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Dr. Bryan Traynor and his team participated in a ground-breaking international study, identifying a causal gene mutation responsible for two dissimilar neurological diseases, ALS and FTD. As members of a worldwide consortium, his research team used next-generation sequencing to identify a large hexanucleotide repeat that disrupts the C9ORF72 gene located on chromosome 9. The mutation accounts for approximately 40% of all familial cases of ALS and FTD in European and North American populations, and also ~1% of Alzheimer’s disease cases. ALS, also known as Lou Gehrig’s disease, is a fatal neurodegenerative disorder that leads to rapidly progressive paralysis and respiratory failure. Frontotemporal dementia (FTD) is the most common form of dementia in the population under the age of 65.
This landmark discovery has impacted how these neurological disorders are diagnosed, investigated and perceived. It also provides a distinct therapeutic target for gene therapy efforts aimed at ameliorating these diseases.
Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious Depression
Reporter and writer: Larry H Bernstein, MD, FCAP
There has been an considerable success in the current state of expanding our knowledge in genomics and therapeutic targets in cancer (although clinical remission targets and relapse are a concern), cardiovascular disease, and infectious disease. Our knowledge of prenatal and perinatal events is still at an early stage. The neurology front is by no means unattended. Here there are two prominent drivers of progress –
among a complex sea of sequence-changes. I indicate some of the current status in this. However, as much as we have know, there is an incredible barrier to formulate working models because:
ligand binding between DNA short-sequences is not predictable over time
binding between proteins and DNA is still largely unknown
specific regulatory roles between nucleotide-sequences and histone proeins are still unclear
the relationship between intracellular as well as extracellular cations and the equilibria between cations and anions in intertitial fluid that bathes the cell and between organelles is virgin territory
Consequently, it is quite an accomplishment to have come as far as we have come, and yet, even with the huge compuational power at our disposal, there is insuficient data to unravel the complexity. This may be especially true in the pathway to understanding of neurological and behavioral disorders.
Broad Map of Brain
John Markoff reports in the Feb 18 front-page of New York Times (Project would construct a broad map of the brain) that the Obama administration envisions a decade-long effort to examine the workings of the human brain and construct a map, comparable to what the Human Genome Project did for genetics. It will be a collaboration between universities, the federal government, private foundations, and teams of scientists (neuro-, nano- and whoever else). The goal is to break through the barrier to understanding the brain’s billions of neurons and gain greater insight into
perception
actions
and consciousness.
Essentially, it holds great promise for understanding
Alzheimer’s disease and Parkinson’s, as well as finding therapies for a variety of mental illnesses. An open-ended question is whether it will also advance artificial intelligence research. It is termed the Brain Activity Map project. http://NYTimes/broad-map-of-brain/
Schizophrenia Genomics
Scientists Reveal Genomic Explanation for Schizophrenia
Two new studies, published in Schizophrenia Research and in Nature Genetics, propose hypotheses in a new mouse model of schizophrenia that demonstrates how gestational brain changes cause behavioural problems later in life.
The first study implicates
A fibroblast growth factor receptor protein, (FGFR1), targets diverse genes implicated in schizophrenia. The research demonstrates how defects in an important neurological pathway in early development
may be responsible for the onset of schizophrenia later in life.
Individuals with sporadic schizophrenia tend to carry more deleterious genetic changes than found in the general population, according to an exome sequencing study that appeared online in Nature Genetics yesterday. “The occurrence of de novo mutations may in part explain the high worldwide incidence of schizophrenia,” according to co-senior author Guy Rouleau, CHU Sainte-Justine Research Center of University of Montreal.
Researchers from Canada and France did exome sequencing on individuals from 14 parent-child trios, each comprised of an individual with schizophrenia and his or her unaffected parents. In the process, they found
15 de novo mutations in coding sequences from eight individuals with the psychiatric condition, including
four nonsense mutations predicted to abbreviate protein sequences.
“They surmise that [de novo mutations] may account for some of the heritability reported for schizophrenia. Recent exome sequencing studies involving parent-child trios have implicated de novo mutations in other brain-related conditions, including
autism spectrum disorder and
mental retardation.
To detect de novo genetic changes specific to schizophrenia, the team compared coding sequences from affected individuals with
the human reference genome, with
both of his or her parents, and
with 26 unrelated control individuals.
Of the 15 de-novo mutations verified by Sager sequencing,
11 were missense mutations predicted to alter the amino acid sequence of the resulting protein and
four were nonsense mutations predicted to truncate it.
Among the genes containing nonsense mutations were the zinc finger protein-coding gene ZNF480, the karyopherin alpha 1 gene KPNA1, the low-density lipoprotein receptor-related gene LRP1, and the ALS-like protein-coding gene ALS2CL.
The 15 mutations were found in coding sequences from eight of the individuals with schizophrenia,
hinting at a higher de novo mutation rate in individuals with sporadic schizophrenia than is predicted in the population overall.
This difference seems to be specific to exomes, and the researchers noted that
de novo mutation rates across the entire genome are likely comparable in those with or without schizophrenia.
They conclude that the enrichment of [de novo mutations] within the coding sequence of individuals with schizophrenia may underlie the pathogenesis of many of these individual. Most of the genes identified in this study have not been previously linked to schizophrenia, thereby providing new potential therapeutic targets.
The second study
identifies the Integrative Nuclear FGFR 1 Signaling (INFS) as a central intersection point for multiple pathways of
as many as 160 different genes believed to be involved in the disorder.
The lead author Dr. Michal Stachowiakthis (UB School of Medicine and Biomedical Sciences) suggests this is the first model that explains schizophrenia
from genes
to development
to brain structure and
finally to behaviour .
A key challenge has been that patients with schizophrenia exhibit mutations in different genes. It is possible to have 100 patients with schizophrenia and each one has a different genetic mutation that causes the disorder. The explanation is possibly because INFS integrates diverse neurological signals that control the development of embryonic stem cell and neural progenitor cells, and
“INFS functions like the conductor of an orchestra,” explains Stachowiak. “It doesn’t matter which musician is playing the wrong note,
it brings down the conductor and the whole orchestra.
With INFS, we propose that
when there is an alteration or mutation in a single schizophrenia-linked gene,
the INFS system that controls development of the whole brain becomes untuned.
Using embryonic stem cells, Stachowiak and colleagues at UB and other institutions found that
some of the genes implicated in schizophrenia bind the FGFR1 (fibroblast growth factor receptor) protein,
which in turn, has a cascading effect on the entire INFS.
“We believe that FGFR1 is the conductor that physically interacts with all genes that affect schizophrenia,” he says. “We think that schizophrenia occurs
when there is a malfunction in the transition from stem cell to neuron, particularly with dopamine neurons.”
The researchers tested their hypothesis by creating an FGFR1 mutation in mice, which produced the hallmarks of the human disease: altered brain anatomy,
behavioural impacts and
overloaded sensory processes.
The researchers would like to devise ways to arrest development of the disease before it presents fully in adolescence or adulthood. The UB work adds to existing evidence that nicotinic agonists, might help improve cognitive function in schizophrenics by acting on the INFS.
childhood-schizophrenia-symptoms (Photo credit: Life Mental Health)
English: Types of point mutations. With examples. (Photo credit: Wikipedia)
Studies of the familial Parkinson disease-related proteins PINK1 and Parkin have demonstrated that these factors promote the fragmentation and turnover of mitochondria following treatment of cultured cells with mitochondrial depolarizing agents. Whether PINK1 or Parkin influence mitochondrial quality control under normal physiological conditions in dopaminergic neurons, a principal cell type that degenerates in Parkinson disease, remains unclear. To address this matter, we developed a method to purify and characterize neural subtypes of interest from the adult Drosophila brain.
Using this method, we find that dopaminergic neurons from Drosophila parkin mutants accumulate enlarged, depolarized mitochondria, and that genetic perturbations that promote mitochondrial fragmentation and turnover rescue the mitochondrial depolarization and neurodegenerative phenotypes of parkin mutants. In contrast, cholinergic neurons from parkin mutants accumulate enlarged depolarized mitochondria to a lesser extent than dopaminergic neurons, suggesting that a higher rate of mitochondrial damage, or a deficiency in alternative mechanisms to repair or eliminate damaged mitochondria explains the selective vulnerability of dopaminergic neurons in Parkinson disease.
Our study validates key tenets of the model that PINK1 and Parkin promote the fragmentation and turnover of depolarized mitochondria in dopaminergic neurons. Moreover, our neural purification method provides a foundation to further explore the pathogenesis of Parkinson disease, and to address other neurobiological questions requiring the analysis of defined neural cell types.
Burmana JL, Yua S, Poole AC, Decala RB , Pallanck L. Analysis of neural subtypes reveals selective mitochondrial dysfunction in dopaminergic neurons from parkin mutants.
Parkinson’s disease is a common neurodegenerative disease in the elderly. To explore the specific role of autophagy and the ubiquitin-proteasome pathway in apoptosis,
a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate
pheochromocytoma (PC12) cell lines
transfected with human mutant (A30P) and wildtype (WT) -synuclein.
The apoptosis ratio was assessed by flow cytometry.
LC3, heat shock protein 70 (hsp70) and caspase-3 expression in cell culture were determined by Western blot.
The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy.
Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy.
The results of Western blots for caspase-3 expression were similar to those of flow cytometry;
hsp70 proteinwas significantly higher in the proteasome inhibitor group than in control, but
in the autophagy inhibitor and stimulator groups, hsp70was similar to control.
