Adding to the list of disease-causing proteins in brain disorders

A multi-institution group of researchers has found new candidate disease proteins for neurodegenerative disorders. James Shorter, Ph.D., assistant professor of Biochemistry and Biophysics at the Perelman School of Medicine, University of Pennsylvania, Paul Taylor, M.D., PhD, St. Jude Children’s Research Hospital, and colleagues describe in an advanced online publication of Nature that mutations in prion-like segments of two RNA-binding proteins are associated with a rare inherited degeneration disorder affecting muscle, brain, motor neurons and bone (called multisystem proteinopathy) and one case of the familial form of amyotrophic lateral sclerosis (ALS).

"This study uses a variety of scientific approaches to provide powerful evidence that unregulated polymerization of proteins involved in RNA metabolism may contribute to ALS and related diseases," said Amelie Gubitz, Ph.D., a program director at the National Institute of Neurological Disorders and Stroke (NINDS).

ALS, or Lou Gehrig’s disease, is a universally fatal neurodegenerative disease. Previous studies found that mutations in two related RNA-binding proteins, TDP-43 and FUS, cause some forms of ALS, but more proteins were suspected of causing other forms of the disease. TDP-43 and FUS regulate how the genetic code is translated for the assembly of proteins.

There are over 200 human RNA-binding proteins, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathology. Computer algorithms, based on protein sequences, designed to identify yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbor a distinctive prion-like segment. These segments are essential for the assembly of certain protein complexes. But, the interplay between human prion-like segments and disease is not well understood.

Using yeast as a model organism, co-author Aaron Gitler, while at Penn in 2011, surveyed 133 of 200-plus candidate human RNA-binding proteins to predict new ALS disease genes, other than TDP-43 and FUS. They further winnowed the candidates to about 10 proteins with prion-like segments, and selected two candidates, TAF15 and EWSR1, for further study. Both TAF15 and EWSR1 aggregated in the test tube and were toxic in yeast.

Remarkably, they also uncovered TAF15 and EWSR1 mutations in ALS patients that were not found in healthy individuals. Based on these findings, they proposed that RNA-binding proteins with prion-like segments might contribute very broadly to the pathology of ALS and related brain disorders.

Characterizing the Top-Ten

Taylor, Gitler, Shorter, and others continued to characterize the top-ten human RNA-binding proteins with prion-like segments. The Nature study describes that two more of the top-ten candidates, called hnRNPA1 and hnRNPA2B1, are mutated and cause familial cases of brain disease. The mutations in hnRNPA1 and hnRNPA2B1 were present in two families with an extremely rare inherited degeneration affecting muscle, brain, motor neuron, and bone and another from a person with familial ALS.

Mutations in these two proteins fell in the prion-like segments and coincided with “sticky” regions in the proteins, making these regions more prone to assemble into self-organizing fibrils. The normal form of the proteins shows a natural tendency to assemble into fibrils, which is exacerbated by the disease mutations.

"The mutations accelerate the formation of the fibrils that recruit normal protein to form more fibrils," noted co-first author Emily Scarborough, from Penn. This dysregulated assembly likely contributes to disease. Indeed, the disease mutations also promote excess incorporation of the proteins into stress granules within a cell and the formation of clumps in the cells of animal models of human neurodegenerative disease.

"Neurodegenerative disease could ensue from unregulated fibril formation initiated spontaneously by environmental stress or another factor that regulates a protein’s assembly," says Scarborough.

"This paper reflects an amazing collaborative effort and provides a great example of how understanding the underlying pure protein biochemistry can help explain how genetic mutations might cause pathology and disease," says Shorter.

"The findings confirm a strong prediction that the disease-causing mutations make the prion-like segment ‘stickier’ and more prone to clump," added co-first author Zamia Diaz, also from Penn.

Diseases associated with fibrils forming from prion-like domains in proteins frequently show “spreading” pathology, in which cellular degeneration via inclusions starts in one center of the brain and “spreads” to neighboring tissue. Although not directly addressed in the Nature study, the findings suggest that cell-to-cell transmission of a self-templating protein could contribute to the spreading pathology that is characteristic of these diseases.

