRNAs complementary to the adjacent area on opposite strands of the target site (Figure 2B). While this induces DSBs in the target DNA, it is expected to create only single nicks in off-target locations and, therefore, result in minimal off-target mutations.

By leveraging computation to reduce off-target mutations, several groups have developed webbased tools to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage. Examples include the CRISPR Design Tool (38) and the ZiFiT Targeter, Version 4.2 (39, 40).

Applications as a Genome-editing and Genome Targeting Tool

Following its initial demonstration in 2012 (9), the CRISPR/Cas9 system has been widely adopted. This has already been successfully used to target important genes in many cell lines and organisms, including human (34), bacteria (41), zebrafish (32), C. elegans (42), plants (34), Xenopus tropicalis (43), yeast (44), Drosophila (45), monkeys (46), rabbits (47), pigs (42), rats (48) and mice (49). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA (14, 21, 29). Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations (50). A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation (26, 51, 52), epigenetic modification (25), and microscopic visualization of specific genome loci (27).

The CRISPR/Cas9 system requires only the redesign of the crRNA to change target specificity. This contrasts with other genome editing tools, including zinc finger and TALENs, where redesign of the protein-DNA interface is required. Furthermore, CRISPR/Cas9 enables rapid genome-wide interrogation of gene function by generating large gRNA libraries (51, 53) for genomic screening.

The Future of CRISPR/Cas9

The rapid progress in developing Cas9 into a set of tools for cell and molecular biology research has been remarkable, likely due to the simplicity, high efficiency and versatility of the system. Of the designer nuclease systems currently available for precision genome engineering, the CRISPR/Cas system is by far the most user friendly. It is now also clear that Cas9’s potential reaches beyond DNA cleavage, and its usefulness for genome locus-specific recruitment of proteins will likely only be limited by our imagination.


Scientists urge caution in using new CRISPR technology to treat human genetic disease

By Robert Sanders, Media relations | MARCH 19, 2015

The bacterial enzyme Cas9 is the engine of RNA-programmed genome engineering in human cells. (Graphic by Jennifer Doudna/UC Berkeley)

A group of 18 scientists and ethicists today warned that a revolutionary new tool to cut and splice DNA should be used cautiously when attempting to fix human genetic disease, and strongly discouraged any attempts at making changes to the human genome that could be passed on to offspring.

Among the authors of this warning is Jennifer Doudna, the co-inventor of the technology, called CRISPR-Cas9, which is driving a new interest in gene therapy, or “genome engineering.” She and colleagues co-authored a perspective piece that appears in the March 20 issue of Science, based on discussions at a meeting that took place in Napa on Jan. 24. The same issue of Science features a collection of recent research papers, commentary and news articles on CRISPR and its implications.    …..

A prudent path forward for genomic engineering and germline gene modification

David Baltimore1,  Paul Berg2, …., Jennifer A. Doudna4,10,*, et al.
Science  19 Mar 2015.


Correcting genetic defects

Scientists today are changing DNA sequences to correct genetic defects in animals as well as cultured tissues generated from stem cells, strategies that could eventually be used to treat human disease. The technology can also be used to engineer animals with genetic diseases mimicking human disease, which could lead to new insights into previously enigmatic disorders.

The CRISPR-Cas9 tool is still being refined to ensure that genetic changes are precisely targeted, Doudna said. Nevertheless, the authors met “… to initiate an informed discussion of the uses of genome engineering technology, and to identify proactively those areas where current action is essential to prepare for future developments. We recommend taking immediate steps toward ensuring that the application of genome engineering technology is performed safely and ethically.”


Amyloid CRISPR Plasmids and si/shRNA Gene Silencers

Santa Cruz Biotechnology, Inc. offers a broad range of gene silencers in the form of siRNAs, shRNA Plasmids and shRNA Lentiviral Particles as well as CRISPR/Cas9 Knockout and CRISPR Double Nickase plasmids. Amyloid gene silencers are available as Amyloid siRNA, Amyloid shRNA Plasmid, Amyloid shRNA Lentiviral Particles and Amyloid CRISPR/Cas9 Knockout plasmids. Amyloid CRISPR/dCas9 Activation Plasmids and CRISPR Lenti Activation Systems for gene activation are also available. Gene silencers and activators are useful for gene studies in combination with antibodies used for protein detection.    Amyloid CRISPR Knockout, HDR and Nickase Knockout Plasmids


CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome

Mehrabian M, Brethour D, MacIsaac S, Kim JK, Gunawardana C.G, Wang H, et al.
PLoS ONE 2014; 9(12): e114594.

The molecular function of the cellular prion protein (PrPC) and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer’s disease are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate guide RNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of>3000 proteins (data deposited to ProteomeXchange Consortium) were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ∼120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins belonging to extracellular components, cell junctions or the cytoskeleton.


Development and Applications of CRISPR-Cas9 for Genome Engineering

Patrick D. Hsu,1,2,3 Eric S. Lander,1 and Feng Zhang1,2,*
Cell. 2014 Jun 5; 157(6): 1262–1278.   doi:  10.1016/j.cell.2014.05.010

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine.

The development of recombinant DNA technology in the 1970s marked the beginning of a new era for biology. For the first time, molecular biologists gained the ability to manipulate DNA molecules, making it possible to study genes and harness them to develop novel medicine and biotechnology. Recent advances in genome engineering technologies are sparking a new revolution in biological research. Rather than studying DNA taken out of the context of the genome, researchers can now directly edit or modulate the function of DNA sequences in their endogenous context in virtually any organism of choice, enabling them to elucidate the functional organization of the genome at the systems level, as well as identify causal genetic variations.

Broadly speaking, genome engineering refers to the process of making targeted modifications to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts). The ability to do so easily and efficiently in eukaryotic and especially mammalian cells holds immense promise to transform basic science, biotechnology, and medicine (Figure 1).

For life sciences research, technologies that can delete, insert, and modify the DNA sequences of cells or organisms enable dissecting the function of specific genes and regulatory elements. Multiplexed editing could further allow the interrogation of gene or protein networks at a larger scale. Similarly, manipulating transcriptional regulation or chromatin states at particular loci can reveal how genetic material is organized and utilized within a cell, illuminating relationships between the architecture of the genome and its functions. In biotechnology, precise manipulation of genetic building blocks and regulatory machinery also facilitates the reverse engineering or reconstruction of useful biological systems, for example, by enhancing biofuel production pathways in industrially relevant organisms or by creating infection-resistant crops. Additionally, genome engineering is stimulating a new generation of drug development processes and medical therapeutics. Perturbation of multiple genes simultaneously could model the additive effects that underlie complex polygenic disorders, leading to new drug targets, while genome editing could directly correct harmful mutations in the context of human gene therapy (Tebas et al., 2014).

Eukaryotic genomes contain billions of DNA bases and are difficult to manipulate. One of the breakthroughs in genome manipulation has been the development of gene targeting by homologous recombination (HR), which integrates exogenous repair templates that contain sequence homology to the donor site (Figure 2A) (Capecchi, 1989). HR-mediated targeting has facilitated the generation of knockin and knockout animal models via manipulation of germline competent stem cells, dramatically advancing many areas of biological research. However, although HR-mediated gene targeting produces highly precise alterations, the desired recombination events occur extremely infrequently (1 in 106–109 cells) (Capecchi, 1989), presenting enormous challenges for large-scale applications of gene-targeting experiments.

Genome Editing Technologies Exploit Endogenous DNA Repair Machinery

To overcome these challenges, a series of programmable nuclease-based genome editing technologies have been developed in recent years, enabling targeted and efficient modification of a variety of eukaryotic and particularly mammalian species. Of the current generation of genome editing technologies, the most rapidly developing is the class of RNA-guided endonucleases known as Cas9 from the microbial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats), which can be easily targeted to virtually any genomic location of choice by a short RNA guide. Here, we review the development and applications of the CRISPR-associated endonuclease Cas9 as a platform technology for achieving targeted perturbation of endogenous genomic elements and also discuss challenges and future avenues for innovation.   ……

Figure 4   Natural Mechanisms of Microbial CRISPR Systems in Adaptive Immunity

……  A key turning point came in 2005, when systematic analysis of the spacer sequences separating the individual direct repeats suggested their extrachromosomal and phage-associated origins (Mojica et al., 2005Pourcel et al., 2005Bolotin et al., 2005). This insight was tremendously exciting, especially given previous studies showing that CRISPR loci are transcribed (Tang et al., 2002) and that viruses are unable to infect archaeal cells carrying spacers corresponding to their own genomes (Mojica et al., 2005). Together, these findings led to the speculation that CRISPR arrays serve as an immune memory and defense mechanism, and individual spacers facilitate defense against bacteriophage infection by exploiting Watson-Crick base-pairing between nucleic acids (Mojica et al., 2005Pourcel et al., 2005). Despite these compelling realizations that CRISPR loci might be involved in microbial immunity, the specific mechanism of how the spacers act to mediate viral defense remained a challenging puzzle. Several hypotheses were raised, including thoughts that CRISPR spacers act as small RNA guides to degrade viral transcripts in a RNAi-like mechanism (Makarova et al., 2006) or that CRISPR spacers direct Cas enzymes to cleave viral DNA at spacer-matching regions (Bolotin et al., 2005).   …..

As the pace of CRISPR research accelerated, researchers quickly unraveled many details of each type of CRISPR system (Figure 4). Building on an earlier speculation that protospacer adjacent motifs (PAMs) may direct the type II Cas9 nuclease to cleave DNA (Bolotin et al., 2005), Moineau and colleagues highlighted the importance of PAM sequences by demonstrating that PAM mutations in phage genomes circumvented CRISPR interference (Deveau et al., 2008). Additionally, for types I and II, the lack of PAM within the direct repeat sequence within the CRISPR array prevents self-targeting by the CRISPR system. In type III systems, however, mismatches between the 5′ end of the crRNA and the DNA target are required for plasmid interference (Marraffini and Sontheimer, 2010).  …..

In 2013, a pair of studies simultaneously showed how to successfully engineer type II CRISPR systems from Streptococcus thermophilus (Cong et al., 2013) andStreptococcus pyogenes (Cong et al., 2013Mali et al., 2013a) to accomplish genome editing in mammalian cells. Heterologous expression of mature crRNA-tracrRNA hybrids (Cong et al., 2013) as well as sgRNAs (Cong et al., 2013Mali et al., 2013a) directs Cas9 cleavage within the mammalian cellular genome to stimulate NHEJ or HDR-mediated genome editing. Multiple guide RNAs can also be used to target several genes at once. Since these initial studies, Cas9 has been used by thousands of laboratories for genome editing applications in a variety of experimental model systems (Sander and Joung, 2014). ……

The majority of CRISPR-based technology development has focused on the signature Cas9 nuclease from type II CRISPR systems. However, there remains a wide diversity of CRISPR types and functions. Cas RAMP module (Cmr) proteins identified in Pyrococcus furiosus and Sulfolobus solfataricus (Hale et al., 2012) constitute an RNA-targeting CRISPR immune system, forming a complex guided by small CRISPR RNAs that target and cleave complementary RNA instead of DNA. Cmr protein homologs can be found throughout bacteria and archaea, typically relying on a 5 site tag sequence on the target-matching crRNA for Cmr-directed cleavage.

