Neuroscience

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Posts tagged ALS

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Mutated Gene Causes Nerve Cell Death

The British astrophysicist Stephen Hawking is likely to be the world’s most famous person living with amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease. ALS is a progressive disease affecting motor neurons, nerve cells that control muscle function, and nearly always leads to death. Researchers at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) in Vienna have now identified a completely new mechanism in the onset of motor neuron diseases. Their findings could be the basis for future treatments for these presently incurable diseases.

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A new principle on motor neuron death
The IMBA scientists, working with an international team of researchers under the leadership of Josef Penninger and Javier Martinez, discovered a completely new fundamental mechanism that triggers the death of motor neurons. Motor neurons are nerve cells responsible for stimulating muscles. The loss of these motor neurons in mice with a genetic mutation in a gene named CLP11 leads to severe and progressive muscular paralysis and, in some cases, to death.
"We’ve been working on resolving the function of the CLP1 gene in a living organism for a long time. To do that, we developed model mice in which the function of CLP1 was genetically inactivated. To our utter surprise we discovered that deactivating CLP1 increases the sensitivity of cell die when exposed to oxidative stress2. That leads to enhanced activity of the p53 protein3 and then to the permanent destruction of motor neurons," says Toshikatsu Hanada, a postdoctoral researcher working in the lab of Josef Penninger and first author of the study along with Stefan Weitzer.

Stephen Hawking - a most renowned patient
Motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are chronic disorders of the neuromuscular system. These diseases are caused by damage in the motor nerve cells in the brain and spinal cord, and the nerves can no longer stimulate motion in the muscles. The primary symptoms are muscular weakness, muscular dystrophy, and problems swallowing or speaking. Stephen Hawking was diagnosed with ALS 50 years ago. But not all ALS patients live so long with the disease: so far there are no treatments for ALS. Nearly all ALS patients die of paralysis of respiratory muscles within a few years.

Completely new disease mechanism
Javier Martinez, an IMBA team leader and co-author of the study, is a specialist in the field of ribonucleic acid (RNA) research. His research group had discovered the CLP1 gene in an earlier study, published in Nature in 2007. Until now, the exact essential function of CLP1 in RNA biology was unclear. “By deactivating CLP1, we have discovered a previously unknown new species of RNA,” says Javier Martinez about the scientific relevance of the work. “The accumulation of this RNA is a consequence of increased oxidative stress in the cell. We see this as one of the triggers for the loss of motor neurons that occurs in ALS and other neuromuscular diseases. Thus our findings describe a completely new mechanism of motor neuron diseases.”

Seminal findings
Josef Penninger, scientific director at the IMBA and last-author of the study, is excited about the researchers’ findings: “This surprising discovery of a role of CLP1 in the onset of motor neuron diseases is an entirely new principle in how RNA talks to oxidative stress. Nearly all genetic mutations found in ALS patients affect either RNA metabolism or oxidative stress, suggesting a possibly unifying principle for these diseases. Our work may have revealed the ‘missing link’ in how these two biological systems communicate and trigger incurable diseases like ALS.”

Stefan Weitzer sees tremendous potential for these findings: “We’ve discovered a new mechanism that leads to the death of motor neurons. If this holds true for other neuronal disease, our results could be one day used to drive the development of treatments for previously incurable diseases. In our work we also describe how the p53 protein regulates the loss of motor neurons. Removing p53 saves mice with CLP1 mutations from certain death.” If scientists are successful in applying these findings to people, the researchers may have discovered a treatment approach to cure ALS and similar diseases. The authors, however, caution that more studies will be needed to translate their findings to human medicine.

This study was performed in collaboration with research groups from the Medical Universities of Vienna and Innsbruck, the University Medical Center at Hamburg-Eppendorf in Germany, the Harvard Medical School, the Harvard Stem Cell Institute, the Boston Children’s and Massachusetts General Hospitals, the Keio University School of Medicine in Tokyo, Oita University in Japan, and the Weizmann Institute of Science in Rehovot in Israel.

Their work, “CLP1 links tRNA metabolism to progressive motor-neuron loss”, was published on March 10, 2013 in “Nature”, an internationally renowned journal.