These findings show that
inhibition of the proteasome and autophagy promotes apoptosis, and
the macroautophagy stimulator rapamycin reduces the apoptosis ratio.
And inhibiting or stimulating autophagy has less impact on hsp70 than the proteasome pathway.
In conclusion,
either stimulation or inhibition of macroautophagy, has less impact on hsp70 than on the proteasome pathway.
rapamycindecreased apoptotic cells in A30P cellsindependent of caspase-3 activity.
Although several lines of evidence recently demonstrated crosstalk between autophagy and caspase-independent apoptosis, we could not confirm that
autophagy activation protects cells from caspase-independent cell death.
Undoubtedly, there are multiple connections between the apoptotic and autophagic processes. Inhibition of autophagy may
subvert the capacity of cells to remove
damaged organelles or to remove misfolded proteins, which
would favor apoptosis.
However, proteasome inhibition activated macroautophagy and accelerated apoptosis. A likely explanation is inhibition of the proteasome favors oxidative reactions that trigger apoptosis, presumably through
a direct effect on mitochondria, and
the absence of NADPH2 and ATP which may
deinhibit the activation of caspase-2 or MOMP.
Another possibility is that aggregated proteins induced by proteasome inhibition increase apoptosis.
Autosomal recessive loss-of-function mutations within the PARK2 genefunctionally inactivate the E3 ubiquitin ligase parkin, resulting
in neurodegeneration of catecholaminergic neurons and a familial form of Parkinson disease.
Current evidence suggests both
a mitochondrial function for parkin and
a neuroprotective role, which may in fact be interrelated.
The antiapoptotic effects of Parkin have been widely reported, and may involve
fundamental changes in the threshold for apoptotic cytochrome c release, but the substrate(s) involved in Parkin dependent protection had not been identified. This study demonstrates
the Parkin-dependent ubiquitination of endogenous Bax
comparing primary cultured neurons from WT and Parkin KO mice and
using multiple Parkin-overexpressing cell culture systems.
The direct ubiquitination of purified Bax was also observed in vitro following incubation with recombinant parkin.
Parkin prevented basal and apoptotic stress induced translocation of Bax to the mitochondria.
an engineered ubiquitination-resistant form of Bax retained its apoptotic function,
but Bax KO cells complemented with lysine-mutant Bax
did not manifest the antiapoptotic effects of Parkin that were observed in cells expressing WT Bax.
The conclusion is that Bax is the primary substrate responsible for the antiapoptotic effects of Parkin, and provides mechanistic insight into at least a subset of the mitochondrial effects of Parkin.
Two international studies published this week point to a link between Alzheimer’s disease and a rare gene mutation that affects the immune system’s inflammation response. The discovery supports an emerging theory about the role of the immune system in the development of Alzheimer’s disease. Both studies were published online this week in the New England Journal of Medicine, one led by John Hardy of University College London, and the other led by the Iceland-based global company deCode Genetics.
Alzheimer’s is a form of distressing brain-wasting disease that gradually robs people of their memories and their ability to lead independent lives. Its main characteristic is the build up of
protein tangles and
plaques inside and between brain cells, which eventually
disrupts their ability to communicate with each other.
Both teams conclude that a rare mutation in a gene called TREM2, which helps trigger immune system responses, raises the risk for developing Alzheimer’s disease. One study suggests it raises it three-fold, the other, four-fold. The UCL-led study included researchers from 44 institutions around the world and data on a total of 25,000 people.
After homing in on the TREM2 gene using new sequencing techniques, they carried out further sequencing that identified a set of
rare mutations that occurred more often in 1,092 Alzheimer’s disease patients than in a group of 1,107 healthy controls.
They evaluated the most common mutation, R47H, and confirmed that this variant of TREM2substantially increases the risk for Alzheimer’s disease. R47H mutation was present in 1.9 percent of the Alzheimer’s patients and in only 0.37 percent of the controls. The researchers on the study led by deCode Genetics indicate that this strong effect is on a par with that of the well-established gene variant known as APOE4. Not all people who have the R47H variant will develop Alzheimer’s and in those who do, other genes and environmental factors will also play a role — but like APOE 4 it does substantially increase risk,” Carrasquillo explains.
The study led by deCode Genetics involved collaborators from Iceland, Holland, Germany and the US, not only found a strong link between the R47H variantand Alzheimer’s disease, but the variant also
predicts poorer cognitive function in older people without Alzheimer’s.
In a statement, lead author Kari Stefánsson, CEO and co-founder of deCODE Genetics says:
The discovery of variant TREM2 is important because
it confers high risk for Alzheimer’s and
because the gene’s normal biological function has been shown to reduce immune response
He surmises that the combined factors make TREM2an attractive target for drug development.
Using deCode’s genome sequencing and genotyping technology, Stefánsson and colleagues identified
approximately 41 million markers, including 191,777 functional variants, from
2,261 Icelandic samples.
They further analyzed these variants against the genomes of
3,550 people with Alzheimer’s disease and
a control group of over-85s who did not have a diagnosis of Alzheimer’s.
This led to them finding the TREM2 variant, and to make sure this was not just a feature of Icelandic people,
they replicated the findings against other control populations in the United States, Germany, the Netherlands and Norway.
Stefánsson says that the results were enabled by having
sophisticated research tools,
access to expanded and high quality genomic data sets, and
investigators with profound analytic skills,
Researching into genetic causes of disease can, thereby, be carried out using an approach that combines sequence data and biological knowledge to find new drug targets.
R47H Variant of TREM2 and Immune Response
Preclinical studies have found that
TREM2 is important for clearing away cell debris and amyloid protein, the protein that is associated with the brain plaques
that are characteristic of Alzheimer’s disease.
The gene helps control the
inflammation response associated with Alzheimer’s and cognitive decline.
Rosa Rademakers, a co-author in the UCL-led study, runs a lab at the Mayo Clinic in Florida that helped to pinpoint the R47H variant of TREM2. Other studies also link the immune system to Alzheimer’s disease, but
studies are needed to establish that R47H acts by altering immune function.
EPIGENETICS, HISTONE PROTEINS, AND ALZHEIMER’S DISEASE
12/10/12 · Emily Humphreys
Epigenetic effects were first described by Conrad Waddington in 1942 as phenotypic changes resulting from an organism interacting with its environment.1 Today, epigenetics is
heritable effects in gene expression that are
not based on the genetic sequence.
One known epigenetic mechanism includes posttranslational modifications of histones that are
found in the nuclei of nearly all eukaryotes and
function to package DNA into nucleosomes.
Histone proteins can be heavily decorated with posttranslational modifications (PTMs), such as
acetyl-,
methyl-, and
phosphoryl- groups at distinct amino acid residues.
These modifications are mainly
located in the N-terminal tails of the histone and
protrude from the core nucleosome structure.
Gene regulation, and the downstream epigenetic effects, can also
depend on the cis or trans orientation of the PTMs.2
One PTM, acetylation, is an important determinant of cell replication, differentiation, and death.3 Zhang, et al. investigated the acetylation of histone proteins in Alzheimer’s disease (AD) pathology found in postmortem human brain tissue compared to neurological controls. To study histone acetylation,
histones were isolated from frozen temporal lobe samples of patients with advanced AD.
Histones were quantified using Selected-reaction-monitoring (SRM)-based targeted proteomics, an LC-MS/MS-based technique demonstrated by the Zhang lab.4 Histones were also analyzed using western blot analysis and LC-MS/MS-TMT (tandem-mass-tagging) quantitative proteomics. The results of these three experimental strategies agreed, further validating the specificity and sensitivity of the targeted proteomics methods. Histone acetylation was reduced throughout in the AD temporal lobe compared to matched controls.
the histone H3 K18/K23 acetylation was significantly reduced.
Alzheimer’s disease and aging have also been associated with loss of histone acetylation in mouse model studies.5 In addition, Francis et al. found
cognitively impaired mice had a 50% reduced H4 acetylation in APP/PS1 mice than wild-type littermates.6
In mice, histone deacetylase inhibitors heve restored histone acetylation and improved memory in mice with age-related impairments or in models for other neurodegenerative diseases.7
Further studies of histone acetylation in AD could lead to target therapies in the disease pathology of neurodegenerative diseases, and
increase our understanding of how epigenetic mechanisms, such as histone acetylation, alter gene regulation.
2. Sidoli, S., Cheng, L., and Jensen O.N. (2012) ‘Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry‘, Journal of Proteomics, 75 (12), (pp. 3419-3433)
3. Zhang. K., et al. (2012) ‘Targeted proteomics for quantification of histone acetylation in Alzheimer’s disease‘, Proteomics, 12 (8), (pp. 1261-1268)
4. Darwanto, A., et al., (2010) ‘A modified “cross-talk” between histone H2B Lys-120 ubiquitination and H3 Lys-K79 methylation‘, The Journal of Biological Chemistry, 285 (28), (pp. 21868-21876)
5. Govindarajan, N., et al. (2011) ‘Sodium butyrate improves memory function in an Alzheimer’s disease model when administered at an advanced stage of disease progression‘, Journal of Alzheimer’s Disease, 26 (1), (pp.187-197)
6. Francis, Y.I., et al., (2009) ‘Dysregulation of histone acetylation in the APP/PS1 mouse model of Alzheimer’s disease‘, Journal of Alzheimer’s Disease, 18 (1), (pp. 131-139)
7. Kilgore, M., et al., (2010) ‘Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of Alzheimer’s disease‘, Neuropsychopharmacology, 35 (4), (pp. 870-880)
November 10, 2012, on page A1 in the U.S. edition of The Wall Street Journal
After years of effort, researcher Dr. Claude Wischikis awaiting the results of new clinical trials that will test his theory on the cause of Alzheimer’s.