Unlike RNAi, which is targeted largely by a 6 nt seed region and to a lesser extent 13 other bases, Cmr crRNAs contain 30–40 nt of target complementarity. Cmr-CRISPR technologies for RNA targeting are thus a promising target for orthogonal engineering and minimal off-target modification. Although the modularity of Cmr systems for RNA-targeting in mammalian cells remains to be investigated, Cmr complexes native to P. furiosus have already been engineered to target novel RNA substrates (Hale et al., 20092012).   ……

Although Cas9 has already been widely used as a research tool, a particularly exciting future direction is the development of Cas9 as a therapeutic technology for treating genetic disorders. For a monogenic recessive disorder due to loss-of-function mutations (such as cystic fibrosis, sickle-cell anemia, or Duchenne muscular dystrophy), Cas9 may be used to correct the causative mutation. This has many advantages over traditional methods of gene augmentation that deliver functional genetic copies via viral vector-mediated overexpression—particularly that the newly functional gene is expressed in its natural context. For dominant-negative disorders in which the affected gene is haplosufficient (such as transthyretin-related hereditary amyloidosis or dominant forms of retinitis pigmentosum), it may also be possible to use NHEJ to inactivate the mutated allele to achieve therapeutic benefit. For allele-specific targeting, one could design guide RNAs capable of distinguishing between single-nucleotide polymorphism (SNP) variations in the target gene, such as when the SNP falls within the PAM sequence.



CRISPR/Cas9: a powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases

Zhuchi Tu, Weili Yang, Sen Yan, Xiangyu Guo and Xiao-Jiang Li

Molecular Neurodegeneration 2015; 10:35

Animal models are extremely valuable to help us understand the pathogenesis of neurodegenerative disorders and to find treatments for them. Since large animals are more like humans than rodents, they make good models to identify the important pathological events that may be seen in humans but not in small animals; large animals are also very important for validating effective treatments or confirming therapeutic targets. Due to the lack of embryonic stem cell lines from large animals, it has been difficult to use traditional gene targeting technology to establish large animal models of neurodegenerative diseases. Recently, CRISPR/Cas9 was used successfully to genetically modify genomes in various species. Here we discuss the use of CRISPR/Cas9 technology to establish large animal models that can more faithfully mimic human neurodegenerative diseases.

Neurodegenerative diseases — Alzheimer’s disease(AD),Parkinson’s disease(PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and frontotemporal dementia (FTD) — are characterized by age-dependent and selective neurodegeneration. As the life expectancy of humans lengthens, there is a greater prevalence of these neurodegenerative diseases; however, the pathogenesis of most of these neurodegenerative diseases remain unclear, and we lack effective treatments for these important brain disorders.

CRISPR/Cas9,  Non-human primates,  Neurodegenerative diseases,  Animal model

There are a number of excellent reviews covering different types of neurodegenerative diseases and their genetic mouse models [812]. Investigations of different mouse models of neurodegenerative diseases have revealed a common pathology shared by these diseases. First, the development of neuropathology and neurological symptoms in genetic mouse models of neurodegenerative diseases is age dependent and progressive. Second, all the mouse models show an accumulation of misfolded or aggregated proteins resulting from the expression of mutant genes. Third, despite the widespread expression of mutant proteins throughout the body and brain, neuronal function appears to be selectively or preferentially affected. All these facts indicate that mouse models of neurodegenerative diseases recapitulate important pathologic features also seen in patients with neurodegenerative diseases.

However, it seems that mouse models can not recapitulate the full range of neuropathology seen in patients with neurodegenerative diseases. Overt neurodegeneration, which is the most important pathological feature in patient brains, is absent in genetic rodent models of AD, PD, and HD. Many rodent models that express transgenic mutant proteins under the control of different promoters do not replicate overt neurodegeneration, which is likely due to their short life spans and the different aging processes of small animals. Also important are the remarkable differences in brain development between rodents and primates. For example, the mouse brain takes 21 days to fully develop, whereas the formation of primate brains requires more than 150 days [13]. The rapid development of the brain in rodents may render neuronal cells resistant to misfolded protein-mediated neurodegeneration. Another difficulty in using rodent models is how to analyze cognitive and emotional abnormalities, which are the early symptoms of most neurodegenerative diseases in humans. Differences in neuronal circuitry, anatomy, and physiology between rodent and primate brains may also account for the behavioral differences between rodent and primate models.


Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases

Hsiuchen Chen and David C. Chan
Human Molec Gen 2009; 18, Review Issue 2 R169–R176

Neurons are metabolically active cells with high energy demands at locations distant from the cell body. As a result, these cells are particularly dependent on mitochondrial function, as reflected by the observation that diseases of mitochondrial dysfunction often have a neurodegenerative component. Recent discoveries have highlighted that neurons are reliant particularly on the dynamic properties of mitochondria. Mitochondria are dynamic organelles by several criteria. They engage in repeated cycles of fusion and fission, which serve to intermix the lipids and contents of a population of mitochondria. In addition, mitochondria are actively recruited to subcellular sites, such as the axonal and dendritic processes of neurons. Finally, the quality of a mitochondrial population is maintained through mitophagy, a form of autophagy in which defective mitochondria are selectively degraded. We review the general features of mitochondrial dynamics, incorporating recent findings on mitochondrial fusion, fission, transport and mitophagy. Defects in these key features are associated with neurodegenerative disease. Charcot-Marie-Tooth type 2A, a peripheral neuropathy, and dominant optic atrophy, an inherited optic neuropathy, result from a primary deficiency of mitochondrial fusion. Moreover, several major neurodegenerative diseases—including Parkinson’s, Alzheimer’s and Huntington’s disease—involve disruption of mitochondrial dynamics. Remarkably, in several disease models, the manipulation of mitochondrial fusion or fission can partially rescue disease phenotypes. We review how mitochondrial dynamics is altered in these neurodegenerative diseases and discuss the reciprocal interactions between mitochondrial fusion, fission, transport and mitophagy.


Applications of CRISPR–Cas systems in Neuroscience

Matthias Heidenreich  & Feng Zhang
Nature Rev Neurosci 2016; 17:36–44

Genome-editing tools, and in particular those based on CRISPR–Cas (clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR–Cas systems has the potential to advance both basic and translational neuroscience research.
Cellular neuroscience
, DNA recombination, Genetic engineering, Molecular neuroscience

Figure 3: In vitro applications of Cas9 in human iPSCs.close

a | Evaluation of disease candidate genes from large-population genome-wide association studies (GWASs). Human primary cells, such as neurons, are not easily available and are difficult to expand in culture. By contrast, induced pluripo…

  1. Genome-editing Technologies for Gene and Cell Therapy

Molecular Therapy 12 Jan 2016

  1. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing

Scientific Reports 31 Mar 2016

  1. Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection

Scientific Reports 12 Nov 2015


Alzheimer’s Disease: Medicine’s Greatest Challenge in the 21st Century

The development of the CRISPR/Cas9 system has made gene editing a relatively simple task.  While CRISPR and other gene editing technologies stand to revolutionize biomedical research and offers many promising therapeutic avenues (such as in the treatment of HIV), a great deal of debate exists over whether CRISPR should be used to modify human embryos. As I discussed in my previous Insight article, we lack enough fundamental biological knowledge to enhance many traits like height or intelligence, so we are not near a future with genetically-enhanced super babies. However, scientists have identified a few rare genetic variants that protect against disease.  One such protective variant is a mutation in the APP gene that protects against Alzheimer’s disease and cognitive decline in old age. If we can perfect gene editing technologies, is this mutation one that we should be regularly introducing into embryos? In this article, I explore the potential for using gene editing as a way to prevent Alzheimer’s disease in future generations. Alzheimer’s Disease: Medicine’s Greatest Challenge in the 21st Century Can gene editing be the missing piece in the battle against Alzheimer’s? (Source: I chose to assess the benefit of germline gene editing in the context of Alzheimer’s disease because this disease is one of the biggest challenges medicine faces in the 21st century. Alzheimer’s disease is a chronic neurodegenerative disease responsible for the majority of the cases of dementia in the elderly. The disease symptoms begins with short term memory loss and causes more severe symptoms – problems with language, disorientation, mood swings, behavioral issues – as it progresses, eventually leading to the loss of bodily functions and death. Because of the dementia the disease causes, Alzheimer’s patients require a great deal of care, and the world spends ~1% of its total GDP on caring for those with Alzheimer’s and related disorders. Because the prevalence of the disease increases with age, the situation will worsen as life expectancies around the globe increase: worldwide cases of Alzheimer’s are expected to grow from 35 million today to over 115 million by 2050.

Despite much research, the exact causes of Alzheimer’s disease remains poorly understood. The disease seems to be related to the accumulation of plaques made of amyloid-β peptides that form on the outside of neurons, as well as the formation of tangles of the protein tau inside of neurons. Although many efforts have been made to target amyloid-β or the enzymes involved in its formation, we have so far been unsuccessful at finding any treatment that stops the disease or reverses its progress. Some researchers believe that most attempts at treating Alzheimer’s have failed because, by the time a patient shows symptoms, the disease has already progressed past the point of no return.

While research towards a cure continues, researchers have sought effective ways to prevent Alzheimer’s disease. Although some studies show that mental and physical exercise may lower ones risk of Alzheimer’s disease, approximately 60-80% of the risk for Alzheimer’s disease appears to be genetic. Thus, if we’re serious about prevention, we may have to act at the genetic level. And because the brain is difficult to access surgically for gene therapy in adults, this means using gene editing on embryos.



Utilising CRISPR to Generate Predictive Disease Models: a Case Study in Neurodegenerative Disorders

Dr. Bhuvaneish.T. Selvaraj  – Scottish Centre for Regenerative Medicine

  • Introducing the latest developments in predictive model generation
  • Discover how CRISPR is being used to develop disease models to study and treat neurodegenerative disorders
  • In depth Q&A session to answer your most pressing questions


Turning On Genes, Systematically, with CRISPR/Cas9


Scientists based at MIT assert that they can reliably turn on any gene of their choosing in living cells. [Feng Zhang and Steve Dixon]

With the latest CRISPR/Cas9 advance, the exhortation “turn on, tune in, drop out” comes to mind. The CRISPR/Cas9 gene-editing system was already a well-known means of “tuning in” (inserting new genes) and “dropping out” (knocking out genes). But when it came to “turning on” genes, CRISPR/Cas9 had little potency. That is, it had demonstrated only limited success as a way to activate specific genes.

A new CRISPR/Cas9 approach, however, appears capable of activating genes more effectively than older approaches. The new approach may allow scientists to more easily determine the function of individual genes, according to Feng Zhang, Ph.D., a researcher at MIT and the Broad Institute. Dr. Zhang and colleagues report that the new approach permits multiplexed gene activation and rapid, large-scale studies of gene function.

The new technique was introduced in the December 10 online edition of Nature, in an article entitled, “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.” The article describes how Dr. Zhang, along with the University of Tokyo’s Osamu Nureki, Ph.D., and Hiroshi Nishimasu, Ph.D., overhauled the CRISPR/Cas9 system. The research team based their work on their analysis (published earlier this year) of the structure formed when Cas9 binds to the guide RNA and its target DNA. Specifically, the team used the structure’s 3D shape to rationally improve the system.

In previous efforts to revamp CRISPR/Cas9 for gene activation purposes, scientists had tried to attach the activation domains to either end of the Cas9 protein, with limited success. From their structural studies, the MIT team realized that two small loops of the RNA guide poke out from the Cas9 complex and could be better points of attachment because they allow the activation domains to have more flexibility in recruiting transcription machinery.

Using their revamped system, the researchers activated about a dozen genes that had proven difficult or impossible to turn on using the previous generation of Cas9 activators. Each gene showed at least a twofold boost in transcription, and for many genes, the researchers found multiple orders of magnitude increase in activation.

After investigating single-guide RNA targeting rules for effective transcriptional activation, demonstrating multiplexed activation of 10 genes simultaneously, and upregulating long intergenic noncoding RNA transcripts, the research team decided to undertake a large-scale screen. This screen was designed to identify genes that confer resistance to a melanoma drug called PLX-4720.