(Source: imba.oeaw.ac.at)

Filed under Lou Gehrig's disease ALS cell death motor neurons muscular paralysis genetics neuroscience science

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Misplaced molecules: New insights into the causes of dementia

A shortage of a protein called TDP-43 caused muscle wasting and stunted nerve cells. This finding supports the idea that malfunction of this protein plays a decisive role in ALS and FTD. The study is published in the “Proceedings of the National Academy of Sciences of the USA" (PNAS).

ALS is an incurable neurological disease which manifests as rapidly progressing muscle wasting. Both limbs and respiratory muscles are affected. This leads to impaired mobility and breathing problems. Patients commonly die within a few years after the symptoms emerged. In rare cases, of which the British physicist Stephen Hawking is the most notable, patients can live with the disease for a long time. In Germany estimates show over 150,000 patients suffering from ALS – an average of 1 in 500 people.

Proteins gone astray

Over the last few years, there has been increasing evidence that ALS and FTD – a form of dementia associated with changes in personality and social behaviour – may have similar or even the same origins. The symptoms overlap and common factors have also been found at the microscopic level. In many cases, particles accumulate and form clumps in the patient’s nerve cells: this applies particularly to the TDP-43 protein.

"Normally, this protein is located in the cell nucleus and is involved in processing genetic information," explains molecular biologist Dr. Bettina Schmid, who works at the DZNE Munich site and at LMU. "However, in cases of disease, TDP-43 accumulates outside the nucleus forming aggregates." Schmid explains that it is not yet clear whether these clumps are harmful. "However, the protein’s normal function is clearly disrupted. It no longer reaches the nucleus to perform its actual task. There seems to be a relationship between this malfunction and the disease."

Studies on zebrafish

However, until now little was known about the function of TDP-43. What are the consequences when this protein becomes non-functional? In order to answer this question, the team led by Bettina Schmid cooperated with the research group of Prof. Christian Haass to investigate the larvae of specially bred zebrafish. Their genetic code had been modified in such a way that no TDP-43 was produced in the organism of the fish. The result: the young fish showed massive muscle wasting and died a few days after hatching. Moreover, the extensions of the nerve cells which control the muscles were abnormal.

"To some extent, these are symptoms typical of ALS and FTD. Therefore, a loss of function of TDP-43 does seem to play a critical role in the disease," says Haass, Site Speaker of the DZNE Munich Site and chair of Metabolic Biochemistry at LMU.

The study revealed one more finding which surprised the researchers: the blood flow of the fish was massively disturbed. “It is well known that circulatory disorders play a part in other forms of dementia, notably in the case of Alzheimer’s,” says Haass. “We now want to investigate whether such problems with blood flow may be a general problem of neurodegenerative diseases and whether such problems occur particularly in patients with ALS and FTD.”

(Source: eurekalert.org)

Filed under neurodegenerative diseases dementia nerve cells proteins zebrafish ALS blood flow neuroscience science

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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.

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.

Filed under ALS Lou Gehrig's disease neurodegenerative diseases brain tissue cell death neuroscience science

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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.

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.

Filed under neurodegenerative diseases Lou Gehrig's disease ALS gene mutation genetics proteins science

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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)

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)

Filed under brain neuron neurodegenerative diseases motor neurons ALS Lou Gehrig's disease science