Dr. Wischik, an Australian in his early 30s in the 1980s, was attempting to answer a riddle: What causes Alzheimer’s disease? He needed to examine brain tissue from Alzheimer’s patients soon after death, which required getting family approvals and enlisting mortuary technicians to extract the brains. He collected more than 300 over about a dozen years.
Alzheimer’s researcher Claude Wischik had a view that a brain protein called tau-not plaque is largely responsible. WSJ’s Shirley Wang spoke with Dr. Wischik about his work on a new drug to treat the devastating disease.
The 63-year-old researcher believes that a protein called tau
forms twisted fibers known as tangles inside the brain cells of Alzheimer’s patients and is largely responsible for driving the disease.
For 20 years, billions of dollars of pharmaceutical investment has placed chief blame on a different protein, beta amyloid, which
forms sticky plaques in the brains of sufferers.
A string of experimental drugs designed to attack beta amyloid have failed recently in clinical trials.
Wherefore Tau thy go?
Dr. Wischik, who now lives in Scotland, sees this as tau’s big moment. The company he co-founded 10 years ago, TauRx Pharmaceuticals Ltd., has developed an experimental Alzheimer’s drug that it will begin testing in the coming weeks in two large clinical trials. Other companies are also investing in tau research. Roche Holding bought the rights to a type of experimental tau drug from Switzerland’s closely held AC Immune SA.
Wischik is a scientist who has struggled against a prevailing orthodoxy. In 1854, British doctor John Snow traced a cholera outbreak in London to a contaminated water supply, but his discovery was rejected. A very infamous example is the discovery of the cause of child-bed fever in Rokitanski’s University of Vienna by Ignaz Semmelweis. In 1982, two Australian scientists declared that bacteria (H. pylori) caused peptic ulcers, later to be awarded the 2005 Nobel Prize in medicine for their discovery.
Dr. Wischik says he and other tau-focused scientists have been shouted down over the years by what he calls the “amyloid orthodoxy.” But Dr. Wischik has been hampered by inconclusive research. A small clinical trial of TauRx’s drug in 2008 produced mixed, results. Of course, influential scientists still think that beta amyloid plays a central role. Although Roche is investing in tau, Richard Scheller, head of drug research at Roche’s biotech unit, Genentech, says the company still has a strong interest in beta amyloid (hedging the bet). He thinks amyloid drugs may have better results if testing on Alzheimer’s patients occurs much earlier in the disease to prove effective; Roche recently announced plans to conduct such a trial. Simply put -“Drugs tied to conventional theories on Alzheimer’s causes haven’t so far been effective.” Scientists Dr. Wischik accuses of wrongly fixating on beta amyloid argue that the evidence for pursuing amyloid is strong. One view expressed is that drugs to attack both beta amyloid and tau will be necessary.
Alzheimer’s disease is the leading cause of dementia in the elderly, and according to the World Health Organization, the cost of caring for dementia sufferers totals about $600 billion each year world-wide. The disease was first identified in 1906 by German physician Alois Alzheimer, who found in the brain of a deceased woman who had suffered from dementia the plaques and tangles that riddled the tissue. In the 1960s, Dr. Martin Roth and colleagues showed that
the degree of clinical dementia was worse for patients with more tangles in the brain.
In the 1980s, Dr. Wischik joined Dr. Roth’s research group at Cambridge University as a Ph.D student, and was quickly assigned the task of
determining what tangles were made of, which launched his brain-collecting mission, and years of examining tissue.
Finally, in 1988, he and colleagues at Cambridge published a paper demonstrating for the first time that
the tangles first observed by Alzheimer were made at least in part of the protein tau, which was supported by later research.
Like all of the body’s proteins, tau has a normal, helpful function—working inside neurons to help
stabilize the fibers that connect nerve cells.
When it misfires, tau clumps together to form harmful tangles that kill brain cells.
Dr. Wischik’s discovery was important news in the Alzheimer’s field:
identifying the makeup of tangles made it possible to start developing ways to stop their formation. But by the early 1990s, tau was overtaken by another protein: beta amyloid.
Signs of Decline
Several pieces of evidence convinced an influential group of scientists that beta amyloidwas the primary cause of Alzheimer’s.
the discovery of several genetic mutations that all but guaranteed a person would develop a hereditary type of the disease.
these appeared to increase the production or accumulation of beta amyloid in the brain,
which led scientists to believe that amyloid deposits were the main cause of the disease.
Athena Neurosciences, a biotech company whose founders included Harvard’s Dr. Selkoe, focused in earnest on developing drugs to attack amyloid. Meanwhile, tau researchers say they found it hard to get research funding or to publish papers in medical journals. It became difficult to have a good publication on tau, because the amyloid cascade was like a dogma. It became the case that if you were not working in the amyloid field you were not working on Alzheimer’s disease. Dr. Wischik and his colleagues fought to keep funding from the UK’s Medical Research Council for the repository of brain tissue they maintained at Cambridge, he says. The brain bank became an important tool. In the early 1990s, Dr. Wischik and his colleagues compared the postmortem brains of Alzheimer’s sufferers against those of people who had died without dementia, to see how their levels of amyloid and taudiffered. They found that both healthy brains and Alzheimer’s brains could be filled with amyloid plaque, but only Alzheimer’s brains contained aggregated tau.
as the levels of aggregated tau in a brain increased, so did the severity of dementia.
In the mid-1990s, Dr. Wischik discovered that
a drug sometimes used to treat psychosis dissolved tangles
Nevertheless, American and British venture capitalists wanted to invest in amyloid projects, not tau.
By 2002, Dr. Wischik scraped together about $5 million from Asian investors with the help of a Singaporean physician who was the father of a classmate of Dr. Wischik’s son in Cambridge. TauRx is based in Singapore but conducts most of its research in Aberdeen, Scotland. As his tau effort launched, early tests of drugs designed to attack amyloid plaques were disappointing. To better understand these results, a team of British scientists largely unaffiliated with Athena or the failed clinical trial decided to examine the brains of patients who had participated in the study. They waited for the patients to die, and then, after probing the brains, concluded that
the vaccine had indeed cleared amyloid plaque but hadn’t prevented further neurodegeneration.
Peter Davies, an Alzheimer’s researcher at the Feinstein Institute for Medical Research in Manhasset, NY, recalls hearing a researcher at a conference in the early 2000s concede that his amyloid research results “don’t fit the hypothesis, but we’ll continue until they do! “I just sat there with my mouth open,” he recalls.
In 2004, TauRx began a clinical trial of its drug, called methylene blue, in 332 Alzheimer’s patients. Around the same time, a drug maker called Elan Corp., which had bought Athena Neurosciences, began a trial of an amyloid-targeted drug called bapineuzumab in 234 patients. A key moment came in 2008, when Dr. Wischik and Elan presented results of their studies at an Alzheimer’s conference in Chicago. The Elan drug
failed to improve cognition any better than a placebo pill, causing Elan shares to plummet by more than 60% over the next few days.
The TauRx results Dr. Wischik presented were more positive, though not unequivocal. The study showed that,
after 50 weeks of treatment, Alzheimer’s patients taking a placebo had fallen 7.8 points on a test of cognitive function,
while people taking 60 mg of TauRx’s drug three times a day had fallen one point—
translating into an 87% reduction in the rate of decline for people taking the TauRx drug.
But TauRx didn’t publish a full set of data from the trial, causing some skepticism among researchers. (Dr. Wischik says it didn’t to protect the company’s commercial interests). What’s more,
a higher, 100-mg dose of the drug didn’t produce the same positive effects in patients;
Dr. Wischik blames this on the way the 100-mg dose was formulated, and says the company is testing a tweaked version of the drug in its new clinical trials, which will begin enrolling patients late this year.
This summer, a trio of companies that now own the rights to bapineuzumab—Elan, Pfizer and Johnson & Johnson—
scrapped development of the drug after it failed to work in two large clinical trials.
Then in August, Eli Lilly & Co. said its experimental medicine targeting beta amyloid,
solanezumab, failed to slow the loss of memory or basic skills like bathing and dressing in two trials
involving 2,050 patients with mild or moderate Alzheimer’s.
Lilly has disclosed that in one of the trials, when moderate patients were stripped away,
the drug slowed cognitive decline only in patients with mild forms of the disease.
Still fervent believers assert that beta amyloid needs to be attacked very early in the disease cycle—
perhaps before symptoms begin.
This spring, the U.S. government said it would help fund a $100 million trial of Roche’s amyloid-targeted drug, crenezumab, in 300 people
who are genetically predisposed to develop early-onset Alzheimer’s but who don’t yet have symptoms.
This trial should help provide a “definitive” answer about the theory.
Scientists and investors are giving more attention to tau. Roche this year said it would pay Switzerland’s AC Immune an undisclosed upfront fee for the rights to a new type of tau-targeted drug, and up to CHF400 million in additional payments if any drugs make it to market.
Dr. Buee, the longtime tau researcher in France, says Johnson & Johnson asked him to provide advice on tau last year, and that he’s currently discussing a tau research contract with a big pharmaceutical company. (A Johnson & Johnson spokeswoman says the company invited Dr. Buee and other scientists to a meeting to discuss a range of approaches to fighting Alzheimer’s.)