“We … synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor,” wrote the authors of the Nature paper. “The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual [single-guide RNA] and complementary DNA overexpression.”

A gene signature based on the top screening hits, the authors added, correlated with a gene expression signature of BRAF inhibitor resistance in cell lines and patient-derived samples. It was also suggested that large-scale screens such as the one demonstrated in the current study could help researchers discover new cancer drugs that prevent tumors from becoming resistant.

More at –


Susceptibility and modifier genes in Portuguese transthyretin V30M amyloid polyneuropathy: complexity in a single-gene disease
Miguel L. Soares1,2, Teresa Coelho3,6, Alda Sousa4,5, …, Maria Joa˜o Saraiva2,5 and Joel N. Buxbaum1
Human Molec Gen 2005; 14(4): 543–553

Familial amyloid polyneuropathy type I is an autosomal dominant disorder caused by mutations in the transthyretin (TTR ) gene; however, carriers of the same mutation exhibit variability in penetrance and clinical expression. We analyzed alleles of candidate genes encoding non-fibrillar components of TTR amyloid deposits and a molecule metabolically interacting with TTR [retinol-binding protein (RBP)], for possible associations with age of disease onset and/or susceptibility in a Portuguese population sample with the TTR V30M mutation and unrelated controls. We show that the V30M carriers represent a distinct subset of the Portuguese population. Estimates of genetic distance indicated that the controls and the classical onset group were furthest apart, whereas the late-onset group appeared to differ from both. Importantly, the data also indicate that genetic interactions among the multiple loci evaluated, rather than single-locus effects, are more likely to determine differences in the age of disease onset. Multifactor dimensionality reduction indicated that the best genetic model for classical onset group versus controls involved the APCS gene, whereas for late-onset cases, one APCS variant (APCSv1) and two RBP variants (RBPv1 and RBPv2) are involved. Thus, although the TTR V30M mutation is required for the disease in Portuguese patients, different genetic factors may govern the age of onset, as well as the occurrence of anticipation.

Autosomal dominant disorders may vary in expression even within a given kindred. The basis of this variability is uncertain and can be attributed to epigenetic factors, environment or epistasis. We have studied familial amyloid polyneuropathy (FAP), an autosomal dominant disorder characterized by peripheral sensorimotor and autonomic neuropathy. It exhibits variation in cardiac, renal, gastrointestinal and ocular involvement, as well as age of onset. Over 80 missense mutations in the transthyretin gene (TTR ) result in autosomal dominant disease The presence of deposits consisting entirely of wild-type TTR molecules in the hearts of 10– 25% of individuals over age 80 reveals its inherent in vivo amyloidogenic potential (1).

FAP was initially described in Portuguese (2) where, until recently, the TTR V30M has been the only pathogenic mutation associated with the disease (3,4). Later reports identified the same mutation in Swedish and Japanese families (5,6). The disorder has since been recognized in other European countries and in North American kindreds in association with V30M, as well as other mutations (7).

TTR V30M produces disease in only 5–10% of Swedish carriers of the allele (8), a much lower degree of penetrance than that seen in Portuguese (80%) (9) or in Japanese with the same mutation. The actual penetrance in Japanese carriers has not been formally established, but appears to resemble that seen in Portuguese. Portuguese and Japanese carriers show considerable variation in the age of clinical onset (10,11). In both populations, the first symptoms had originally been described as typically occurring before age 40 (so-called ‘classical’ or early-onset); however, in recent years, more individuals developing symptoms late in life have been identified (11,12). Hence, present data indicate that the distribution of the age of onset in Portuguese is continuous, but asymmetric with a mean around age 35 and a long tail into the older age group (Fig. 1) (9,13). Further, DNA testing in Portugal has identified asymptomatic carriers over age 70 belonging to a subset of very late-onset kindreds in whose descendants genetic anticipation is frequent. The molecular basis of anticipation in FAP, which is not mediated by trinucleotide repeat expansions in the TTR or any other gene (14), remains elusive.

Variation in penetrance, age of onset and clinical features are hallmarks of many autosomal dominant disorders including the human TTR amyloidoses (7). Some of these clearly reflect specific biological effects of a particular mutation or a class of mutants. However, when such phenotypic variability is seen with a single mutation in the gene encoding the same protein, it suggests an effect of modifying genetic loci and/or environmental factors contributing differentially to the course of disease. We have chosen to examine age of onset as an example of a discrete phenotypic variation in the presence of the particular autosomal dominant disease-associated mutation TTR V30M. Although the role of environmental factors cannot be excluded, the existence of modifier genes involved in TTR amyloidogenesis is an attractive hypothesis to explain the phenotypic variability in FAP. ….

ATTR (TTR amyloid), like all amyloid deposits, contains several molecular components, in addition to the quantitatively dominant fibril-forming amyloid protein, including heparan sulfate proteoglycan 2 (HSPG2 or perlecan), SAP, a plasma glycoprotein of the pentraxin family (encoded by the APCS gene) that undergoes specific calcium-dependent binding to all types of amyloid fibrils, and apolipoprotein E (ApoE), also found in all amyloid deposits (15). The ApoE4 isoform is associated with an increased frequency and earlier onset of Alzheimer’s disease (Ab), the most common form of brain amyloid, whereas the ApoE2 isoform appears to be protective (16). ApoE variants could exert a similar modulatory effect in the onset of FAP, although early studies on a limited number of patients suggested this was not the case (17).

In at least one instance of senile systemic amyloidosis, small amounts of AA-related material were found in TTR deposits (18). These could reflect either a passive co-aggregation or a contributory involvement of protein AA, encoded by the serum amyloid A (SAA ) genes and the main component of secondary (reactive) amyloid fibrils, in the formation of ATTR.

Retinol-binding protein (RBP), the serum carrier of vitamin A, circulates in plasma bound to TTR. Vitamin A-loaded RBP and L-thyroxine, the two natural ligands of TTR, can act alone or synergistically to inhibit the rate and extent of TTR fibrillogenesis in vitro, suggesting that RBP may influence the course of FAP pathology in vivo (19). We have analyzed coding and non-coding sequence polymorphisms in the RBP4 (serum RBP, 10q24), HSPG2 (1p36.1), APCS (1q22), APOE (19q13.2), SAA1 and SAA2 (11p15.1) genes with the goal of identifying chromosomes carrying common and functionally significant variants. At the time these studies were performed, the full human genome sequence was not completed and systematic singlenucleotide polymorphism (SNP) analyses were not available for any of the suspected candidate genes. We identified new SNPs in APCS and RBP4 and utilized polymorphisms in SAA, HSPG2 and APOE that had already been characterized and shown to have potential pathophysiologic significance in other disorders (16,20–22). The genotyping data were analyzed for association with the presence of the V30M amyloidogenic allele (FAP patients versus controls) and with the age of onset (classical- versus late-onset patients). Multilocus analyses were also performed to examine the effects of simultaneous contributions of the six loci for determining the onset of the first symptoms.  …..

The potential for different underlying models for classical and late onset is supported by the MDR analysis, which produces two distinct models when comparing each class with the controls. One could view the two onset classes as unique diseases. If this is the case, then the failure to detect a single predictive genetic model is consistent with two related, but different, diseases. This is exactly what would be expected in such a case of genetic heterogeneity (28). Using this approach, a major gene effect can be viewed as a necessary, but not sufficient, condition to explain the course of the disease. Analyzing the cases but omitting from the analysis of phenotype the necessary allele, in this case TTR V30M, can then reveal a variety of important modifiers that are distinct between the phenotypes.

The significant comparisons obtained in our study cohort indicate that the combined effects mainly result from two and three-locus interactions involving all loci except SAA1 and SAA2 for susceptibility to disease. A considerable number of four-site combinations modulate the age of onset with SAA1 appearing in a majority of significant combinations in late-onset disease, perhaps indicating a greater role of the SAA variants in the age of onset of FAP.

The correlation between genotype and phenotype in socalled simple Mendelian disorders is often incomplete, as only a subset of all mutations can reliably predict specific phenotypes (34). This is because non-allelic genetic variations and/or environmental influences underlie these disorders whose phenotypes behave as complex traits. A few examples include the identification of the role of homozygozity for the SAA1.1 allele in conferring the genetic susceptibility to renal amyloidosis in FMF (20) and the association of an insertion/deletion polymorphism in the ACE gene with disease severity in familial hypertrophic cardiomyopathy (35). In these disorders, the phenotypes arise from mutations in MEFV and b-MHC, but are modulated by independently inherited genetic variation. In this report, we show that interactions among multiple genes, whose products are confirmed or putative constituents of ATTR deposits, or metabolically interact with TTR, modulate the onset of the first symptoms and predispose individuals to disease in the presence of the V30M mutation in TTR. The exact nature of the effects identified here requires further study with potential application in the development of genetic screening with prognostic value pertaining to the onset of disease in the TTR V30M carriers.

If the effects of additional single or interacting genes dictate the heterogeneity of phenotype, as reflected in variability of onset and clinical expression (with the same TTR mutation), the products encoded by alleles at such loci could contribute to the process of wild-type TTR deposition in elderly individuals without a mutation (senile systemic amyloidosis), a phenomenon not readily recognized as having a genetic basis because of the insensitivity of family history in the elderly.


Safety and Efficacy of RNAi Therapy for Transthyretin Amyloidosis

Coelho T, Adams D, Silva A, et al.
N Engl J Med 2013;369:819-29.

Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin.

Methods We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers.

Results Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively.

ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene.


Alnylam May Seek Approval for TTR Amyloidosis Rx in 2017 as Other Programs Advance

Officials from Alnylam Pharmaceuticals last week provided updates on the two drug candidates from the company’s flagship transthyretin-mediated amyloidosis program, stating that the intravenously delivered agent patisiran is proceeding toward a possible market approval in three years, while a subcutaneously administered version called ALN-TTRsc is poised to enter Phase III testing before the end of the year.

Meanwhile, Alnylam is set to advance a handful of preclinical therapies into human studies in short order, including ones for complement-mediated diseases, hypercholesterolemia, and porphyria.

The officials made their comments during a conference call held to discuss Alnylam’s second-quarter financial results.

ATTR is caused by a mutation in the TTR gene, which normally produces a protein that acts as a carrier for retinol binding protein and is characterized by the accumulation of amyloid deposits in various tissues. Alnylam’s drugs are designed to silence both the mutant and wild-type forms of TTR.

Patisiran, which is delivered using lipid nanoparticles developed by Tekmira Pharmaceuticals, is currently in a Phase III study in patients with a form of ATTR called familial amyloid polyneuropathy (FAP) affecting the peripheral nervous system. Running at over 20 sites in nine countries, that study is set to enroll up to 200 patients and compare treatment to placebo based on improvements in neuropathy symptoms.

According to Alnylam Chief Medical Officer Akshay Vaishnaw, Alnylam expects to have final data from the study in two to three years, which would put patisiran on track for a new drug application filing in 2017.

Meanwhile, ALN-TTRsc, which is under development for a version of ATTR that affects cardiac tissue called familial amyloidotic cardiomyopathy (FAC) and uses Alnylam’s proprietary GalNAc conjugate delivery technology, is set to enter Phase III by year-end as Alnylam holds “active discussions” with US and European regulators on the design of that study, CEO John Maraganore noted during the call.

In the interim, Alnylam continues to enroll patients in a pilot Phase II study of ALN-TTRsc, which is designed to test the drug’s efficacy for FAC or senile systemic amyloidosis (SSA), a condition caused by the idiopathic accumulation of wild-type TTR protein in the heart.