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Scientists Discover Structure of Protein Essential for Quality Control, Nerve Function Using an innovative approach, scientists at The Scripps Research Institute (TSRI) have determined the structure of Ltn1, a recently discovered “quality-control” protein that is found in the cells of all plants, fungi and animals. Ltn1 appears to be essential for keeping cells’ protein-making machinery working smoothly. It may also be relevant to human neurodegenerative diseases, for an Ltn1 mutation in mice leads to a motor-neuron disease resembling amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease). “To better understand Ltn1’s mechanism of action, we needed to solve its structure, and that’s what we’ve done here,” said TSRI Associate Professor Claudio Joazeiro. “In addition, this project has brought us a set of structural analysis techniques that we can apply to other exciting problems in biology,” said TSRI Professor Bridget Carragher. Joazeiro and Carragher, along with Clint Potter, also a TSRI professor, are senior authors of the new report, which appears in the online Early Edition of the Proceedings of the National Academy of Sciences the week of January 14, 2013. Links to Neurodegenerative Disease Ltn1 first turned up on biologists’ radar screens several years ago when a joint Novartis-Phenomix research team noted that mice with an unknown gene mutation were born normal but suffered from progressive paralysis. The scientists dubbed the animals lister mice, because they listed to one side as they walked. Collaborating with Joazeiro, the Novartis team reported in a 2009 paper that the mutated gene normally codes for a type of enzyme known as an E3 ubiquitin ligase, and that the mouse phenotype was due to a neurodegenerative syndrome resembling ALS. In a study published in the journal Nature the following year, Joazeiro and his postdoctoral research associate Mario H. Bengtson found that the enzyme serves as a crucial quality-control manager for the cellular protein-making factories called ribosomes. Occasionally a ribosome receives miscoded genetic instructions and produces certain types of abnormal proteins, known as “nonstop proteins”— jamming the ribosomal machinery like a wrinkled sheet of paper in an office printer. Bengtson and Joazeiro found that Ltn1 fixes jammed ribosomes by tagging nonstop proteins with ubiquitin molecules, thereby marking them for quick destruction by roving cellular garbage-disposers called proteasomes. “The question for us then was, ‘How does Ltn1 do this?’” said Joazeiro. Pushing the Boundaries of Electron Microscopy To help find out, he began a collaboration with Carragher and Potter, who run the National Resource for Automated Molecular Microscopy (NRAMM), an advanced electron microscope facility at TSRI that is funded by the National Institutes of Health’s National Center for Research Resources. Ltn1 was deemed too large for its structure to be determined by current nuclear magnetic resonance (NMR) technology, and, as the scientists know now, too flexible to allow the highly regular crystalline packing needed by X-ray crystallographers. “It’s a very floppy molecule, so it would be hard to crystallize,” said Potter. Advanced electron microscopy offered a way, however. Dmitry Lyumkis, a graduate student in the NRAMM laboratory and first author of the study, took high-resolution images of yeast Ltn1 with an electron microscope. He then used sophisticated image and data processing software to align and average individual images. The technique eliminates much of the random “noise” that obscures single images and produces a sharp 3D picture of the protein. No one has ever used electron microscopy to distinguish so many—more than 20—conformations of such a small protein. “Usually electron microscopists determine no more than two or three conformational states, and they work with protein complexes whose size is in the megadalton range, but Ltn1 is only 180 kilodaltons, an order of magnitude smaller,” Lyumkis said. An Unusually Flexible Structure The analysis revealed that Ltn1 has an elongated, double-jointed and extraordinarily flexible structure with two working ends—the N-terminus and C-terminus. “We anticipate that the N-terminus is responsible for association with the ribosome and know that the C-terminus is responsible for the ubiquitylation of nonstop proteins,” said Lyumkis. “We suspect that the high flexibility of this structure is needed for it to work on the variety of nonstop proteins that can get stuck in ribosomes.” One of the next steps for the team is to evaluate Ltn1’s individual segments, which appear to be more rigid, using X-ray crystallography, in order to develop a piece-by-piece atomic-resolution model of the enzyme. Another is to determine the structure of Ltn1 when it is attached to a ribosome and operating on a nonstop protein. Joazeiro notes that a typical yeast cell has nearly 200,000 ribosomes but requires only 200 Ltn1 copies for adequate quality control under normal growth conditions. “Somehow this enzyme can efficiently sense which ribosomes are jammed, and we expect that by solving the joint structure of Ltn1 and a ribosome, we’ll be able to understand how it does this,” he says. Lyumkis, Carragher, Potter and their colleagues at NRAMM also plan to use a similar electron microscopy-based approach to find the structures of other important proteins with highly variable “heterogeneous” conformations. “Heterogeneity has been a big challenge,” said Potter, “and being able to collect this large dataset and do all of this data processing successfully has been a critical breakthrough.”

Scientists Discover Structure of Protein Essential for Quality Control, Nerve Function

Using an innovative approach, scientists at The Scripps Research Institute (TSRI) have determined the structure of Ltn1, a recently discovered “quality-control” protein that is found in the cells of all plants, fungi and animals.

Ltn1 appears to be essential for keeping cells’ protein-making machinery working smoothly. It may also be relevant to human neurodegenerative diseases, for an Ltn1 mutation in mice leads to a motor-neuron disease resembling amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease).

“To better understand Ltn1’s mechanism of action, we needed to solve its structure, and that’s what we’ve done here,” said TSRI Associate Professor Claudio Joazeiro.