With its new clinical trial program under way, TauRx is the first company to test a tau-targeted drug against Alzheimer’s in a large human study, known in the industry as a phase 3 trial. Dr. Wischik
During autophagy, cytosol, protein aggregates, and organelles
are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components.
In all eukaryotes this pathway is important for
adaptation to stress conditions such as nutrient deprivation, as well as
to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures.
Starvation-induced autophagy has been viewed as a nonselective transport pathway; but recent studies have revealed that
autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles.
In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy:
ribophagy, the specific degradation of ribosomes, and
reticulophagy, the selective elimination of portions of the ER.
Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show
that macroautophagy requires the Alzheimer’s disease (AD)-related protein presenilin-1 (PS1).
In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1,
substrate proteolysis and autophagosome clearance during macroautophagy are prevented
as a result of a selective impairment of autolysosome acidification and cathepsin activation.
These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit,
essential for its efficient ER-to-lysosome delivery,
requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the sec61alpha/ oligosaccharyltransferase complex.
PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.
Transposons have been barging into genomes and crossing species boundaries throughout evolution. Rapidly evolving bacterial species often use them to transmit antibiotic resistance to one another. Nearly half of the DNA in the human genome consists of transposons, and the percentage can potentially creep upward with every generation. That’s because nearly 20 percent of transposons are capable of replicating in a way that is unconstrained by the normal rules of DNA replication during cell division ― although through generations over time, most have become inactivated and no longer pose a threat.
While humans are riddled with transposons, compared to some organisms, they’ve gotten off easy, according to Madhani, a professor of biochemistry and biophysics at UCSF. The water lily’s genome is 99 percent derived from transposons. The lowly salamander has about the same number of genes as humans, but in some species the genome is nearly 40 times bigger, due to all the inserted, replicating transposons.
The scientists’ discovery of SCANR and how it targets transposons in the yeast Cryptococcus neoformans builds upon the Nobel-Prize-winning discovery of jumping genes by maize geneticist Barbara McClintock, and the Nobel-prize-winning discovery by molecular biologists Richard Roberts and Phillip Sharp that parts of a single gene may be separated along chromosomes by intervening bits of DNA, called introns. Introns are transcribed into RNA from DNA but then are spliced out of the instructions for building proteins.
In the current study, the researchers discovered that the cell’s splicing machinery stalls when it gets to transposon introns. SCANR recognizes this glitch and
prevents transposon replication by
triggering the production of “small interfering RNA” molecules, which
neutralize the transposon RNA.
The earlier discovery by biologists Andrew Fire and Craig Mello of the phenomenon of RNA interference, a feature of this newly identified transposon targeting, also led to a Nobel Prize. “Scientists might find that many of the peculiar ways in which genes are expressed differently in higher organisms are, like
intron splicing in the case of SCANR, useful
in distinguishing and defending ‘self’ genes from ‘non-self’ genes,” Madhani said.
Researchers at UCSF ( Phillip Dumesic, an MD/PhD student and first author of the study, graduate students Prashanthi Natarajan and Benjamin Schiller, and postdoctoral fellow Changbin Chen, PhD.) and collaborators at the Whitehead Institute of Medical Research in Cambridge, Mass., and from the Scripps Research Institute in La Jolla, Calif., contributed to the research.
Researchers Discover Gene Invaders Are Stymied by a Cell’s Genome Defense
If unrestrained, transposons replicate and insert themselves randomly throughout the genome.
San Francisco, CA (Scicasts) – Gene wars rage inside our cells, with invading DNA regularly threatening to subvert our human blueprint. Now, building on Nobel-Prize-winning findings, UC San Francisco researchers have discovered a molecular machine that helps protect a cell’s genes against these DNA interlopers.
The machine, named SCANR, recognizes and targets foreign DNA. The UCSF team identified it in yeast, but comparable mechanisms might also be found in humans. The targets of SCANR are
small stretches of DNA called transposons, a name that conjures images of alien scourges.
But transposons are real, and to some newborns, life threatening. Found inside the genomes
of organisms as simple as bacteria and
as complex as humans,
they are in a way alien ― at some point,
each was imported into its host’s genome from another species.
Unlike an organism’s native genes, which are reproduced a single time during cell division, transposons ― also called jumping genes ― replicate multiple times, and
insert themselves at random places within the DNA of the host cell.
When transposons insert themselves in the middle of an important gene, they may cause malfunction, disease or birth defects.
But just as the immune system has ways of distinguishing what is part of the body and what is foreign and does not belong, researchers led by UCSF’s Dr. Hiten Madhani, discovered in
SCANR a novel way through which the genetic machinery within a cell’s nucleus recognizes and targets transposons.
“We’ve known that only a fraction of human-inherited diseases are caused by these mobile genetic elements,” Madhani said. “Now we’ve found that cells use a step in gene expression to distinguish ‘self’ from ‘non-self’ and to halt the spread of transposons.” The study was published online Feb. 13 in the journal Cell (http://www.cell.com/abstract/S0092-8674%2813%2900138-4).
Epigenetics of brain and brawn
Study Shows Epigenetics Shapes Fate of Brain vs. Brawn Castes in Carpenter Ants
Philadelphia, PA (Scicasts) – The recently published genome sequences of seven well-studied ant species are opening up new vistas for biology and medicine. A detailed look at molecular mechanisms that underlie the complex behavioural differences in two worker castes in the Florida carpenter ant, Camponotus floridanus, has revealed a link to epigenetics. This is the study of how the expression or suppression of particular genes by chemical modifications affects an organism’s
physical characteristics,
development, and
behaviour.
Epigenetic processes not only play a significant role in many diseases, but are also involved in longevity and aging. Interdisciplinary research teams led by Dr. Shelley Berger, from the Perelman School of Medicine at the University of Pennsylvania, in collaboration with teams led by Danny Reinberg from New York University and Juergen Liebig from Arizona State University, describe their work in Genome Research. The group found that epigenetic regulation is key to
distinguishing one caste, the “majors”, as brawny Amazons of the carpenter ant colony,
compared to the “minors”, their smaller, brainier sisters.
These two castes have the same genes, but strikingly distinct behaviours and shape.
Ants, as well as termites and some bees and wasps, are eusocial species that organize themselves into rigid caste-based societies, or colonies, in which only one queen and a small contingent of male ants are usually fertile and reproduce. The rest of a colony is composed of functionally sterile females that are divided into worker castes that perform specialized roles such as
foragers,
soldiers, and
caretakers.
In Camponotus floridanus, there are two worker castes that are physically and behaviourally different, yet genetically very similar. “For all intents and purposes, those two castes are identical when it comes to their gene sequences,” notes senior author Berger, professor of Cell and Developmental Biology. “The two castes are a perfect situation to understand
how epigenetics,
how regulation ‘above’ genes,
plays a role in establishing these dramatic differences in a whole organism.”
To understand how caste differences arise, the team examined the role of modifications of histones throughout the genome. They produced the first genome-wide epigenetic maps of genome structure in a social insect. Histones can be altered by the addition of small chemical groups, which affect the expression of genes. Therefore, specific histone modifications can create dramatic differences between genetically similar individuals, such as the physical and behavioural differences between ant castes. “These chemical modifications of histones alter how compact the genome is in a certain region,” Simola explains. “Certain modifications allow DNA to open up more, and some of them to close DNA more. This, in turn, affects how genes get expressed, or turned on, to make proteins.
In examining several different histone modifications, the team found a number of distinct differences between the major and minor castes. Simola states that the most notable modification,
discriminates the two castes from each other and
correlates well with the expression levels of different genes between the castes.
And if you look at which genes are being expressed between these two castes, these genes correspond very nicely to the brainy versus brawny idea. In the majors we find that genes that are involved in muscle development are expressed at a higher level, whereas in the minors, many genes involved in brain development and neurotransmission are expressed at a higher level.”
These changes in histone modifications between ant castes are likely caused by a regulator gene, called CBP, that has “already been implicated in aspects of learning and behaviour by genetic studies in mice and in certain human diseases,” Berger says. “The idea is that the same CBP regulator and histone modification are involved in a learned behaviour in ants – foraging – mainly in the brainy minor caste, to establish a pattern of gene regulation that leads to neuronal patterning for figuring out where food is and being able to bring the food back to the nest.” Simola notes that “we know from mouse studies that if you inactivate or delete the CBP regulator, it actually leads to significant learning deficits in addition to craniofacial muscular malformations. So from mammalian studies, it’s clear this is an important protein involved in learning and memory.”
The research team is looking ahead to expand the work by manipulating the expression of the CBP regulator in ants to observe effects on caste development and behaviour. Berger observes that all of the genes known to be major epigenetic regulators in mammals are conserved in ants, which makes them a good model for studying behaviour and longevity.
Research Reveals Mechanism of Epigenetic Reprogramming
Cambridge, UK (Scicasts) – New research reveals a potential way for how parents’ experiences could be passed to their offspring’s genes.
Epigenetics is a system that turns our genes on and off. The process works by chemical tags, known as epigenetic marks, attaching to DNA and telling a cell to either use or ignore a particular gene. The most common epigenetic mark is a methyl group.
When these groups fasten to DNA through a process called methylation
they block the attachment of proteins which normally turn the genes on.
As a result, the gene is turned off.