Based on “encouraging” data thus far, Vaishnaw said that Alnylam has upped the expected enrollment in this study to 25 patients from 15. Available data from the trial is slated for release in November, he noted, stressing that “any clinical endpoint result needs to be considered exploratory given the small sample size and the very limited duration of treatment of only six weeks” in the trial.

Vaishnaw added that an open-label extension (OLE) study for patients in the ALN-TTRsc study will kick off in the coming weeks, allowing the company to gather long-term dosing tolerability and clinical activity data on the drug.

Enrollment in an OLE study of patisiran has been completed with 27 patients, he said, and, “as of today, with up to nine months of therapy … there have been no study drug discontinuations.” Clinical endpoint data from approximately 20 patients in this study will be presented at the American Neurological Association meeting in October.

As part of its ATTR efforts, Alnylam has also been conducting natural history of disease studies in both FAP and FAC patients. Data from the 283-patient FAP study was presented earlier this year and showed a rapid progression in neuropathy impairment scores and a high correlation of this measurement with disease severity.

During last week’s conference call, Vaishnaw said that clinical endpoint and biomarker data on about 400 patients with either FAC or SSA have already been collected in a nature history study on cardiac ATTR. Maraganore said that these findings would likely be released sometime next year.

Alnylam Presents New Phase II, Preclinical Data from TTR Amyloidosis Programs


Amyloid disease drug approved

Nature Biotechnology 2012; (3

The first medication for a rare and often fatal protein misfolding disorder has been approved in Europe. On November 16, the E gave a green light to Pfizer’s Vyndaqel (tafamidis) for treating transthyretin amyloidosis in adult patients with stage 1 polyneuropathy symptoms. [Jeffery Kelly, La Jolla]


Safety and Efficacy of RNAi Therapy for Transthyretin ……/NEJMoa1208760?&#8230;

The New England Journal of Medicine

Aug 29, 2013 – Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart.


Alnylam’s RNAi therapy targets amyloid disease

Ken Garber
Nature Biotechnology 2015; 33(577)

RNA interference’s silencing of target genes could result in potent therapeutics.

The most clinically advanced RNA interference (RNAi) therapeutic achieved a milestone in April when Alnylam Pharmaceuticals in Cambridge, Massachusetts, reported positive results for patisiran, a small interfering RNA (siRNA) oligonucleotide targeting transthyretin for treating familial amyloidotic polyneuropathy (FAP).  …

  1. Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases

Nature Biotechnology 11 April 2016

  1. CRISPR-Cas systems for editing, regulating and targeting genomes

Nature Biotechnology 02 March 2014

  1. Near-optimal probabilistic RNA-seq quantification

Nature Biotechnology 04 April 2016


Translational Neuroscience: Toward New Therapies

Karoly Nikolich, ‎Steven E. Hyman – 2015 – ‎Medical

Tafamidis for Transthyretin Familial Amyloid Polyneuropathy: A Randomized, Controlled Trial. … Multiplex Genome Engineering Using CRISPR/Cas Systems.


Is CRISPR a Solution to Familial Amyloid Polyneuropathy?

Author and Curator: Larry H. Bernstein, MD, FCAP

Originally published as

FAP is characterized by the systemic deposition of amyloidogenic variants of the transthyretin protein, especially in the peripheral nervous system, causing a progressive sensory and motor polyneuropathy.

FAP is caused by a mutation of the TTR gene, located on human chromosome 18q12.1-11.2.[5] A replacement of valine by methionine at position 30 (TTR V30M) is the mutation most commonly found in FAP.[1] The variant TTR is mostly produced by the liver.[citation needed] The transthyretin protein is a tetramer.    ….



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Natural Killer Cell Response: Treatment of Cancer

Curator: Larry H. Bernstein, MD, FCAP


Molecular mechanisms of natural killer cell activation in response to cellular stress

C J Chan1,2,3, M J Smyth1,2,3,4,5 and L Martinet1,2,4,5        Edited by M Piacentini

Cell Death and Differentiation (2014) 21, 5–14;

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress.


  • Stress induces specific intrinsic and extrinsic physiological mechanisms within cells that lead to their identification as functionally abnormal
  • Sources of cellular stress can be nutritional, physical, pathogenic, or oncogenic
  • Intrinsic responses to cellular stress include activation of the DNA-damage response, tumor-suppressor genes, and senescence
  • The extrinsic response to cellular stress is activation of the immune system, such as natural killer cells
  • Intrinsic responses to cellular stress can directly upregulate factors that can activate the immune system, and the immune system been shown to be indispensable for the efficacy of some chemotherapy

Further critical determinants of intrinsic responses to stress and cell death that can activate the immune system must be identified

  • Identification of the different cellular pathways and molecular determinants controlling the immunogenicity of different cancer therapies is required
  • How can we harness the ability of therapeutic agents to activate both the intrinsic and extrinsic responses to cellular stress to achieve more specific and safer approaches to cancer treatment?

Any insult to a cell that leads to its abnormal behavior or premature death can be defined as a source of stress. As the turnover and maintenance of cells in all multi-cellular organisms is tightly regulated, it is essential that stressed cells be rapidly identified to avoid widespread tissue damage and to maintain tissue homeostasis. Various intrinsic cellular mechanisms exist within cells that become activated when they are exposed to stress. These include activation of DNA-damage response proteins, senescence programs, and tumor-suppressor genes.1 Extrinsic mechanisms also exist that combat cellular stress, through the upregulation of mediators that can activate different components of the immune system.2 Although frequently discussed separately, much recent evidence has indicated that intrinsic and extrinsic responses to cellular stress are intimately linked.3

As the link between cell intrinsic and extrinsic responses to stress have been uncovered, these observations are now being harnessed therapeutically, particularly in the context of cancer.4 Indeed, various chemotherapeutic agents and radiotherapy are critically dependent on the immune system to elicit their full therapeutic benefit.5, 6 The mechanisms by which this occurs may be twofold: (i) the induction of intrinsic cellular stress mechanisms activates innate immunity and (ii) the release and presentation of tumor-specific antigens engages an inflammatory adaptive immune response.

NK cells are the major effector lymphocyte of innate immunity found in all the primary and secondary immune compartments as well as various mucosal tissues.7 Through their ability to induce direct cytotoxicity of target cells and produce pro-inflammatory cytokines such as interferon-gamma, NK cells are critically involved in the immune surveillance of tumors8, 9, 10 and microbial infections.11, 12 The major mechanism that regulates NK cell contact-dependent functions (such as cytotoxicity and recognition of targets) is the relative contribution of inhibitory and activating receptors that bind to cognate ligands.

Under normal physiological conditions, NK cell activity is inhibited through the interaction of their inhibitory receptors with major histocompatibility complex (MHC) class I.13, 14 However, upon instances of cellular stress that are frequently associated with viral infection and malignant transformation, ligands for activating receptors are often upregulated and MHC class I expression may be downregulated. The upregulation of these activating ligands and downregulation of MHC class I thus provides a signal for NK cells to become activated and display effector functions. Activating receptors are able to provide NK cells with a strong stimulus in the absence of co-stimulation due to the presence of adaptor molecules such as DAP10, DAP12, FcRγ, and CD3ζ that contain immunoreceptor tyrosine-based activating motifs (ITAMs).15, 16,17 By contrast, inhibitory receptors contain inhibitory motifs (ITIMs) within their cytoplasmic tails that can activate downstream targets such as SHP-1 and SHP-2 and directly antagonize those signaling pathways activated through ITAMs.18, 19, 20 The specific details of individual classes of inhibitory and activating receptors and their ligands are summarized in Figure 1 and have been extensively reviewed elsewhere.14, 21 Instead, this review will more focus on the relevant activating receptors that are primarily involved in the direct regulation of NK cell-mediated recognition of cellular stress: natural killer group 2D (NKG2D) and DNAX accessory molecule-1 (DNAM-1).

Figure 1.

Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the authorNK cell receptors and their cognate ligands. Major inhibitory and activating receptors on NK cells and their cognate ligands on targets are depicted. BAT3, human leukocyte antigen (HLA)-B-associated transcript 3; CRTAM, class I-restricted T-cell-associated molecule; HA, hemagglutinin; HLA-E, HLA class I histocompatibility antigen, alpha chain E; IgG, immunoglobulin G; LFA-1, leukocyte function-associated antigen-1; LLT1, lectin-like transcript 1; TIGIT, T cell immunoglobulin and ITIM domain

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NK Cell-Mediated Recognition of Cellular Stress by NKG2D and DNAM-1

NKG2D is a lectin-like type 2 transmembrane receptor expressed as a homodimer in both mice and humans by virtually all NK cells.22, 23 Upon interaction with its ligands, NKG2D can trigger NK cell-mediated cytotoxicity against their targets. The ligands for NKG2D are self proteins related to MHC class I molecules.24 In humans, these ligands consist of the MHC class I chain-related protein (MIC) family (e.g., MICA and MICB) and the UL16-binding protein (ULBP1-6) family.25, 26 In mice, ligands for NKG2D include the retinoic acid early inducible (Rae) gene family, the H60 family, and mouse ULBP-like transcript-1 (MULT-1).27, 28, 29 NKG2D ligands are generally absent on the cell surface of healthy cells but are frequently upregulated upon cellular stress associated with viral infection and malignant transformation.3, 30 Indeed, NKG2D ligand expression has been found on many transformed cell lines, and NKG2D-dependent elimination of tumor cells expressing NKG2D ligands has been well documented in vitro and in tumor transplant experiments.25, 30, 31, 32, 33 In humans, NKG2D ligands have been described on different primary tumors34, 35 and specific NKG2D gene polymorphisms are associated with susceptibility to cancer.36 Finally, blocking NKG2D through gene inactivation or monoclonal antibodies leads to an increased susceptibility to tumor development in mouse models,37, 38demonstrating the key role played by NKG2D in immune surveillance of tumors. NKG2D can also contribute to shape tumor immunogenicity, a process called immunoediting, as demonstrated by the frequent ability of tumor cells to avoid NKG2D-mediated recognition through NKG2D ligand shedding, as discussed later in this review.38, 39, 40

DNAM-1 is a transmembrane adhesion molecule constitutively expressed on T cells, NK cells, macrophages, and a small subset of B cells in mice and humans.41, 42, 43 DNAM-1 contains an extracellular region with two IgV-like domains, a transmembrane region and a cytoplasmic region containing tyrosine- and serine-phosphorylated sites that is able to initiate downstream activation cascades.41, 44 There is accumulating evidence showing that DNAM-1 not only promotes adhesion of NK cells and CTLs but also greatly enhances their cytotoxicity toward ligand-expressing targets.41, 45, 46, 47, 48, 49, 50 The ligands for DNAM-1 are the nectin/nectin-like family members CD155 (PVR, necl-5) and CD112 (PVRL2, nectin-2).45, 46 Like NKG2D ligands, DNAM-1 ligands are frequently expressed on virus-infected and transformed cells.51, 52DNAM-1 ligands, especially CD155, are overexpressed by many types of solid and hematological malignancies and blocking DNAM-1 interactions with its ligands reduces the ability of NK cells to kill tumor cells in vitro.41, 49, 53, 54, 55, 56, 57 Further evidence of the role of DNAM-1 in tumor immune surveillance is provided by studies using experimental and spontaneous models of cancer in vivo showing enhanced tumor spread in the absence of DNAM-1.47, 48, 49, 50, 58

As NKG2D and DNAM-1 ligands are frequently expressed on stressed cells, many studies have sought to determine the mechanisms that underpin these observations. The guiding hypothesis for these studies is that cell-intrinsic responses to stress are directly linked to cell-extrinsic responses that can trigger rapid NK cell surveillance and elimination of stressed cells. Indeed, major cell-intrinsic responses to cellular stress can directly lead to NK cell-activating ligand upregulation and are outlined in the following sections.