“In addition, this project has brought us a set of structural analysis techniques that we can apply to other exciting problems in biology,” said TSRI Professor Bridget Carragher.

Joazeiro and Carragher, along with Clint Potter, also a TSRI professor, are senior authors of the new report, which appears in the online Early Edition of the Proceedings of the National Academy of Sciences the week of January 14, 2013.

Links to Neurodegenerative Disease

Ltn1 first turned up on biologists’ radar screens several years ago when a joint Novartis-Phenomix research team noted that mice with an unknown gene mutation were born normal but suffered from progressive paralysis. The scientists dubbed the animals lister mice, because they listed to one side as they walked. Collaborating with Joazeiro, the Novartis team reported in a 2009 paper that the mutated gene normally codes for a type of enzyme known as an E3 ubiquitin ligase, and that the mouse phenotype was due to a neurodegenerative syndrome resembling ALS.

In a study published in the journal Nature the following year, Joazeiro and his postdoctoral research associate Mario H. Bengtson found that the enzyme serves as a crucial quality-control manager for the cellular protein-making factories called ribosomes. Occasionally a ribosome receives miscoded genetic instructions and produces certain types of abnormal proteins, known as “nonstop proteins”— jamming the ribosomal machinery like a wrinkled sheet of paper in an office printer. Bengtson and Joazeiro found that Ltn1 fixes jammed ribosomes by tagging nonstop proteins with ubiquitin molecules, thereby marking them for quick destruction by roving cellular garbage-disposers called proteasomes.

“The question for us then was, ‘How does Ltn1 do this?’” said Joazeiro.

Pushing the Boundaries of Electron Microscopy

To help find out, he began a collaboration with Carragher and Potter, who run the National Resource for Automated Molecular Microscopy (NRAMM), an advanced electron microscope facility at TSRI that is funded by the National Institutes of Health’s National Center for Research Resources.

Ltn1 was deemed too large for its structure to be determined by current nuclear magnetic resonance (NMR) technology, and, as the scientists know now, too flexible to allow the highly regular crystalline packing needed by X-ray crystallographers. “It’s a very floppy molecule, so it would be hard to crystallize,” said Potter.

Advanced electron microscopy offered a way, however. Dmitry Lyumkis, a graduate student in the NRAMM laboratory and first author of the study, took high-resolution images of yeast Ltn1 with an electron microscope. He then used sophisticated image and data processing software to align and average individual images. The technique eliminates much of the random “noise” that obscures single images and produces a sharp 3D picture of the protein.

No one has ever used electron microscopy to distinguish so many—more than 20—conformations of such a small protein. “Usually electron microscopists determine no more than two or three conformational states, and they work with protein complexes whose size is in the megadalton range, but Ltn1 is only 180 kilodaltons, an order of magnitude smaller,” Lyumkis said.

An Unusually Flexible Structure

The analysis revealed that Ltn1 has an elongated, double-jointed and extraordinarily flexible structure with two working ends—the N-terminus and C-terminus. “We anticipate that the N-terminus is responsible for association with the ribosome and know that the C-terminus is responsible for the ubiquitylation of nonstop proteins,” said Lyumkis. “We suspect that the high flexibility of this structure is needed for it to work on the variety of nonstop proteins that can get stuck in ribosomes.”

One of the next steps for the team is to evaluate Ltn1’s individual segments, which appear to be more rigid, using X-ray crystallography, in order to develop a piece-by-piece atomic-resolution model of the enzyme. Another is to determine the structure of Ltn1 when it is attached to a ribosome and operating on a nonstop protein. Joazeiro notes that a typical yeast cell has nearly 200,000 ribosomes but requires only 200 Ltn1 copies for adequate quality control under normal growth conditions. “Somehow this enzyme can efficiently sense which ribosomes are jammed, and we expect that by solving the joint structure of Ltn1 and a ribosome, we’ll be able to understand how it does this,” he says.

Lyumkis, Carragher, Potter and their colleagues at NRAMM also plan to use a similar electron microscopy-based approach to find the structures of other important proteins with highly variable “heterogeneous” conformations. “Heterogeneity has been a big challenge,” said Potter, “and being able to collect this large dataset and do all of this data processing successfully has been a critical breakthrough.”