Scientists have witnessed epigenetic inheritance, the observation that offspring may inherit altered traits due to their parents’ past experiences. For example, historical incidences of famine have resulted in health effects on the children and grandchildren of individuals who had restricted diets,
possibly because of inheritance of altered epigenetic marks caused by a restricted diet.
However, it is thought that between each generation
the epigenetic marks are erased in cells called primordial gene cells (PGC), the precursors to sperm and eggs.
This ‘reprogramming’ allows all genes to be read afresh for each new person – leaving scientists to question how epigenetic inheritance could occur.
The new Cambridge study initially discovered how the DNA methylation marks are erased in PGCs. The methylation marks are converted to hydroxymethylation which is then
progressively diluted out as the cells divide.
This process turns out to be remarkably efficient and seems to reset the genes for each new generation.
The researchers, also found that some rare methylation can ‘escape’ the reprogramming process and can thus be passed on to offspring – revealing how epigenetic inheritance could occur. This is important because aberrant methylation could accumulate at genes during a lifetime in response to environmental factors, such as chemical exposure or nutrition, and can cause abnormal use of genes, leading to disease. If these marks are then inherited by offspring, their genes could also be affected. The research demonstrates how genes could retain some memory of their past experiences, indicating that the idea that epigenetic information is erased between generations – should be reassessed. The precursors to sperm and eggs are very effective in erasing most methylation marks, but they are fallible and at a low frequency may allow some epigenetic information to be transmitted to subsequent generations.
Professor Azim Surani from the University of Cambridge, principal investigator of the research, said: “The new study has the potential to be exploited in two distinct ways.
how to erase aberrant epigenetic marks that may underlie some diseases in adults.
address whether germ cells can acquire new epigenetic marks through environmental or dietary influences on parents that may evade erasure and be transmitted to subsequent generations
The research was published 25 January, in the journal Science. Story adapted from the University of Cambridge.
Study Suggests Expanding the Genetic Alphabet May Be Easier than Previously Thought
Featured In: Academia News | Genomics
Monday, June 4, 2012
A new study led by scientists at The Scripps Research Institute suggests that the replication process for DNA—the genetic instructions for living organisms that is composed of four bases (C, G, A and T)—is more open to unnatural letters than had previously been thought. An expanded “DNA alphabet” could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications, from precise molecular probes and nanomachines to useful new life forms.
The new study, which appears in the June 3, 2012 issue of Nature Chemical Biology, solves the mystery of how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases.
“We now know that the efficient replication of our unnatural base pair isn’t a fluke, and also that the replication process is more flexible than had been assumed,” said Floyd E. Romesberg, associate professor at Scripps Research, principal developer of the new DNA bases, and a senior author of the new study. The Romesberg laboratory collaborated on the new study with the laboratory of co-senior author Andreas Marx at the University of Konstanz in Germany, and the laboratory of Tammy J. Dwyer at the University of San Diego.
Adding to the DNA Alphabet
Romesberg and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).
The following year, Romesberg and colleagues showed that NaM and 5SICS could be efficiently transcribed into RNA in the lab dish. But these bases’ success in mimicking the functionality of natural bases was a bit mysterious. They had been found simply by screening thousands of synthetic nucleotide-like molecules for the ones that were replicated most efficiently. And it had been clear immediately that their chemical structures lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication‑—a process in which a large enzyme, DNA polymerase, moves along a single, unwrapped DNA strand and stitches together the opposing strand, one complementary base at a time.
An early structural study of a very similar base pair in double-helix DNA added to Romesberg’s concerns. The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, “intercalated” fashion. “Their pairing resembles a ‘mispair,’ such as two identical bases together, which normally wouldn’t be recognized as a valid base pair by the DNA polymerase,” said Denis Malyshev, a graduate student in Romesberg’s lab who was lead author along with Karin Betz of Marx’s lab.
Yet in test after test, the NaM-5SICS pair was efficiently replicable. “We wondered whether we were somehow tricking the DNA polymerase into recognizing it,” said Romesberg. “I didn’t want to pursue the development of applications until we had a clearer picture of what was going on during replication.”
Edge to Edge
To get that clearer picture, Romesberg and his lab turned to Dwyer’s and Marx’s laboratories, which have expertise in finding the atomic structures of DNA in complex with DNA polymerase. Their structural data showed plainly that the NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, “Watson-Crick” positioning when gripped by the polymerase during the crucial moments of DNA replication.
“The DNA polymerase apparently induces this unnatural base pair to form a structure that’s virtually indistinguishable from that of a natural base pair,” said Malyshev.
NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by “hydrophobic” forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium. “It’s very possible that these hydrophobic forces have characteristics that enable the flexibility and thus the replicability of the NaM-5SICS base pair,” said Romesberg. “Certainly if their aberrant structure in the double helix were held together by more rigid covalent bonds, they wouldn’t have been able to pop into the correct structure during DNA replication.”
An Arbitrary Choice?
The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could some day be used to extend the DNA alphabet. It also hints that Evolution’s choice of the existing four-letter DNA alphabet—on this planet—may have been somewhat arbitrary. “It seems that life could have been based on many other genetic systems,” said Romesberg.
He and his laboratory colleagues are now trying to optimize the basic functionality of NaM and 5SICS, and to show that these new bases can work alongside natural bases in the DNA of a living cell.
“If we can get this new base pair to replicate with high efficiency and fidelity in vivo, we’ll have a semi-synthetic organism,” Romesberg said. “The things that one could do with that are pretty mind blowing.”
The other contributors to the paper, “KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry,” are Thomas Lavergne of the Romesberg lab, Wolfram Welte and Kay Diederichs of the Marx lab, and Phillip Ordoukhanian of the Center for Protein and Nucleic Acid Research at The Scripps Research Institute.
Studies of the familial Parkinson disease-related proteins PINK1 and Parkin have demonstrated that these factors promote the fragmentation and turnover of mitochondria following treatment of cultured cells with mitochondrial depolarizing agents. Whether PINK1 or Parkin influence mitochondrial quality control under normal physiological conditions in dopaminergic neurons, a principal cell type that degenerates in Parkinson disease, remains unclear. To address this matter, we developed a method to purify and characterize neural subtypes of interest from the adult Drosophila brain.
Using this method, we find that dopaminergic neurons from Drosophila parkin mutants accumulate enlarged, depolarized mitochondria, and that genetic perturbations that promote mitochondrial fragmentation and turnover rescue the mitochondrial depolarization and neurodegenerative phenotypes of parkin mutants. In contrast, cholinergic neurons from parkin mutants accumulate enlarged depolarized mitochondria to a lesser extent than dopaminergic neurons, suggesting that a higher rate of mitochondrial damage, or a deficiency in alternative mechanisms to repair or eliminate damaged mitochondria explains the selective vulnerability of dopaminergic neurons in Parkinson disease.
Our study validates key tenets of the model that PINK1 and Parkin promote the fragmentation and turnover of depolarized mitochondria in dopaminergic neurons. Moreover, our neural purification method provides a foundation to further explore the pathogenesis of Parkinson disease, and to address other neurobiological questions requiring the analysis of defined neural cell types.
Burmana JL, Yua S, Poole AC, Decala RB , Pallanck L. Analysis of neural subtypes reveals selective mitochondrial dysfunction in dopaminergic neurons from parkin mutants.
Autophagy in Parkinson’s Disease.
Parkinson’s disease is a common neurodegenerative disease in the elderly. To explore the specific role of autophagy and the ubiquitin-proteasome pathway in apoptosis, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P) and wildtype (WT) -synuclein.
The apoptosis ratio was assessed by flow cytometry. LC3, heat shock protein 70 (hsp70) and caspase-3 expression in cell culture were determined by Western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy. The results of Western blots for caspase-3 expression were similar to those of flow cytometry; hsp70 protein was significantly higher in the proteasome inhibitor group than in control, but in the autophagy inhibitor and stimulator groups, hsp70 was similar to control. These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio. And inhibiting or stimulating autophagy has less impact on hsp70 than the proteasome pathway.
In conclusion, either stimulation or inhibition of macroautophagy, has less impact on hsp70 than on the proteasome pathway. This study found that rapamycin decreased apoptotic cells in A30P cells independent of caspase-3 activity. Although several lines of evidence recently demonstrated crosstalk between autophagy and caspase-independent apoptosis, we could not confirm that autophagy activation protects cells from caspase-independent cell death. Undoubtedly, there are multiple connections between the apoptotic and autophagic processes.
Inhibition of autophagy may subvert the capacity of cells to remove damaged organelles or to remove misfolded proteins, which would favor apoptosis. However, proteasome inhibition activated macroautophagy and accelerated apoptosis. A likely explanation is inhibition of the proteasome favors oxidative reactions that trigger apoptosis, presumably through
a direct effect on mitochondria, and
the absence of NADPH2 and ATP
which may deinhibit the activation of caspase-2 or MOMP. Another possibility is that aggregated proteins induced by proteasome inhibition increase apoptosis.