The DNA-Damage Response

Cellular stress caused by the activation of the DNA-damage response leads to downstream apoptosis or cell-cycle arrest. The activation of DNA-damage checkpoints occurs when there are excessive DNA strand breaks and replication errors, thereby representing an important tumorigenesis barrier that can slow or inhibit the progression of malignant transformation.59, 60 Two major transducers of the DNA-damage response are the PI3-kinase-related protein kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). ATM and ATR can modulate numerous signaling pathways such as checkpoint kinases (Chk1 and Chk2, which inhibit cell-cycle progression and promote DNA repair) and p53 (which mediates cell-cycle arrest and apoptosis).61

In addition to the induction of cell-cycle arrest and apoptosis, activation of the DNA-damage response has been shown to promote the expression of several activating ligands that are specific for NK cell receptors, primarily those of the NKG2D receptor. These findings have shown a critical direct link between cellular transformation, apoptosis, and surveillance by the immune system.62 The first evidence of this link between DNA damage and immune cell activation was provided by Raulet and colleagues who showed that NKG2D ligands were upregulated by genotoxic stress and stalled DNA replication conditions known to activate either ATM or ATR.63 These observations have now been extended by several other studies that have defined further DNA-damaging conditions (e.g., genotoxic drugs/chemotherapy, deregulated proliferation, or oxidative stress) that can promote NKG2D ligand upregulation.64, 65, 66, 67

The role of the DNA-damage response in controlling NKG2D ligand expression and subsequent NK cell activation has also been demonstrated in the context of anti-viral immunity, specifically in Abelson murine leukemia virus infection.68 This pathogen was shown to induce activation-induced cytidine deaminase (AID) expression outside the germinal center, resulting in generalized hypermutation, DNA-damage checkpoint activation, and Chk1 phosphorylation. The genotoxic activity of virally induced AID not only restricted the proliferation of infected cells but also induced the expression of NKG2D ligands. More recently, another member of APOBEC-AID family of cytidine deaminases, A3G, has been shown to promote the recognition of HIV-infected cells by NK cells after DNA-damage response activation.69 In this study, viral protein Vpr-mediated repair processes, which generate nicks, gaps, and breaks of DNA, activate an ATM/ATR DNA-damage response that leads to NKG2D ligand expression.

The DNA-damage sensors ATM and ATR have also been shown to regulate other key NK cell-activating ligands such as the DNAM-1 ligand, CD155.58, 65, 70 For example, in the Eμ-myc spontaneous B-cell lymphoma model, activation of the DNA-damage response leads to the upregulation of CD155 in the early-stage transformed B cells, subsequently activating spontaneous tumor regression in an NK cell- and T-cell-dependent manner.58 The DNA-damage response can also regulate the expression of the death receptor DR5.71 The engagement of DR5 by the effector molecule TRAIL, which is expressed by NK cells and T cells, can induce apoptosis of target cells and has been shown to have a key role in immune surveillance against tumors.72 Collectively, these results suggest that the detection of DNA damage, primarily through ATM and ATR, may represent a conserved protection mechanism governing the immunogenicity of infected or transformed cells, leading to direct recognition by NK cells (Figure 2).

Figure 2.

Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the authorOverview of the molecular pathways leading to NK cell recognition of intrinsic cellular stress. Oncogenic transformation and viral infection can activate intrinsic cellular responses to stress. These responses include activation of the DNA-damage response, senescence, tumor suppressors, and the presentation and/or release of HSPs that, in turn, can activate NK cells through various receptor–ligand interactions. Senescent cells can also release pro-inflammatory cytokines that can recruit NK cells and other innate immunity, such as macrophages. CCL2, C-C motif chemokine ligand 2; CXCL11, C-X-C motif chemokine ligand 11; DR, death receptor 5; IFN, interferon; IL, interleukin; LFA-1, leukocyte function-associated antigen-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand

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As a result of these studies, many therapeutic agents known to induce DNA damage have been evaluated for their ability to increase the immunogenicity of cancer cells for a more targeted therapeutic approach using NK cells.64, 65 For example, treatment of multiple myeloma cells with doxorubicin, melphalan, or bortezomib can lead to DNAM-1 and NKG2D ligand upregulation.65Indeed, many chemotherapeutic agents commonly used, especially in hematological malignancies, can trigger the DNA-damage pathway. Therefore, it is reasonable to speculate that there is a general role of ATM and ATR in the induction of NK cell activation as a therapeutic effect of these agents.


Cellular senescence is generally defined as a growth-arrest program in mammalian cells that limits their lifespan.73 The major type of cellular senescence is replicative senescence that occurs due to telomere shortening. However, it is now generally accepted that premature senescence can also occur due to oncogene activation (oncogene-induced senescence) and/or the loss/gain of tumor-suppressor gene function, in the absence of telomere shortening.74 Thus, premature senescence is an important barrier against malignant transformation.59 Upon engagement of the senescence program, although cells are in growth arrest, they remain metabolically active and can produce many pro-inflammatory cytokines, as well as upregulate adhesion molecules and activating ligands to alert the immune system.75, 76, 77Activation of the immune system, in particular innate immunity, has a critical role in the clearance of senescent cells.78, 79, 80, 81More specifically, in a model of hepatocellular carcinoma, it has been shown that reactivation of p53 can induce a senescence program, resulting in tumor regression through the activation of NK cells, macrophages, and neutrophils. Of note, intercellular adhesion molecule (ICAM)-1, which can trigger both adhesion and cytotoxicity of NK cells,82 and interleukin-15, a cytokine that can promote NK cell effector function,83 were both upregulated in senescent tumors. More recently, the potential contribution of NK cells was also shown in the clearance of senescent hepatic stellate cells, a mechanism important in limiting liver fibrosis in response to a fibrogenic agent.80 ICAM-1, NKG2D ligands (MICA and ULPB2), and DNAM-1 ligands (CD155) were all upregulated on senescent hepatic stellate cells.

The specific mechanisms linking the senescence program to immune activation are not yet fully understood. However, the intracellular molecular mechanisms that govern induction of senescence may provide possible indications. Both replicative senescence and premature senescence (e.g., oncogene-induced senescence) have been shown to have common molecular determinants, such as the activation of the DNA-damage response pathway (e.g., ATM and ATR) and downstream activation of p53 and p16INK4A.1, 59, 84, 85, 86 Activation of the DNA-damage response would presumably initiate the upregulation of NK cell-activating ligands as previously discussed. However, how senescence may be linked to the induction of pro-inflammatory cytokine release is a more compelling question and requires further investigation (Figure 2). Nevertheless, induction of pro-inflammatory cytokines is an important protective mechanism in order to recruit immune cells that can rapidly recognize and remove senescent cells. Interestingly, activation of NK cells by senescent cells has been observed in a clinical context when multiple myeloma cells were treated with chemotherapy and genotoxic agents.65 In this setting, NKG2D and DNAM-1 ligands were both upregulated through a mechanism that required activation of the DNA-damage pathway initiated by ATM and ATR.65

Tumor Suppressors: p53

p53 is a potent tumor suppressor and central regulator of apoptosis, DNA repair, and cell proliferation, that is activated in response to DNA damage, oncogene activation, and other cellular stress.87 The number of identified cellular functions that p53 regulates has greatly increased over the past few years, and there is now a vast array of evidence that shows that p53 can be induced by viral infection88 to limit pathogen spread by inducing apoptosis.89, 90 Furthermore, p53 not only acts as an intrinsic barrier against tumorigenesis or pathogenic spread but can also lead to increased cellular immunogenicity. For example, p53 reactivation in a hepatocellular carcinoma can promote tumor regression mediated by innate immunity.78 A direct link between p53 expression and immune cell recognition was recently provided by Textor et al.91 where expression of p53 in lung cancer cell lines strongly upregulated the NKG2D ligands ULBP1 and 2, resulting in NK cell activation. Subsequently, p53-responsive elements were found to directly regulate ULBP1 and 2 expression, the deletion of which abolished the capacity of p53 to mediate ULBP1 and 2 upregulation. Another recent report that used a pharmacological activator of p53 confirmed the ability of p53 to directly induce ULBP2 expression that was independent of ATM/ATR.92 However, it has also been shown that miR34a and miR34C microRNAs (miRNAs) induced by p53 can target ULBP2 mRNA and reduce its cell-surface expression, suggesting that p53 may have a dual role in regulating ULBP2 expression.93 Finally, early work showed that NKG2D ligands can be upregulated by ATR/ATM in the total absence of p53 in tumor cell lines,62, 63 suggesting the existence of ATM/ATR-dependent and p53-independent pathways that regulate NKG2D ligand expression in response to cellular stress.

In addition to regulating NK cell ligand expression, genetic reactivation of p53 in tumors can also induce a wide array of pro-inflammatory mediators ranging from adhesion receptor (ICAM-1) expression to the production of various chemokines (CXCL11 and monocyte chemoattractant protein-1) and cytokines (interleukin-15).78 Furthermore, recent studies in anti-viral immunity indicate that several interferon-inducible genes and Toll-like receptor-3 expression are direct transcriptional targets of p53 and that p53 contributes to production of type I interferon by virally infected cells.94, 95, 96 All together, these studies suggest that p53 accumulation could represent a key determinant of the immunogenicity of stressed cells that are infected or undergoing malignant transformation through its ability to regulate innate immune activation.


Malignant transformation is a complex process that frequently involves the activation of one or more oncogenes in addition to the inactivation or mutation of tumor-suppressor genes (e.g., p53). Oncogene activation is a powerful inducer of cellular stress that is able to activate intrinsic cellular programs that lead to cell apoptosis or senescence (e.g., activation of the DNA-damage response and p53).1 In addition, many recent reports have also shown that major oncogenes can activate extrinsic responses to cellular stress through inducing the upregulation of NK cell-activating ligands.63, 97, 98 This suggests that oncogene activation can represent a key cellular event in alerting the immune system to ongoing cellular transformation (Figure 3).

Figure 3.

Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the authorMolecular mechanisms that regulate the cell surface expression of NKG2D ligands. The major group of NK cell-activating ligands that are upregulated by intrinsic cellular responses to stress are those that bind the NKG2D receptor. Activation of the DNA-damage response, senescence, oncogenes, tumor suppressors, or sensing of deregulated proliferation can induce NKG2D ligand gene transcription and increase mRNA translation, leading to extracellular protein expression. MMP, matrix metalloproteases

Full figure and legend (183K)

The enhanced expression of the proto-oncogene Myc has been described as a critical event leading to cellular transformation and is a frequently found genetic alteration in cancer.99 In a recent study, again using the Eμ-myc model, Medzhitov and colleagues demonstrated the ability of c-Myc to alert NK cells to early oncogenic transformation through the upregulation of Rae-1.97 In this study, the induction of Rae-1 was dependent on the direct regulation of Rae-1 transcription by Myc through its interaction with the Raet1 epsilon gene. Collectively, these results provide a possible direct molecular mechanism to explain the increased susceptibility of NKG2D gene-targeted mice to lymphoma development in the Eμ-myc model.38

Recent evidence suggests that several oncogenic mutations of Ras (H-Ras, N-Ras, and K-Ras) can also regulate NKG2D ligand expression in both mice and humans.98 Interestingly, in this case, NKG2D ligands were regulated through MAPK/MEK and PI3K pathways downstream of oncogenic H-RasV12. The activation of PI3K pathways, and more particularly the p110α subunits by virus-encoded proteins, has also been shown to induce the Rae-1 family of ligands.100 As many viruses can manipulate the PI3K pathway101 and tumors often bear Ras and p110α oncogene mutations,102 collectively, this data suggests that there is the existence of a common molecular mechanism by which NK cells sense cellular stress mediated by PI3K-dependent regulation of NKG2D ligands.