"Related proteins with prion-like domains must be considered candidates for initiating and perhaps propagating similar pathologies in muscle, brain, motor neurons, and bone," concluded Shorter.

Identification of abnormal protein may help diagnose, treat ALS and frontotemporal dementia

Amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease, and frontotemporal dementia (FTD) are devastating neurodegenerative diseases with no effective treatment. Researchers are beginning to recognize ALS and FTD as part of a spectrum disorder with overlapping symptoms. Now investigators reporting online February 12 in the Cell Press journal Neuron have discovered an abnormal protein that first forms as a result of genetic abnormalities and later builds up in the brains of many patients with either disease.

"In identifying the novel protein that abnormally accumulates in the brains of affected patients, we have uncovered a potentially new therapeutic target and biomarker that would allow clinicians to confirm diagnosis of the diseases," says senior author Dr. Leonard Petrucelli, Chair of Neuroscience at Mayo Clinic in Florida.

By analyzing brain tissue from patients with ALS or FTD, Dr. Petrucelli and his team discovered that the abnormal protein, which they call C9RANT, is generated as a result of repeat expansions of nucleotides in the noncoding region of the C9ORF72 gene. These expansions are the most common cause of ALS and FTD. “Simply put, an error in the highly regulated cellular process through which proteins are generated causes the abnormal production of C9RANT,” explains Dr. Petrucelli.

The researchers discovered the protein C9RANT after creating a novel antibody to specifically detect it. The ability to detect C9RANT in individuals’ cerebrospinal fluid may provide a valuable diagnostic and prognostic tool for identifying patients carrying the C9ORF72 repeat expansion and for then tracking the progression of the disease in these at-risk individuals.

"Although it remains to be shown whether C9RANT is causing the cell death or toxicity associated with disease symptoms, our discovery offers a potential target to prevent neuronal loss in patients carrying the C9ORF72 repeat expansion," says Dr. Petrucelli.

The concept that abnormal proteins accumulate and can be toxic to cells is not new. In fact, tau protein forms tangles in Alzheimer’s disease and alpha-synuclein forms clumps in Parkinson’s disease. Just as new therapies are being developed to break down the protein aggregates associated with these diseases, developing a therapeutic strategy to target C9RANT aggregates may also prove beneficial.

What Causes Lou Gehrig’s Sticky Masses?

Globs of protein clustered in the neurons that control muscles have long been the hallmark of amyotrophic lateral sclerosis (ALS), the fatal neurodegenerative disease also commonly known as Lou Gehrig’s disease. Now, a study of the most commonly found mutant gene in people with ALS reveals an unexpected origin of some of those sticky masses, a finding that may offer drug developers a new target for treatments.

Located on the ninth chromosome, which explains part of its unwieldy name, the C9orf72 gene has a bit of a stutter. A typical version in healthy people contains a stretch of DNA where a string of six genetic letters—GGGGCC—repeats up to 25 times. Scientists have recently found that in a sizable share of people with ALS and frontotemporal dementia (FTD), a less common neurological disease characterized by language, memory, and emotional problems, this repeat occurs many more times; some people have thousands of copies.

Since these C9orf72 mutations were discovered in 2011, some researchers have speculated that the repeats interrupt production of the gene’s normal protein, which serves some as-yet unknown, but vital function in motor neurons or other brain cells. Others have hypothesized that the mutation spawns a large, misshapen strand of RNA that grabs on to proteins such as TDP-43, which normally help process RNA, creating protein tangles that starve the cell of the machinery it needs to function.

Molecular biologists at the Ludwig Maximilians University Munich in Germany and the University of Antwerp in Belgium, however, wondered whether the genetic stutters themselves coded for proteins that became tangled in the cell. Few scientists had considered this because the stutters don’t contain the “start signal” that allows proteins to be made. Still, in a few other diseases caused by genetic repeats, the cell manages to produce proteins from the abnormal gene despite lacking this signal. Sometimes these proteins are toxic and ultimately kill the cell.