Autosomal recessive loss-of-function mutations within the PARK2 gene functionally inactivate the E3 ubiquitin ligase parkin, resulting in neurodegeneration of catecholaminergic neurons and a familial form of Parkinson disease. Current evidence suggests both a mitochondrial function for parkin and a neuroprotective role, which may in fact be interrelated. The antiapoptotic effects of Parkin have been widely reported, and may involve fundamental changes in the threshold for apoptotic cytochrome c release, but the substrate(s) involved in Parkin dependent protection had not been identified. Here, we demonstrate the Parkin-dependent ubiquitination of endogenous Bax comparing primary cultured neurons from WT and Parkin KO mice and using multiple Parkin-overexpressing cell culture systems. The direct ubiquitination of purified Bax was also observed in vitro following incubation with recombinant parkin. The authors found that Parkin prevented basal and apoptotic stress induced translocation of Bax to the mitochondria. Moreover, an engineered ubiquitination-resistant form of Bax retained its apoptotic function, but Bax KO cells complemented with lysine-mutant Bax did not manifest the antiapoptotic effects of Parkin that were observed in cells expressing WT Bax. These data suggest that Bax is the primary substrate responsible for the antiapoptotic effects of Parkin, and provide mechanistic insight into at least a subset of the mitochondrial effects of Parkin.
Parkin, an E3 ubiquitin ligase implicated in Parkinson’s disease, promotes degradation of dysfunctional mitochondria by autophagy. Using proteomic and cellular approaches, we show that upon translocation to mitochondria, Parkin activates the ubiquitin–proteasome system (UPS) for widespread degradation of outer membrane proteins. This is evidenced by an increase in K48-linked polyubiquitin on mitochondria, recruitment of the 26S proteasome and rapid degradation of multiple outer membrane proteins. The degradation of proteins by the UPS occurs independently of the autophagy pathway, and inhibition of the 26S proteasome completely abrogates Parkin-mediated mitophagy in HeLa, SH-SY5Y and mouse cells. Although the mitofusins Mfn1 and Mfn2 are rapid degradation targets of Parkin, degradation of additional targets is essential for mitophagy. These results indicate that remodeling of the mitochondrial outer membrane proteome is important for mitophagy, and reveal a causal link between the UPS and autophagy, the major pathways for degradation of intracellular substrates.
Aloy P. Shaping the future of interactome networks. (A report of the third Interactome Networks Conference, Hinxton, UK, 29 August-1 September 2007). Genome Biology 2007; 8:316 (doi:10.1186/gb-2007-8-10-316)
Complex systems are often networked, and biology is no exception. Following on from the genome sequencing projects, experiments show that proteins in living organisms are highly connected, which helps to explain how such great complexity can be achieved by a comparatively small set of gene products. At a recent conference on interactome networks held outside Cambridge, UK, the most recent advances in research on cellular networks were discussed. This year’s conference focused on identifying the strengths and weaknesses of currently resolved interaction networks and the techniques used to determine them – reflecting the fact that the field of mapping interaction networks is maturing.
The preparation of sufficient amounts of high-quality protein samples is the major bottleneck for structural proteomics. The use of recombinant proteins has increased significantly during the past decades. The most commonly used host, Escherichia coli, presents many challenges including protein misfolding, protein degradation, and low solubility. A novel SUMO fusion technology appears to enhance protein expression and solubility (www.lifesensors.com). Efficient removal of the SUMO tag by SUMO protease in vitro facilitates the generation of target protein with a native N-terminus. In addition to its physiological relevance in eukaryotes, SUMO can be used as a powerful biotechnology tool forenhanced functional protein expression in prokaryotes and eukaryotes.
IL-6 regulation on mitochondrial remodeling/dysfunction
Muscle protein turnover regulation during cancer cachexia is being rapidly defined, and skeletal muscle mitochondria function appears coupled to processes regulating muscle wasting. Skeletal muscle oxidative capacity and the expression of proteins regulating mitochondrial biogenesis and dynamics are disrupted in severely cachectic ApcMin/+ mice. It has not been determined if these changes occur at the onset of cachexia and are necessary for the progression of muscle wasting. Exercise and anti-cytokine therapies have proven effective in preventing cachexia development in tumor bearing mice, while their effect on mitochondrial content, biogenesis and dynamics is not well understood.
The purposes of this study were to
1) determine IL-6 regulation on mitochondrial remodeling/dysfunction during the progression of cancer cachexia and
2) to determine if exercise training can attenuate mitochondrial dysfunction and the induction of proteolytic pathways during IL-6 induced cancer cachexia.
ApcMin/+ mice were examined during the progression of cachexia, after systemic interleukin (IL)-6r antibody treatment, or after IL-6 over-expression with or without exercise. Direct effects of IL-6 on mitochondrial remodeling were examined in cultured C2C12 myoblasts.
Mitochondrial content was not reduced during the initial development of cachexia, while muscle PGC-1α and fusion (Mfn1, Mfn2) protein expression was repressed.
With progressive weight loss mitochondrial content decreased, PGC-1α and fusion proteins were further suppressed, and fission protein (FIS1) was induced.
IL-6 receptor antibody administration after the onset of cachexia improved mitochondrial content,
PGC-1α,
Mfn1/Mfn2 and
FIS1 protein expression.
IL-6 over-expression in pre-cachectic mice accelerated body weight loss and muscle wasting, without reducing mitochondrial content, while PGC-1α and Mfn1/Mfn2 protein expression was suppressed and FIS1 protein expression induced. Exercise normalized these IL-6 induced effects. C2C12 myotubes administered IL-6 had
increased FIS1 protein expression,
increased oxidative stress, and
reduced PGC-1α gene expression
without altered mitochondrial protein expression.
Altered expression of proteins regulating mitochondrial biogenesis and fusion are early events in the initiation of cachexia regulated by IL-6, which precede the loss of muscle mitochondrial content. Furthermore, IL-6 induced mitochondrial remodeling and proteolysis can be rescued with moderate exercise training even in the presence of high circulating IL-6 levels.
White JP, Puppa MJ, Sato S, Gao S. IL-6 regulation on skeletal muscle mitochondrial remodeling during cancer cachexia in the ApcMin/+ mouse. Skeletal Muscle 2012; 2:14-30. http://www.skeletalmusclejournal.com/content/2/1/14
Starvation-induced Autophagy
Upon starvation cells undergo autophagy, a cellular degradation pathway important in the turnover of whole organelles and long lived proteins. Starvation-induced protein degradation has been regarded as an unspecific bulk degradation process. We studied global protein dynamics during amino acid starvation-induced autophagy by quantitative mass spectrometry and were able to record nearly 1500 protein profiles during 36 h of starvation. Cluster analysis of the recorded protein profiles revealed that cytosolic proteins were degraded rapidly, whereas proteins annotated to various complexes and organelles were degraded later at different time periods. Inhibition of protein degradation pathways identified the lysosomal/autophagosomal system as the main degradative route.
Thus, starvation induces degradation via autophagy, which appears to be selective and to degrade proteins in an ordered fashion and not completely arbitrarily as anticipated so far.
Skeletal muscles are the agent of motion and one of the most important tissues responsible for the control of metabolism. Coordinated movements are allowed by the highly organized structure of the cytosol of muscle fibers (or myofibers), the multinucleated and highly specialized cells of skeletal muscles involved in contraction. Contractile proteins are assembled into repetitive structures, the basal unit of which is the sarcomere, that are well packed into the myofiber cytosol. Myonuclei are located at the edge of the myofibers, whereas the various organelles such as mitochondria and sarcoplasmic reticulum are embedded among the myofibrils. Many different changes take place in the cytosol of myofibers during catabolic conditions:
proteins are mobilized
organelles networks are reorganized for energy needs
the setting of myonuclei can be modified.
Further,
strenuous physical activity,
improper dietary regimens and
aging
lead to mechanical and metabolic damages of
myofiber organelles,
especially mitochondria, and
contractile proteins.
During aging the protein turnover is slowed down, therefore it is easier to accumulate aggregates of dysfunctional proteins. Therefore, a highly dynamic tissue such as skeletal muscle requires a rapid and efficient system for the removal of altered organelles, the elimination of protein aggregates, and the disposal of toxic products.
The two major proteolytic systems in muscle are the ubiquitin-proteasome and the autophagy-lysosome pathways. The proteasome system requires
the transcription of the two ubiquitin ligases (atrogin-1 and MuRF1) and
the ubiquitination of the substrates.
Therefore, the ubiquitin-proteasome system can provide the rapid elimination of single proteins or small aggregates. Conversely, the autophagic system is able to degrade entire organelles and large proteins aggregates. In the autophagy-lysosome system, double-membrane vesicles named autophagosomes are able to engulf a portion of the cytosol and fuse with lysosomes, where their content is completely degraded by lytic enzymes.
The autophagy flux can be biochemicaly monitored following LC3 lipidation and p62 degradation. LC3 is the mammalian homolog of the yeast Atg8 gene, which is lipidated when recruited for the double-membrane commitment and growth. p62 (SQSTM-1) is a polyubiquitin-binding protein involved in the proteasome system and that can either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomes. The GFP-LC3 transgenic mouse model allows easy detection of autophagosomes by simply monitoring the presence of bright GFP-positive puncta inside the myofibrils and beneath the plasma membrane of the myofibers, thus investigate the activation of autophagy in skeletal muscles with different contents of slow and fast-twitching myofibers and in response to stimuli such as fasting. For example, in the fast-twiching extensor digitorum longus muscle few GFP-LC3 dots were observed before starvation, while many small GFP-LC3 puncta appeared between myofibrils and in the perinuclear regions after 24 h starvation. Conversely, in the slow-twitching soleus muscle, autophagic puncta were almost absent in standard condition and scarcely induced after 24 h starvation.