Interestingly, whereas Myc was involved in the transcriptional regulation of NKG2D ligands, PI3K can increase NKG2D ligand expression by increasing the translation of Rae-1 mRNA.98 This involved the induction of eIF4E, a protein that enhances the translation of mRNA.103 As number of tumors and viruses can upregulate host translation initiation machinery through the overexpression of eIF4E,104, 105 this may represent an important means by which NK cells can discriminate tumor- and virus-infected cells from normal cells.

Heat-Shock Proteins (HSPs)

HSPs are highly conserved intracellular chaperone molecules that are present in most prokaryotic and eukaryotic cells that mediate protection against cellular damage under conditions of stress. HSPs are distributed in most intracellular compartments of cells where they support the correct folding of nascent polypeptides, prevent protein aggregation, and assist in protein transport across membranes.106 Many tumors display overexpression of HSPs as a response to cellular stress induced by oncogenic transformation.107, 108 HSPs can also be mobilized to the plasma membrane, or even released from cells, under conditions of stress.109

Although intracellular HSPs can promote cell survival by interfering with different apoptosis components, many studies have reported that membrane-bound or soluble HSPs can directly stimulate innate immunity.110 A major immunostimulatory function of HSPs is to promote the presentation of tumor-specific antigens by MHC class I to CD8 T cells.111, 112, 113 Soluble and membrane-bound HSPs can also induce antigen-presenting cell maturation and the resultant secretion of pro-inflammatory cytokines.114, 115, 116Finally, HSPs may directly activate NK cells as HSP70, when overexpressed on tumor cells, can induce a selective dose-dependent increase in NK cell-mediated cytotoxicity in vitro.117 NK cells may directly recognize HSP70 through a 14-amino-acid oligomer (TKD) that is localized in the C-terminal domain of the protein through CD94.118, 119 Tumor-specific HSP70 that is either presented at the cell surface or secreted on exosomes can also enhance NK cell activity against diverse types of cancer in vivo.120, 121 Most importantly, hepatocellular carcinoma cells that are treated with various chemotherapeutic agents can become more susceptible to NK cell-mediated cytotoxicity through their release of HSP-containing exosomes, giving the aforementioned findings a therapeutic context.122 Collectively, these results suggest that HSP translocation to the plasma membrane or secretion during cellular stress may represent a potent danger signal that can stimulate NK cell activity, particularly in the context of cancer.


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Alzheimer Disease Developments – Spring 2015

Larry H. Bernstein, MD, FCAP, Curator




Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment


JNK: A Putative Link Between Insulin Signaling and VGLUT1 in Alzheimer’s Disease

Omega-3 Fatty Acid Status Enhances the Prevention of Cognitive Decline by B Vitamins in Mild Cognitive ImpairmentOpenly Available
Oulhaj, Abderrahim | Jernerén, Fredrik | Refsum, Helga | Smith, A. David | de Jager, Celeste A.

Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic EncephalopathyOpenly Available
Stern, Robert A. | Tripodis, Yorghos | Baugh, Christine M. | Fritts, Nathan G. | Martin, Brett M. | Chaisson, Christine | Cantu, Robert C. | Joyce, James A. | Shah, Sahil | Ikezu, Tsuneya | Zhang, Jing | Gercel-Taylor, Cicek | Taylor, Douglas D

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate KineticsOpenly Available
Eketjäll, Susanna | Janson, Juliette | Kaspersson, Karin | Bogstedt, Anna | Jeppsson, Fredrik | Fälting, Johanna | Haeberlein, Samantha Budd | Kugler, Alan R. | Alexander, Robert C. | Cebers, Gvido

Predictive Value of Cerebrospinal Fluid Visinin-Like Protein-1 Levels for Alzheimer’s Disease Early Detection and Differential Diagnosis in Patients with Mild Cognitive Impairment
Babić Leko, Mirjana | Borovečki, Fran | Dejanović, Nenad | Hof, Patrick R. | Šimić, Goran

Plasma Phospholipid and Sphingolipid Alterations in Presenilin1 Mutation Carriers: A Pilot Study
Chatterjee, Pratishtha | Lim, Wei L.F. | Shui, Guanghou | Gupta, Veer B. | James, Ian | …… | Wenk, Marcus R. | Bateman, Randall J. | Morris, John C. | Martins, Ralph N.

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Metallothioneins in Prion- and Amyloid-Related Diseases


Microglia are the immune cells of the CNS and account for approximately 10% of the CNS cellpopulation, with regional variation in density [27, 28]. During embryonic development, microglia originate from yolk sac progenitor cells that migrate into the developing CNS during early embryogenesis [29,30].Following construction of the blood-brain barrier (BBB), microglia are renewed by local turnover [31]. In the healthy brain, microglia actively support neurons through the release of insulin-like growth factor 1, nerve growth factor, ciliary neurotrophic factor, and brain-derived neurotrophic factor (BDNF) [32–34]. Microglia also provide indirect support to neurons by clearance of debris to maintain the extracellular environment, and phagocytosis of apoptotic cells to facilitate neurogenesis [35, 36]. In the adult brain, microglia coordinate much of their activity with astrocytes and activate in response to similar stimuli [37, 38]. Dysfunctional signaling between microglia and astrocytes often results in chronic inflammation, a characteristic of many neurodegenerative diseases [39, 40].

Historically, it has been thought that microglia ‘rest’ when not responding to inflammatory stimuli or damage [41, 42]. However, this notion is being increasingly recognized as inaccurate [43]. When not involved in active inflammatory signaling, microglia constantly patrol the neuropil by extension and retraction of their finely branched processes [44]. Microglial activation is often broadly classified into two states; pro-inflammatory (M1) or anti-inflammatory (M2) [36, 45], based on similar phenotypes in peripheral macrophages [46]. M1 activated microglia are characterized by increased expression of pro-inflammatory mediators and cytokines, including inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β, often under the control of the transcription factor nuclear factor-κB [45]. Pro-inflammatory microglia rapidly retract their processes and adopt an amoeboid morphology and often migrate closer to the site of injury [47]. Anti-inflammatory M2 activation of microglia, often referred to as alternative activation, represents the other side of microglial behavior. Anti-inflammatory activation is characterized by increased expression of cytokines including arginase 1 and interleukin-10, and is associated with increased ramification of processes [45]. The polarization of microglia into M1 or M2 throughout the brain is well characterized, especially in neurodegenerative diseases [48]. In the AD brain, microglia expressing markers of M1 activation are typically localized to brain regions such as the hippocampus that are most heavily affected in the disease [49]. However, it is important to note that M1 and M2 classifications of microglia may over-simplify microglial phenotypes and may only represent the extremes of microglial activation [50]. It has been more recently proposed that microglia likely occupy a continuum between these phenotypes [39, 51].

Do microglia have multiple roles in AD?

Classical pro-inflammatory activation of microglia has long been associated with AD [39, 49]. Samples taken from late-stage AD brains contain characteristic signs of inflammation, including amoeboid morphology of microglia, high levels of pro-inflammatory cytokines in the cerebrospinal fluid, and evidence of neuronal damage due to chronic exposure to pro-inflammatory cytokines and oxidative stress [52, 53]. The cause of this inflammation may be in response to direct toxicity of Aβ to neurons resulting in activation of nearby microglia and astrocytes [53, 54]. However, Aβ may also induce inflammatory activation of microglia and astrocytes. Activated immune cells are typically present surrounding amyloid plaques [55–57], with such peri-plaque cells exhibiting strong evidence of pro-inflammatory activation [56, 58–60]. The presence of undigested Aβ particles within these activated microglia may suggest that the Aβ peptide itself is a pro-inflammatory signal for microglia [61–64]. In vitro experiments provide supporting evidence for the in vivo studies, with Aβ promoting pro-inflammatory microglial activation [65, 66], and also acting as a potent chemotactic signal [67].

However, it is important to note that although widespread inflammation is characteristic of late-stage AD, it remains unclear what role inflammation could play in early stages of the disease. Some evidence suggests that reducing inflammation through the long-term use of some non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of AD [68]. However, these findings have not yet been verified in clinical trials [69, 70]. Little is understood about how NSAIDs and related compounds affect the delicate balance of pro- versus anti-inflammatory microglial activity within the brain. Although there is considerable evidence to suggest that chronic inflammation may contribute to pathology in the later stages of AD, it is important to note that inflammation normally only represents a small aspect of microglial function. The non-inflammatory functions of microglia may play a more important role in early disease; specifically, microglial functions relating to maintenance of the CNS.

Phagocytosis: A vital role of microglia that may be lost in AD    


Recently, a new function has been proposed for microglia. A number of studies have provided evidence that microglia prune synapses throughout life. Microglia are known to remove extraneous synapses during development to ensure that only meaningful connections remain [43]. It was, however, thought that differentiated astrocytes performed the majority of synaptic pruning in the adult brain [91]. The discovery that microglial processes are constantly active within the brain and are often positioned near synapses raised the question of whether microglial synaptic pruning continued throughout life [44, 47, 92–94]. This question was answered in 2014 in a study that demonstrated that microglia do prune synapses into adulthood, and that this activity is important for normal brain function [95]. These findings supported those found a year earlier in a study reporting that ablation of microglia from brain slices increases synapse density and results in abnormal firing of hippocampalneurons [96].

Altered microglial behavior may underlie altered neuronal firing in AD  

Altered neuronal activity is an early phenomenon in AD

The cause of DMN hypoactivity in AD is not yet clear; however studies performed in cohorts that are genetically predisposed to AD suggest that DMN hypoactivity is preceded by a period of hyperactivity and increased functional connectivity [123, 136], often manifesting as an absence of normal DMN deactivation during external tasks [137–140]. DMN hyperactivity may interfere with hippocampal memory encoding, leading to the memory deficits that are present in mild cognitive impairment [141, 142]. It has been proposed that hippocampal hyperexcitability in AD may develop as a protective mechanism against increased input from the DMN [142–144]. As AD progresses, the initial hyperexcitability of the DMN and hippocampus may result in hypoactivity due to exhaustion of compensatory mechanisms [123, 136]. Evidence from both transgenic AD mice and longitudinal human studies supports an exhaustion model of hyperactivation leading to later hypoactivation [143, 145–147]. Interestingly, a number of studies report a lower incidence of AD among those who regularly practice meditation which specifically ‘calms’ the DMN [148].

Our understanding of AD as a disease is changing. Historically considered to be primarily a disease of neuronal degeneration, this neurocentric view has widened to encompass non-neuronal cells such as astrocytes into our understanding of the disease process and pathogenesis. A proposed model for microglia in AD is shown in Fig. 2. Microglia perform a wide range of functions in the CNS and although this includes induction of an inflammatory reaction in response to damage, they also have critical roles for maintaining normal function in the brain. Recent evidence shows that microglia regulate neuronal activity through synaptic pruning throughout life as an extension on their normal phagocytosis behavior. The discovery of a large number of AD risk genes associated with reduced immune cell function suggests that perturbed microglial phagocytosis could lead to AD. In our model, altered microglial phagocytosis of synapses results in network dysfunction and onset of AD, occurring downstream of Aβ.

The immune system and microglia represent a novel target for intervention in AD. Importantly, a large number of anti-inflammatory drugs are already in use for other conditions. What is important to know at this stage is exactly how to best target immune cell function. The studies outlined here provide evidence that an indiscriminate dampening down of all microglial activity may result in a worse outcome for individuals by suppressing normal microglial regulatory functions. We currently do not know whether future microglial-based therapies should focus on reducing chronic inflammation or conversely, whether they should be aimed at boosting microglial phagocytosis. It is also likely that future treatment strategies may use a combination of approaches to target Aβ, immune cell phagocytosis and network activity. An increasing view in the AD field is that any drug or therapy needs to be provided very early in the disease process to maximize its beneficial effects. Although we are currently unable to effectively target those at risk of AD at such an early stage, advances in neuroimaging for subtle changes in network activity, or in assays for immune cell function, may provide new avenues for identification of early damage and risk of disease.