Filed under neurodegenerative diseases ALS protein nerve cells nerve function science

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Stem Cells May Hold Promise for Lou Gehrig’s Disease (ALS) Apparent stem cell transplant success in mice may hold promise for people with amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease. The results of the study were released today and will be presented at the American Academy of Neurology’s 65th Annual Meeting in San Diego, March 16 to 23, 2013. “There have been remarkable strides in stem cell transplantation when it comes to other diseases, such as cancer and heart failure,” said study author Stefania Corti, MD, PhD, with the University of Milan in Italy and a member of the American Academy of Neurology. “ALS is a fatal, progressive, degenerative disease that currently has no cure. Stem cell transplants may represent a promising avenue for effective cell-based treatment for ALS and other neurodegenerative diseases.” For the study, mice with an animal model of ALS were injected with human neural stem cells taken from human induced pluripotent stem cells (iPSCs). iPSC are adult cells such as skin cells that have been genetically reprogrammed to an embryonic stem cell-like state. Neurons are a basic building block of the nervous system, which is affected by ALS. After injection, the stem cells migrated to the spinal cord of the mice, matured and multiplied. The study found that stem cell transplantation significantly extended the lifespan of the mice by 20 days and improved their neuromuscular function by 15 percent. “Our study shows promise for testing stem cell transplantation in human clinical trials,” said Corti. (Image: ALAMY)

Stem Cells May Hold Promise for Lou Gehrig’s Disease (ALS)

Apparent stem cell transplant success in mice may hold promise for people with amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease. The results of the study were released today and will be presented at the American Academy of Neurology’s 65th Annual Meeting in San Diego, March 16 to 23, 2013. “There have been remarkable strides in stem cell transplantation when it comes to other diseases, such as cancer and heart failure,” said study author Stefania Corti, MD, PhD, with the University of Milan in Italy and a member of the American Academy of Neurology. “ALS is a fatal, progressive, degenerative disease that currently has no cure. Stem cell transplants may represent a promising avenue for effective cell-based treatment for ALS and other neurodegenerative diseases.”

For the study, mice with an animal model of ALS were injected with human neural stem cells taken from human induced pluripotent stem cells (iPSCs). iPSC are adult cells such as skin cells that have been genetically reprogrammed to an embryonic stem cell-like state. Neurons are a basic building block of the nervous system, which is affected by ALS. After injection, the stem cells migrated to the spinal cord of the mice, matured and multiplied.

The study found that stem cell transplantation significantly extended the lifespan of the mice by 20 days and improved their neuromuscular function by 15 percent. “Our study shows promise for testing stem cell transplantation in human clinical trials,” said Corti.

(Image: ALAMY)

Filed under ALS Lou Gehrig’s disease embryonic stem cells neuroscience science stem cell transplantation stem cells American Academy of Neurology