Autophagy in Muscle Homeostasis
The autophagic flux was found to be increased during certain catabolic conditions, such as fasting, atrophy , and denervation , thus contributing to protein breakdown. Food deprivation is one of the strongest stimuli known to induce autophagy in muscle. Indeed skeletal muscle, after the liver, is the most responsive tissue to autophagy activation during food deprivation. Since muscles are the biggest reserve of amino acids in the body, during fasting autophagy has the vital role to maintain the amino acid pool by digesting muscular protein and organelles. In mammalian cells, mTORC1, which consists of
mTOR and
Raptor,
is the nutrient sensor that negatively regulates autophagy.
During atrophy, protein breakdown is mediated by atrogenes, which are under the forkhead box O (FoxO) transcription factors control, and activation of autophagy seems to aggravate muscle loss during atrophy. In vivo and in vitro studies demonstrated that several genes coding for components of the autophagic machinery, such as
LC3,
GABARAP,
Vps34,
Atg12 and
Bnip3,
are controlled by FoxO3 transcription factor. FoxO3 is able to regulate independently
the ubiquitin-proteasome system and
the autophagy-lysosome machinery in vivo and in vitro.
Denervation is also able to induce autophagy in skeletal muscle, although at a slower rate than fasting. This effect is mediated by RUNX1, a transcription factor upregulated during autophagy; the lack of RUNX1 results in
excessive autophagic flux in denervated muscle and leads to atrophy.
The generation of Atg5 and Atg7 muscle-specific knockout mice have shown that
with suppression of autophagy both models display muscle weakness and atrophy and
a significant reduction of weight, which is
correlated with the important loss of muscle tissue due to an atrophic condition.
An unbalanced autophagy flux is highly detrimental for muscle, as too much induces atrophy whereas too little leads to muscle weakness and degeneration. Muscle wasting associated with autophagy inhibition becomes evident and symptomatic only after a number of altered proteins and dysfunctional organelles are accumulated, a condition that becomes evident after months or even years. On the other hand, the excessive increase of autophagy flux is able to induce a rapid loss of muscle mass (within days or weeks). Alterations of autophagy are involved in the pathogenesis of several myopathies and dystrophies.
The maintenance of muscle homeostasis is finely regulated by the balance between catabolic and anabolic process. Macroautophagy (or autophagy) is a catabolic process that provides the degradation of protein aggregation and damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagy flux is fundamental for
the homeostasis of skeletal muscles during physiological situations and
in response to stress.
Defective as well as excessive autophagy is harmful for muscle health and has a pathogenic role in several forms of muscle diseases.
Mutations in parkin, a ubiquitin ligase, cause early-onset familial Parkinson’s disease (AR-JP). How Parkin suppresses Parkinsonism remains unknown. Parkin was recently shown to promote the clearance of impaired mitochondria by autophagy, termed mitophagy. Here, we show that Parkin promotes mitophagy by catalyzing mitochondrial ubiquitination, which in turn recruits ubiquitin-binding autophagic components, HDAC6 and p62, leading to mitochondrial clearance.
During the process, juxtanuclear mitochondrial aggregates resembling a protein aggregate-induced aggresome are formed. The formation of these “mito-aggresome” structures requires microtubule motor-dependent transport and is essential for efficient mitophagy. Importantly, we show that AR-JP–causing Parkin mutations are defective in supporting mitophagy due to distinct defects at
recognition,
transportation, or
ubiquitination of impaired mitochondria,
thereby implicating mitophagy defects in the development of Parkinsonism. Our results show that impaired mitochondria and protein aggregates are processed by common ubiquitin-selective autophagy machinery connected to the aggresomal pathway, thus identifying a mechanistic basis for the prevalence of these toxic entities in Parkinson’s disease.
Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson’s disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including
protein degradation,
mitochondrial homeostasis, and
autophagy;
however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting
mitochondrial fission and/or fusion,
to mediate their autophagic turnover.
The precise mechanism of recruitment and the ubiquitination target are unclear. Here we show in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. We hypothesize that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy.
Mutations in Parkin, an E3 ubiquitin ligase that regulates protein turnover, represent one of the major causes of familial Parkinson’s disease (PD), a neurodegenerative disorder characterized by the loss of dopaminergic neurons and impaired mitochondrial functions. The underlying mechanism by which pathogenic parkin mutations induce mitochondrial abnormality is not fully understood. Here we demonstrate that Parkin interacts with and subsequently ubiquitinates dynamin-related protein 1 (Drp1), for promoting its proteasome-dependent degradation. Pathogenic mutation or knockdown of Parkin inhibits the ubiquitination and degradation of Drp1, leading to an increased level of Drp1 for mitochondrial fragmentation. These results identify Drp1 as a novel substrate of Parkin and suggest a potential mechanism linking abnormal Parkin expression to mitochondrial dysfunction in the pathogenesis of PD.
Wang H, Song P, Du L, Tian W. Parkin ubiquitinates Drp1 for proteasome-dependent degradation: implication of dysregulated mitochondrial dynamics in Parkinson’s disease.
Mutations in the genes PTEN-induced putative kinase 1 (PINK1), PARKIN, and DJ-1 cause autosomal recessive forms of Parkinson disease (PD), and the Pink1/Parkin pathway regulates mitochondrial integrity and function. An important question is whether the proteins encoded by these genes function to regulate activities of other cellular compartments. A study in mice, reported by Xiong et al. in this issue of the JCI, demonstrates that Pink1, Parkin, and DJ-1 can form a complex in the cytoplasm, with Pink1 and DJ-1 promoting the E3 ubiquitin ligase activity of Parkin to degrade substrates via the proteasome (see the related article, doi:10.1172/ JCI37617).
This protein complex in the cytosol may or may not be related to the role of these proteins in regulating mitochondrial function or oxidative stress in vivo. Three models for the role of the PPD complex. In this issue of the JCI, Xiong et al. report that Pink1, Parkin, and DJ-1 bind to each other and form a PPD E3 ligase complex in which Pink1 and DJ-1 modulate Parkin-dependent ubiquitination and subsequent degradation of substrates via the proteasome. Previous work suggests that the Pink1/Parkin pathway regulates mitochondrial integrity and promotes mitochondrial fission in Drosophila.
(A) Parkin and DJ-1 may be recruited to the mitochondrial outer membrane during stress and interact with Pink1. These interactions may facilitate the ligase activity of Parkin, thereby facilitating the turnover of molecules that regulate mitochondrial dynamics and mitophagy. The PPD complex may have other roles in the cytosol that result in degradative ubiquitination and/or relay information from mitochondria to other cellular compartments.
(B) Alternatively, Pink1 may be released from mitochondria after cleavage to interact with DJ-1 and Parkin in the cytosol.
A and B differ in the site of action of the PPD complex and the cleavage status of Pink1.
The complex forms on the mitochondrial outer membrane potentially containing full-length Pink1 in A, and in the cytosol with cleaved Pink1 in B.
Lack of DJ-1 function results in phenotypes that are distinct from the mitochondrial phenotypes observed in null mutants of Pink1 or Parkin in Drosophila. Thus, although the PPD complex is illustrated here as regulating mitochondrial fission, the role of DJ-1 in vivo remains to be clarified.
(C) It is also possible that the action occurs in the cytosol and is independent of the function of Pink1/Parkin in regulating mitochondrial integrity and function.
The Xiong et al. study offers an entry point for explorations of the role of Pink1, Parkin, and DJ-1 in the cytoplasm. It remains to be shown whether Parkin, in complex with Pink1 and DJ-1, carries out protein degradation in vivo.
Nitric oxide (NO) is implicated in neuronal cell survival. However, excessive NO production mediates neuronal cell death, in part via mitochondrial dysfunction. Here, we report that the mitochondrial ubiquitin ligase, MITOL, protects neuronal cells from mitochondrial damage caused by accumulation of S-nitrosylated microtubule associated protein 1B-light chain 1 (LC1). S-nitrosylation of LC1 induces a conformational change that serves both to activate LC1 and to promote its ubiquination by MITOL, indicating that microtubule stabilization by LC1 is regulated through its interaction with MITOL. Excessive NO production can inhibit MITOL, and MITOL inhibition resulted in accumulation of S-nitrosylated LC1 following stimulation of NO production by calcimycin and N-methyl-D-aspartate. LC1 accumulation under these conditions resulted in mitochondrial dysfunction and neuronal cell death. Thus, the balance between LC1 activation by S-nitrosylation and down-regulation by MITOL is critical for neuronal cell survival. Our findings may contribute significantly to an understanding of the mechanisms of neurological diseases caused by nitrosative stress-mediated mitochondrial dysfunction.
A common histopathological hallmark of most neurodegenerative diseases is the presence of aberrant proteinaceous inclusions inside affected neurons. Because these protein aggregates are detected using antibodies against components of the ubiquitin–proteasome system (UPS), impairment of this machinery for regulated proteolysis has been suggested to be at the root of neurodegeneration. This hypothesis has been difficult to prove in vivo owing to the lack of appropriate tools. The recent report of transgenic mice with ubiquitous expression of a UPS-reporter protein should finally make it possible to test in vivo the role of the UPS in neurodegeneration.
The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are the two most important mechanisms that normally repair or remove abnormal proteins. Alterations in the function of these systems to degrade misfolded and aggregated proteins are being increasingly recognized as playing a pivotal role in the pathogenesis of many neurodegenerative disorders such as Parkinson’s disease. Dysfunction of the UPS has been already strongly implicated in the pathogenesis of this disease and, more recently, growing interest has been shown in identifying the role of ALP in neurodegeneration. Mutations of a-synuclein and the increase of intracellular concentrations of non-mutant a-synuclein have been associated with Parkinson’s disease phenotype.