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Late-Onset Metachromatic Leukodystrophy with Early Onset Dementia Associated with a Novel Missense Mutation in the Arylsulfatase A Gene

Microbes and Alzheimer’s DiseaseOpenly Available
Itzhaki, Ruth F. | Lathe, Richard | Balin, Brian J. | Ball, Melvyn J. | Bearer, Elaine L. | Braak, Heiko | Bullido, Maria J. | Carter, Chris | Clerici, Mario | Cosby, S. Louise | Del Tredici, Kelly | Field, Hugh | Fulop, Tamas | Grassi, Claudio | Griffin, W. Sue T. | Haas, Jürgen | Hudson, Alan P. | Kamer, Angela R. | Kell, Douglas B. | Licastro, Federico | Letenneur, Luc | Lövheim, Hugo | Mancuso, Roberta | Miklossy, Judith | Otth, Carola | Palamara, Anna Teresa | Perry, George | Preston, Christopher | Pretorius, Etheresia | Strandberg, Timo | Tabet, Naji | Taylor-Robinson, Simon D. | Whittum-Hudson, Judith A.

Longitudinal Relationships between Caloric Expenditure and Gray Matter in the Cardiovascular Health StudyOpenly Available
Raji, Cyrus A. | Merrill, David A. | Eyre, Harris | Mallam, Sravya | Torosyan, Nare | Erickson, Kirk I. | Lopez, Oscar L. | Becker, James T. | Carmichael, Owen T. | Gach, H. Michael | Thompson, Paul M. | Longstreth Jr., W.T. | Kuller, Lewis H.

Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic EncephalopathyOpenly Available
Stern, Robert A. | Tripodis, Yorghos | Baugh, Christine M. | Fritts, Nathan G. | Martin, Brett M. | Chaisson, Christine | Cantu, Robert C. | Joyce, James A. | Shah, Sahil | Ikezu, Tsuneya | Zhang, Jing | Gercel-Taylor, Cicek | Taylor, Douglas D.

Unraveling Alzheimer’s: Making Sense of the Relationship between Diabetes and Alzheimer’s Disease1Openly Available
Schilling, Melissa A.

Pain Assessment in Elderly with Behavioral and Psychological Symptoms of DementiaOpenly Available
Malara, Alba | De Biase, Giuseppe Andrea | Bettarini, Francesco | Ceravolo, Francesco | Di Cello, Serena | Garo, Michele | Praino, Francesco | Settembrini, Vincenzo | Sgrò, Giovanni | Spadea, Fausto | Rispoli, Vincenzo

Editor’s Choice from Volume 50, Number 4 / 2016

Post Hoc Analyses of ApoE Genotype-Defined Subgroups in Clinical Trials
Kennedy, Richard E. | Cutter, Gary R. | Wang, Guoqiao | Schneider, Lon S.

Protective Effect of Amyloid-β Peptides Against Herpes Simplex Virus-1 Infection in a Neuronal Cell Culture Model
Bourgade, Karine | Le Page, Aurélie | Bocti, Christian | Witkowski, Jacek M. | Dupuis, Gilles | Frost, Eric H. | Fülöp, Tamás

Association Between Serum Ceruloplasmin Specific Activity and Risk of Alzheimer’s Disease
Siotto, Mariacristina | Simonelli, Ilaria | Pasqualetti, Patrizio | Mariani, Stefania | Caprara, Deborah | Bucossi, Serena | Ventriglia, Mariacarla | Molinario, Rossana | Antenucci, Mirca | Rongioletti, Mauro | Rossini, Paolo Maria | Squitti, Rosanna

Effects of Hypertension and Anti-Hypertensive Treatment on Amyloid-β (Aβ) Plaque Load and Aβ-Synthesizing and Aβ-Degrading Enzymes in Frontal Cortex
Ashby, Emma L. | Miners, James S. | Kehoe , Patrick G. | Love, Seth

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate KineticsOpenly Available
Eketjäll, Susanna | Janson, Juliette | Kaspersson, Karin | Bogstedt, Anna | Jeppsson, Fredrik | Fälting, Johannad | Haeberlein, Samantha Budd | Kugler, Alan R. | Alexander, Robert C. | Cebers, Gvido

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Avoiding chemotherapy toxicities

Larry H. Bernstein, MD, FCAP, Curator



Nanoparticle ‘cluster bombs’ destroy cancer cells

New delivery method directly penetrates tumor cells, avoiding toxic side effects of cisplatin chemotherapy drug
The nanoparticles start out relatively large (100 nm) (large blue circle, upper left) to enable smooth transport into the tumor through leaky blood vessels. Then, in acidic conditions found close to tumors, the particles discharge “bomblets” (right, small blue circles) just 5 nm in size. Once inside tumor cells, a second chemical step activates the platinum-based drug cisplatin (bottom) to attack the cancer directly. (credit: Emory Health Sciences)

Scientists have devised a triple-stage stealth “cluster bomb” system for delivering the anti-cancer chemotherapy drug cisplatin, using nanoparticles designed to break up when they reach a tumor:

  1. The nanoparticles start out relatively large  — 100 nanometers wide — so that they can move through the bloodstream and smoothly transport into the tumor through leaky blood vessels.
  2. As they detect acidic conditions close to tumors, the nanoparticles discharge “bomblets” just 5 nanometers in size to penetrate tumor cells.
  3. Once inside tumor cells, the bomblets release the platinum-based cisplatin, which kills by crosslinking and damaging DNA.

Doctors have used cisplatin to fight several types of cancer for decades, but toxic side effects — to the kidneys, nerves and inner ear — have limited its effectiveness. But in research with three different mouse tumor models*, the researchers have now shown that their nanoparticles can enhance cisplatin drug accumulation in tumor tissues for several types of cancer.

Details of the research — by teams led by professor Jun Wang, PhD, at the University of Science and Technology of China and by professor Shuming Nie, PhD, in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory — were published this week in the journal PNAS.

* When mice bearing human pancreatic tumors were given the same doses of free cisplatin or cisplatin clothed in pH-sensitive nanoparticles, the level of platinum in tumor tissues was seven times higher with the nanoparticles. This suggests the possibility that nanoparticle delivery of a limited dose of cisplatin could restrain the toxic side effects during cancer treatment.

The researchers also showed that the nanoparticles were effective against a cisplatin-resistant lung cancer model and an invasive metastatic breast cancer model in mice. In the lung cancer model, a dose of free cisplatin yielded just 10 percent growth inhibition, while the same dose clothed in nanoparticles yielded 95 percent growth inhibition, the researchers report. In the metastatic breast cancer model, treating mice with cisplatin clothed in nanoparticles prolonged animal survival by weeks; 50 percent of the mice were surviving at 54 days with nanoparticles compared with 37 days for the same dose of free cisplatin.

Abstract of Stimuli-responsive clustered nanoparticles for improved tumor penetration and therapeutic efficacy

A principal goal of cancer nanomedicine is to deliver therapeutics effectively to cancer cells within solid tumors. However, there are a series of biological barriers that impede nanomedicine from reaching target cells. Here, we report a stimuli-responsive clustered nanoparticle to systematically overcome these multiple barriers by sequentially responding to the endogenous attributes of the tumor microenvironment. The smart polymeric clustered nanoparticle (iCluster) has an initial size of ∼100 nm, which is favorable for long blood circulation and high propensity of extravasation through tumor vascular fenestrations. Once iCluster accumulates at tumor sites, the intrinsic tumor extracellular acidity would trigger the discharge of platinum prodrug-conjugated poly(amidoamine) dendrimers (diameter ∼5 nm). Such a structural alteration greatly facilitates tumor penetration and cell internalization of the therapeutics. The internalized dendrimer prodrugs are further reduced intracellularly to release cisplatin to kill cancer cells. The superior in vivo antitumor activities of iCluster are validated in varying intractable tumor models including poorly permeable pancreatic cancer, drug-resistant cancer, and metastatic cancer, demonstrating its versatility and broad applicability.

The facts suggest that big pharma represents only a few companies in most fields of disease. They spend an enormous amount of money in lobbying congress and doctors to get them to do their bidding.They wouldn’t spend the money if they didn’t need to do so.The profit motive is central with patient well being only being practiced if it pays off.Cancer is a superb example, with new drugs being offered usually at astronomical prices in this country. Like wise the FDA is controlled by them and it is in their best interests to make the cost of developing new drugs outrageously expensive.Only big pharma can afford to get new drugs approved.
After the phase 3 trials are completed usually the documentation to ask for approval to market a drug is at least 100,000 pages long. The legal talent needed to compile such documents ( and this is only one of many documents produced in the process) is extremely expensive. The time taken for approval stretches into many years and then the drugs are often not approved.(only a small percentage are approved).
Antibiotics were one example of a group of drugs that really did cure many diseases. Big pharma found it didn’t pay to develop new antibiotics because the treatment was short and so successful that patients used the drugs only for a short time.
Over time, as Alexander Fleming forsaw, the bacteria would develop resistance, especially if they were extensively used indiscriminantly. Now many dangerous bacteria are resistant to many or all antibiotics and there is no treatment available. Since bacteria can pass this resistance to specific antibiotics to almost any species of bacteria, its only a matter of time before we will be back in the pre-antibiotic era.

“In the metastatic breast cancer model, treating mice with cisplatin clothed in nanoparticles prolonged animal survival by weeks; 50 percent of the mice were surviving at 54 days with nanoparticles compared with 37 days for the same dose of free cisplatin.”

I’m not so convinced after all. But this is perfectly in line with big pharma goals. Only an idiot would kill its main source of income.


It is almost impossible to set up a conspiracy against big pharma’s abusive practices.Every avenue their high priced lawyers can think of to stop budding conspiracies has been blocked by law where possible. One possible road might be to do research and development in other countries outside US legal juristiction, however most drugs without FDA approval can and are stopped at the border and confiscated even if as in Canada the same drug produced in the US is being manufactured in Canada.Almost certainly Cisplatin is under patent in the US and the patent holder has the right to refuse the use of the drug for any reason they want, including being used in this cluster bomb drug. The manufacturer is almost certainly making huge profits from selling Cisplatin and I doubt they want to see a cheap drug cure many cancers. I guess the only way to go is to try and turn to a country like India.A number of cancer drugs were being sold by US patent holders at wholesale prices that were to high for most Indians. The government of India refused to allow these companies to patent their medicines in India and forced them to license the drugs and much cheaper prices.Most US patents are not operative in India, they can produce US style insulin pumps at a fraction of our cost as they can in China and Vietnam or Mexico. It would be difficult to send these pumps to buyers in the US from India but by shipping them from another country, say Canada or Mexico most would make it past customs. As for Cancer treatment, India and china have some very fine trained biochemist and doctors, who could easily apply many of the immunological treatments against cancer. All arms of the immune system have been used to produce miracle treatments that have cured some patients that were on their death beds.The treatments can be tested carefully in these countries, and improved by any methods including some I have suggested.By advertising in the US to cancer patients that they can inexpensively have these working treatments cheaply as a medical tourist, it is only a matter of time before they will cure the disease wholesale and break the medical industrial complex down. As far as generics that are not being produced here, by setting up a non profit corporation that produces any and all drugs that come off patent as a goal, at the cheapest price less a reasonable markup for cost of manufacture etc. one by one they will end the abuse of not producing or overpricing generics.