Based on the DNA sequence of the GGGGCC-laden C9orf72 seen in ALS and FTD patients, the European team determined that if translated, the gene would produce various proteins containing strings of repeat amino acids. Dubbed dipeptide repeat (DPR) proteins, these molecules don’t normally appear in humans and should be prone to clumping, the scientists concluded. Indeed, when they began to search for DPR protein clusters in actual human brain tissues, they found them in tissue from FTD and ALS patients with the C9orf72 mutation. No such lumps showed up in the brain tissue of healthy controls or ALS and FTD patients without the C9orf72 mutation, increasing the likelihood that the mutation produced them, Dieter Edbauer, a molecular biologist at Ludwig Maximilians, and his co-authors report online today in Science.

Researchers turn one form of neuron into another in the brain

A new finding by Harvard stem cell biologists turns one of the basics of neurobiology on its head – demonstrating that it is possible to turn one type of already differentiated neuron into another within the brain.

The discovery by Paola Arlotta and Caroline Rouaux “tells you that maybe the brain is not as immutable as we always thought, because at least during an early window of time one can reprogram the identity of one neuronal class into another,” said Arlotta, an Associate Professor in Harvard’s Department of Stem Cell and Regenerative Biology (SCRB).

The principle of direct lineage reprogramming of differentiated cells within the body was first proven by SCRB co-chair and Harvard Stem Cell Institute (HSCI) co-director Doug Melton and colleagues five years ago, when they reprogrammed exocrine pancreatic cells directly into insulin producing beta cells.

Arlotta and Rouaux now have proven that neurons too can change their mind. The work is being published on-line by the journal Nature Cell Biology.

In their experiments, Arlotta targeted callosal projection neurons, which connect the two hemispheres of the brain, and turned them into neurons similar to corticospinal motor neurons, one of two populations of neurons destroyed in Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease. To achieve such reprogramming of neuronal identity, the researchers used a transcription factor called Fezf2, which long as been known for playing a central role in the development of corticospinal neurons in the embryo.

What makes the finding even more significant is that the work was done in the brains of living mice, rather than in collections of cells in laboratory dishes. The mice were young, so researchers still do not know if neuronal reprogramming will be possible in older laboratory animals – and humans. If it is possible, this has enormous implications for the treatment of neurodegenerative diseases.

"Neurodegenerative diseases typically effect a specific population of neurons, leaving many others untouched. For example, in ALS it is corticospinal motor neurons in the brain and motor neurons in the spinal cord, among the many neurons of the nervous system, that selectively die," Arlotta said. "What if one could take neurons that are spared in a given disease and turn them directly into the neurons that die off? In ALS, if you could generate even a small percentage of corticospinal motor neurons, it would likely be sufficient to recover basic functioning," she said.

The experiments that led to the new finding began five years ago, when “we wondered: in nature you never seen a neuron change identity; are we just not seeing it, or is this the reality? Can we take one type of neuron and turn it into another?” Arlotta and Rouaux asked themselves.

Over the course of the five years, the researchers analyzed “thousands and thousands of neurons, looking for many molecular markers as well as new connectivity that would indicate that reprogramming was occurring,” Arlotta said. “We could have had this two years ago, but while this was a conceptually very simple set of experiments, it was technically difficult. The work was meant to test important dogmas on the irreversible nature of neurons in vivo. We had to prove, without a shadow of a doubt, that this was happening.”

The work in Arlotta’s lab is focused on the cerebral cortex, but “it opens the door to reprogramming in other areas of the central nervous system,” she said.

Arlotta, an HSCI principal faculty member, is now working with colleague Takao Hensch, of Harvard’s Department of Molecular and Cellular Biology, to explicate the physiology of the reprogrammed neurons, and learn how they communicate within pre-existing neuronal networks.

"My hope is that this will facilitate work in a new field of neurobiology that explores the boundaries and power of neuronal reprogramming to re-engineer circuits relevant to disease," said Paola Arlotta.

(Image courtesy Tulane University)