The demonstration that a-synuclein is degraded by both proteasome and autophagy indicates a possible linkage between the dysfunction of the UPS or ALP and the occurrence of this disorder.The fact that mutant a-synucleins inhibit ALP functioning by tightly binding to the receptor on the lysosomal membrane for autophagy pathway further supports the assumption that impairment of the ALP may be related to the development of Parkinson’s disease. In this review, we summarize the recent findings related to this topic and discuss the unique role of the ALP in this neurogenerative disorder and the putative therapeutic potential through ALP enhancement.
There is growing evidence that dysfunction of the mitochondrial respiratory chain and failure of the cellular protein degradation machinery, specifically the ubiquitin-proteasome system, play an important role in the pathogenesis of Parkinson’s disease. We now show that the corresponding pathways of these two systems are linked at the transcriptomic level in Parkinsonian substantia nigra. We examined gene expression in medial and lateral substantia nigra (SN) as well as in frontal cortex using whole genome DNA oligonucleotide microarrays. In this study, we use a hypothesis-driven approach in analysing microarray data to describe the expression of mitochondrial and ubiquitin-proteasomal system (UPS) genes in Parkinson’s disease (PD).
Although a number of genes showed up-regulation, we found an overall decrease in expression affecting the majority of mitochondrial and UPS sequences. The down-regulated genes include genes that encode subunits of complex I and the Parkinson’s-disease-linked UCHL1. The observed changes in expression were very similar for both medial and lateral SN and also affected the PD cerebral cortex. As revealed by “gene shaving” clustering analysis, there was a very significant correlation between the transcriptomic profiles of both systems including in control brains.
Therefore, the mitochondria and the proteasome form a higher-order gene regulatory network that is severely perturbed in Parkinson’s disease. Our quantitative results also suggest that Parkinson’s disease is a disease of more than one cell class, i.e. that it goes beyond the catecholaminergic neuron and involves glia as well.
The causes of various neurodegenerative diseases, particularly sporadic cases, remain unknown, but increasing evidence suggests that these diseases may share similar molecular and cellular mechanisms of pathogenesis. One prominent feature common to most neurodegenerative diseases is the accumulation of misfolded proteins in the form of insoluble protein aggregates or inclusion bodies. Although these aggregates have different protein compositions, they all contain ubiquitin and proteasome subunits, implying a failure of the ubiquitin-proteasome system (UPS) in the removal of misfolded proteins.
A direct link between UPS dysfunction and neurodegeneration has been provided by recent findings that genetic mutations in UPS components cause several rare, familial forms of neurodegenerative diseases. Furthermore, it is becoming increasingly clear that oxidative stress, which results from aging or exposure to environmental toxins, can directly damage UPS components, thereby contributing to the pathogenesis of sporadic forms of neurodegenerative diseases.
Aberrations in the UPS often result in defective proteasome-mediated protein degradation, leading to accumulation of toxic proteins and eventually to neuronal cell death. Interestingly, emerging evidence has begun to suggest that impairment in substrate-specific components of the UPS, such as E3 ubiquitin-protein ligases, may cause aberrant ubiquitination and neurodegeneration in a proteasome-independent manner. This provides an overview of the molecular components of the UPS and their impairment in familial and sporadic forms of neurodegenerative diseases, and summarizes present knowledge about the pathogenic mechanisms of UPS dysfunction in neurodegeneration.
Molecular mechanisms of protein ubiquitination and degradation by the UPS. Ubiquitination involves a highly specific enzyme cascade in which
ubiquitin (Ub) is first activated by the ubiquitinactivating enzyme (E1),
then transferred to an ubiquitin-conjugating enzyme (E2), and
finally covalently attached to the substrate by an ubiquitin-protein ligase (E3).
Ubiquitination is a reversible posttranslational modification in which the removal of Ub is mediated by a deubiquitinating enzyme (DUB).
Substrate proteins can be either monoubiquitinated or polyubiquitinated through successive conjugation of Ub moieties to an internal lysine residue in Ub.
K48-linked poly-Ub chains are recognized by the 26S proteasome, resulting in degradation of the substrate and recycling of Ub.
Monoubiquitination or K63-linked polyubiquitination plays a number of regulatory roles in cells that are proteasome-independent.
Parkin
Loss-of-function mutations in parkin, a 465-amino-acid RING-type E3 ligase, were first identified as the cause for autosomal recessive juvenile Parkinsonism (AR-JP) and subsequently found to account for ~50% of all recessively transmitted early-onset PD cases. Interestingly, patients with parkin mutations do not exhibit Lewy body pathology.
Possible pathogenic mechanisms by which impaired UPS components cause neurodegeneration. Genetic mutations or oxidative stress from aging and/or exposure to environmental toxins have been shown to impair the ubiquitination machinery (particularly E3 ubiquitin-protein ligases) and deubiquitinating enzymes (DUBs), resulting in abnormal ubiquitination. Depending on the type of ubiquitination affected, the impairment could cause neurodegeneration through two different mechanisms.
aberrant K48-linked polyubiquitination resulting from impaired E3s or DUBs alters protein degradation by the proteasome, leading to accumulation of toxic proteins and subsequent neurodegeneration. The proteasomes could be directly damaged by oxidative stress or might be inhibited by protein aggregation, which exacerbates the neurotoxicity.
aberrant monoubiquitination or K63-linked polyubiquitination resulting from impaired E3s or DUBs alters crucial non-proteasomal functions, such as gene transcription and protein trafficking, thereby causing neurodegeneration without protein aggregation.
These two models are not mutually exclusive because a single E3 or DUB enzyme, such as parkin or UCH-L1, could regulate more than one type of ubiquitination. In addition, abnormal ubiquitination and neurodegeneration could also result from mutation or oxidative stress-induced structural changes in the protein substrates that alter their recognition and degradation by the UPS.
filedesc Schematic diagram of the ubiquitylation system. Created by Roger B. Dodd (Photo credit: Wikipedia)
Current Noteworthy Work
Statins inhibit HMG-CoA reductase, a key enzyme in cholesterol synthesis, and are widely used to treat hypercholesterolemia.
These drugs can lead to a number of side effects in muscle, including muscle fiber breakdown; however, the mechanisms of muscle injury by statins are poorly understood. We report that lovastatin induced the expression of atrogin-1, a key gene involved in skeletal muscle atrophy, in humans with statin myopathy, in zebrafish embryos, and in vitro in murine skeletal muscle cells. In cultured mouse myotubes, atrogin-1 induction following lovastatin treatment was accompanied by distinct morphological changes, largely absent in atrogin-1 null cells. In zebrafish embryos, lovastatin promoted muscle fiber damage, an effect that was closely mimicked by knockdown of zebrafish HMG-CoA reductase. Moreover, atrogin-1 knockdown in zebrafish embryos prevented lovastatin-induced muscle injury. Finally, overexpression of PGC-1α, a transcriptional coactivator that induces mitochondrial biogenesis and protects against the development of muscle atrophy, dramatically prevented lovastatin-induced muscle damage and abrogated atrogin-1 induction both in fish and in cultured mouse myotubes. Collectively, our human, animal, and in vitro findings shed light on the molecular mechanism of statin-induced myopathy and suggest that atrogin-1 may be a critical mediator of the muscle damage induced by statins.
Macroautophagy (hereafter referred to as autophagy) is a cellular degradation system in which cytoplasmic components, including organelles, are sequestered by double membrane structures called autophagosomes and the sequestered materials are degraded by lysosomal hydrolases for supply of amino acids and for cellular homeostasis. Although autophagy has generally been considered nonselective, recent studies have shed light on another indispensable role for basal autophagy in cellular homeostasis, which is mediated by selective degradation of a specific substrate(s). p62 is a ubiquitously expressed cellular protein that is conserved in metazoa but not in plants and fungi, and recently it has been known as one of the selective substrates for autophagy.
This protein is localized at the autophagosome formation site and directly interacts with LC3, an autophagosome localizing protein . Subsequently, the p62 is incorporated into the autophagosome and then degraded. Therefore, impaired autophagy is accompanied by accumulation of p62 followed by the formation of p62 and ubiquitinated protein aggregates because of the nature of both self- oligomerization and ubiquitin binding of p62.
Epicrisis
This extensive review leaves little left unopened. We have seen the central role that the UPS system plays in normal organelle proteolysis in concert with autophagy. Impaired ubiquitination occurs from aging, and/or toxins, under oxidative stress involving E3s or DUBs.
This leads to altered gene transcripton, altered protein trafficking, and plays a role in neurodegenative disease, and muscle malfunction.
English: A cartoon representation of a lysine 48-linked diubiquitin molecule. The two ubiquitin chains are shown as green cartoons with each chain labelled. The components of the linkage are indicated and shown as orange sticks. Image was created using PyMOL from PDB id 1aar. (Photo credit: Wikipedia)
Different forms of protein ubiquitylation (Photo credit: Wikipedia)
filedesc Schematic diagram of the ubiquitylation system. Created by Roger B. Dodd (Photo credit: Wikipedia)
Autophagy (Photo credit: Wikipedia)
English: Structure of the PARK2 protein. Based on PyMOL rendering of PDB 1iyf. (Photo credit: Wikipedia)
Comparison of the process of macroautophagy versus microautophagy. (Photo credit: Wikipedia)
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