Successively overcoming a series of biological barriers that cancer nanotherapeutics would encounter upon intravenous administration is required for achieving positive treatment outcomes. A hurdle to this goal is the inherently unfavorable tumor penetration of nanoparticles due to their relatively large sizes. We developed a stimuli-responsive clustered nanoparticle (iCluster) and justified that its adaptive alterations of physicochemical properties (e.g. size, zeta potential, and drug release rate) in accordance with the endogenous stimuli of the tumor microenvironment made possible the ultimate overcoming of these barriers, especially the bottleneck of tumor penetration. Results in varying intractable tumor models demonstrated significantly improved antitumor efficacy of iCluster than its control groups, demonstrating that overcoming these delivery barriers can be achieved by innovative nanoparticle design.


  1. Engineering of self-assembled nanoparticle platform for precisely controlled combination drug therapy.
    Nagesh Kolishetti et al., Proc Natl Acad Sci U S A, 2010
  2. Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety.
    Lin Zhu et al., Proc Natl Acad Sci U S A, 2013
  3. Protein-assisted self-assembly of multifunctional nanoparticles.
    Maxim P Nikitin et al., Proc Natl Acad Sci U S A, 2010
  4. Photoswitchable nanoparticles for in vivo cancer chemotherapy.
    Rong Tong et al., Proc Natl Acad Sci U S A, 2013
  5. Investigating the optimal size of anticancer nanomedicine.
    Li Tang et al., Proc Natl Acad Sci U S A, 2014 
  6. Nanoparticles seek and destroy glioblastoma in mice
    Sanford-Burnham Medical Research Institute,ScienceDaily, 2011
  7. Nanoparticle ‘alarm clock’ tested to awaken immune systems put to sleep by cancer
    Norris Cotton Cancer CenterDartmouth-Hitchcock Medical Center, ScienceDaily, 2014
  8. Injectable nanoparticle generator could radically transform metastatic cancer treatment
    Houston Methodist, ScienceDaily, 2016
  9. Introducing the multi-tasking nanoparticle
    UC Davis Comprehensive Cancer Center,ScienceDaily, 2014
  10. First-of-its-kind self-assembled nanoparticle for targeted and triggered thermo-chemotherapy
    Brigham and Women’s Hospital, ScienceDaily, 2012
 Researchers use optogenetic light to block tumor development
Uses light-triggered bioelectric current

Tufts University biologists have demonstrated (using a frog model*) for the first time that it is possible to prevent tumors from forming (and to normalize tumors after they have formed) by using optogenetics (light) to control bioelectrical signalling among cells.

Light/bioelectric control of tumors

Virtually all healthy cells maintain a more negative voltage in the cell interior compared with the cell exterior. But the opening and closing of ion channels in the cell membrane can cause the voltage to become more positive (depolarizing the cell) or more negative (polarizing the cell). That makes it possible to detect tumors by their abnormal bioelectrical signature before they are otherwise apparent.

The study was published online in an open-access paper in Oncotarget on March 16.

The use of light to control ion channels has been a ground-breaking tool in research on the nervous system and brain, but optogenetics had not yet been applied to cancer.

The researchers first injected  cells in Xenopus laevis (frog) embryos with RNA that encoded a mutant RAS oncogene known to cause cancer-like growths.

The researchers then used blue light to activate positively charged ion channels,which induced an electric current that caused the cells to go from a cancer-like depolarized state to a normal, more negative polarized state. The did the same with a green light-activated proton pump, Archaerhodopsin (Arch). Activation of both agents significantly lowered the incidence of tumor formation and also increased the frequency with which tumors regressed into normal tissue.

“These electrical properties are not merely byproducts of oncogenic processes. They actively regulate the deviations of cells from their normal anatomical roles towards tumor growth and metastatic spread,” said senior and corresponding author Michael Levin, Ph.D., who holds the Vannevar Bush chair in biology and directs the Center for Regenerative and Developmental Biology at Tufts School of Arts and Sciences.

“Discovering new ways to specifically control this bioelectrical signaling could be an important path towards new biomedical approaches to cancer. This provides proof of principle for a novel class of therapies which use light to override the action of oncogenic mutations,” said Levin. “Using light to specifically target tumors would avoid subjecting the whole body to toxic chemotherapy or similar reagents.”

This work was supported by the G. Harold and Leila Y. Mathers Charitable Foundation.

* Frogs are a good model for basic science research into cancer because tumors in frogs and mammals share many of the same characteristics. These include rapid cell division, tissue disorganization, increased vascular growth, invasiveness and cells that have an abnormally positive internal electric voltage.

Abstract of Use of genetically encoded, light-gated ion translocators to control tumorigenesis

It has long been known that the resting potential of tumor cells is depolarized relative to their normal counterparts. More recent work has provided evidence that resting potential is not just a readout of cell state: it regulates cell behavior as well. Thus, the ability to control resting potential in vivo would provide a powerful new tool for the study and treatment of tumors, a tool capable of revealing living-state physiological information impossible to obtain using molecular tools applied to isolated cell components. Here we describe the first use of optogenetics to manipulate ion-flux mediated regulation of membrane potential specifically to prevent and cause regression of oncogene-induced tumors. Injection of mutant-KRAS mRNA induces tumor-like structures with many documented similarities to tumors, in Xenopus tadpoles. We show that expression and activation of either ChR2D156A, a blue-light activated cation channel, or Arch, a green-light activated proton pump, both of which hyperpolarize cells, significantly lowers the incidence of KRAS tumor formation. Excitingly, we also demonstrate that activation of co-expressed light-activated ion translocators after tumor formation significantly increases the frequency with which the tumors regress in a process called normalization. These data demonstrate an optogenetic approach to dissect the biophysics of cancer. Moreover, they provide proof-of-principle for a novel class of interventions, directed at regulating cell state by targeting physiological regulators that can over-ride the presence of mutations.

A biosensor that’s 1 million times more sensitive

Aims at detecting cancers earlier, improving treatment and outcomes
A schematic representation of the miniaturized gold-aluminum oxide hyperbolic metamaterial (HMM) sensor device with a fluid flow channel, showing a scanning electron microscope (SEM) image [gray inset] of the 2D subwavelength gold diffraction grating on top of the hyperbolic metamaterials layers (scale bar, 2 µm) (credit: Kandammathe Valiyaveedu Sreekanth et al./Nature Materials
An optical sensor that’s 1 million times more sensitive than the current best available has been developed by Case Western Reserve University researchers. Based on nanostructured metamaterials, it can identify a single lightweight molecule in a highly dilute solution.The research goal is to provide oncologists a way to detect a single molecule of an enzyme produced by circulating cancer cells. That could allow doctors to diagnose and monitor patients with certain cancers far earlier than possible today.

“The prognosis of many cancers depends on the stage of the cancer at diagnosis,” said Giuseppe “Pino” Strangi, professor of physics at Case Western Reserve and research leader. “Very early, most circulating tumor cells express proteins of a very low molecular weight, less than 500 Daltons,” Strangi explained. “These proteins are usually too small and in too low a concentration to detect with current test methods, yielding false negative results.

“With this platform, we’ve detected proteins of 244 Daltons, which should enable doctors to detect cancers earlier — we don’t know how much earlier yet,” he said. “This biosensing platform may help to unlock the next era of initial cancer detection.”

The researchers believe the sensing technology will also be useful in diagnosing and monitoring other diseases.

A biological sieve

The nanosensor, which fits in the palm of a hand, acts like a biological sieve, capable of isolating a small protein molecule weighing less than 800 quadrillionths of a nanogram from an extremely dilute solution.

To make the device so sensitive, Strangi’s team faced two long-standing barriers: Light waves cannot detect objects smaller than their own physical dimensions (about 500 nanometers, depending on wavelength). And molecules in dilute solutions float in Brownian (random) motion and are unlikely to land on the sensor’s surface.

The solution was to use a microfluidic channel to restrict the molecules’ ability to float around and a plasmon-based metamaterial made of 16 nanostructured layers of reflective and conductive gold and transparent aluminum oxide, a dielectric, each 10s of atoms thick. Light directed onto and through the layers is concentrated into a very small volume much smaller than the wavelength of light.*

“It’s extremely sensitive,” Strangi said. “When a small molecule lands on the surface, it results in a large local modification, causing the light to shift.” Depending on the size of the molecule, the reflecting light shifts different amounts. The researchers hope to learn to identify specific biomarker and other molecules for different cancers by their light shifts.

To add specificity to the sensor, the team added a layer of trap molecules — molecules that bind specifically with the molecules they hunt. In tests, the researchers used two trap molecules to catch two different biomolecules: bovine serum albumin, with a molecular weight of 66,430 Daltons, and biotin, with a molecular weight of 244 Daltons. Each produced a signature light shift.

Other researchers have reported using plasmon-based biosensors to detect biotin in solutions at concentrations ranging from more than 100 micromoles per liter to 10 micromoles per liter. This device proved 1 million times more sensitive, finding and identifying biotin at a concentration of 10 picomoles per liter.

Testing and clinical use in process

Strangi’s lab is working with other oncologists worldwide to test the device and begin moving the sensor toward clinical use.

In Cleveland, Strangi and Nima Sharifi, MD, co-leader of the Genitourinary Cancer Program for the Case Comprehensive Cancer Center, have begun testing the sensor with proteins related to prostate cancers.

“For some cancers, such as colorectal and pancreatic cancer, early detection is essential,” said Sharifi, who is also the Kendrick Family Chair for Prostate Cancer Research at Cleveland Clinic. “High sensitivity detection of cancer-specific proteins in blood should enable detection of tumors when they are at an earlier disease stage.

“This new sensing technology may help us not only detect cancers, but what subset of cancer, what’s driving its growth and spread, and what it’s sensitive to,” he said. “The sensor, for example, may help us determine markers of aggressive prostate cancers, which require treatments, or indolent forms that don’t.”

The research is published online in the journal Nature Materials.

* The top gold layer is perforated with holes, creating a grating that diffuses light shone on the surface into two dimensions. The incoming light, which is several hundreds of nanometers in wavelength, appears to be confined and concentrated in a few nanometers at the interface between the gold and the dielectric layer.  As the light strikes the sensing area, it excites free electrons causing them to oscillate and generate a highly confined propagating surface wave, called a surface plasmon polariton. This propagating surface wave will in turn excite a bulk wave propagating across the sensing platform. The presence of the waves cause deep sharp dips in the spectrum of reflecting light. The combination and the interplay of surface plasmon and bulk plasmon waves are what make the sensor so sensitive. Strangi said. By exciting these waves through the eight bilayers of the metamaterial, they create remarkably sharp resonant modes. Extremely sharp and sensitive resonances can be used to detect smaller objects.

Abstract of Extreme sensitivity biosensing platform based on hyperbolic metamaterials

Optical sensor technology offers significant opportunities in the field of medical research and clinical diagnostics, particularly for the detection of small numbers of molecules in highly diluted solutions. Several methods have been developed for this purpose, including label-free plasmonic biosensors based on metamaterials. However, the detection of lower-molecular-weight (<500 Da) biomolecules in highly diluted solutions is still a challenging issue owing to their lower polarizability. In this context, we have developed a miniaturized plasmonic biosensor platform based on a hyperbolic metamaterial that can support highly confined bulk plasmon guided modes over a broad wavelength range from visible to near infrared. By exciting these modes using a grating-coupling technique, we achieved different extreme sensitivity modes with a maximum of 30,000 nm per refractive index unit (RIU) and a record figure of merit (FOM) of 590. We report the ability of the metamaterial platform to detect ultralow-molecular-weight (244 Da) biomolecules at picomolar concentrations using a standard affinity model streptavidin–biotin.

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