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Removing protein ‘garbage’ in nerve cells may help control 2 neurodegenerative diseases Neuroscientists at Georgetown University Medical Center say they have new evidence that challenges scientific dogma involving two fatal neurodegenerative diseases — amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) — and, in the process, have uncovered a possible therapeutic target as a novel strategy to treat both disorders. The study, posted online in the Journal of Biological Chemistry, found that the issue in both diseases is the inability of the cell’s protein garbage disposal system to “pull out” and destroy TDP-43, a powerful, sometimes mutated gene that produces excess amounts of protein inside the nucleus of a nerve cell, or neuron. "This finding suggests that if we’re able to ‘rev up’ that clearance machinery and help the cell get rid of the bad actors, it could possibly reduce or slow the development of ALS and FTD," says the study’s lead investigator, neuroscientist Charbel E-H Moussa, MB, PhD. "The potential of such an advance is very exciting." He cautions, though, that determining if this strategy is possible in humans could take many years and will involve teams of researchers. The way to rev up protein disposal is to add parkin — the cell’s natural disposal units — to brain cells. In this study, Moussa and his colleagues demonstrated in two animal experiments that delivering parkin genes to neurons slowed down ALS pathologies linked to TDP-43.” Moussa says that his study further demonstrates that clumps known as “inclusions” of TDP-43 protein found inside neuron bodies in both disorders do not promote these diseases, as some researchers have argued. What happens in both diseases is that this protein, which is a potent regulator of thousands of genes, leaves the nucleus and collects inside the gel-like cytoplasm of the neuron. In ALS, also known as Lou Gehrig’s disease, this occurs in motor neurons, affecting movement; in FTD, it occurs in the frontal lobe of the brain, leading to dementia. "In both diseases, TDP-43 is over-expressed or mutated, and the scientific debate has been whether loss of TDP-43 in the nucleus or gain of TDP-43 in the cytoplasm is the problem," Moussa says. "Our study suggests TDP-43 in the cell cytoplasm is deposited there in order to eventually be destroyed — without contributing to disease — and that TDP-43 in the nucleus is causing the damage," he says. "Because so much protein is being produced, the cell can’t keep up with removing these toxic particles in the nucleus and the dumping of them in the cytoplasm. There may be a way to fix this problem." Moussa has long studied parkin, a molecule best known, when mutated and inactive, for its role in a familial form of Parkinson’s disease. He has studied it in Alzheimer’s disease and other forms of dementia. His hypothesis, which he has demonstrated in several recently published studies, is that parkin could help remove the toxic fragments of amyloid beta protein that builds up in the brains of Alzheimer’s disease patients. What’s more, he developed a method to clear this amyloid beta when it begins to build up in neurons — a gene therapy strategy he has shown works in rodents. Work continues on this potential therapy. In this study, Moussa found that parkin “tags” TDP-43 protein in the nucleus with a molecule that takes it from the nucleus and into the cytoplasm of the cell. “This is good. If TDP-43 is in the cytoplasm, it will prevent further nuclear damage and deregulation of genetic materials that determine protein identity,” he says. "This discovery challenges the dogma that accumulation of TDP-43 in the cytoplasm is," Moussa says. "We think parkin is tagging proteins in the nucleus for destruction, but there just isn’t enough parkin around — compared with over-production of TDP-43 — to do the job." Moussa says his next research steps will be to inject a drug that activates parkin to see whether that can prolong the lifespan and reduce motor defects in mice with ALS. (Image: iStock)

Removing protein ‘garbage’ in nerve cells may help control 2 neurodegenerative diseases

Neuroscientists at Georgetown University Medical Center say they have new evidence that challenges scientific dogma involving two fatal neurodegenerative diseases — amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) — and, in the process, have uncovered a possible therapeutic target as a novel strategy to treat both disorders.

The study, posted online in the Journal of Biological Chemistry, found that the issue in both diseases is the inability of the cell’s protein garbage disposal system to “pull out” and destroy TDP-43, a powerful, sometimes mutated gene that produces excess amounts of protein inside the nucleus of a nerve cell, or neuron.

"This finding suggests that if we’re able to ‘rev up’ that clearance machinery and help the cell get rid of the bad actors, it could possibly reduce or slow the development of ALS and FTD," says the study’s lead investigator, neuroscientist Charbel E-H Moussa, MB, PhD. "The potential of such an advance is very exciting." He cautions, though, that determining if this strategy is possible in humans could take many years and will involve teams of researchers.

The way to rev up protein disposal is to add parkin — the cell’s natural disposal units — to brain cells. In this study, Moussa and his colleagues demonstrated in two animal experiments that delivering parkin genes to neurons slowed down ALS pathologies linked to TDP-43.”

Moussa says that his study further demonstrates that clumps known as “inclusions” of TDP-43 protein found inside neuron bodies in both disorders do not promote these diseases, as some researchers have argued.

What happens in both diseases is that this protein, which is a potent regulator of thousands of genes, leaves the nucleus and collects inside the gel-like cytoplasm of the neuron. In ALS, also known as Lou Gehrig’s disease, this occurs in motor neurons, affecting movement; in FTD, it occurs in the frontal lobe of the brain, leading to dementia.

"In both diseases, TDP-43 is over-expressed or mutated, and the scientific debate has been whether loss of TDP-43 in the nucleus or gain of TDP-43 in the cytoplasm is the problem," Moussa says.

"Our study suggests TDP-43 in the cell cytoplasm is deposited there in order to eventually be destroyed — without contributing to disease — and that TDP-43 in the nucleus is causing the damage," he says. "Because so much protein is being produced, the cell can’t keep up with removing these toxic particles in the nucleus and the dumping of them in the cytoplasm. There may be a way to fix this problem."

Moussa has long studied parkin, a molecule best known, when mutated and inactive, for its role in a familial form of Parkinson’s disease. He has studied it in Alzheimer’s disease and other forms of dementia. His hypothesis, which he has demonstrated in several recently published studies, is that parkin could help remove the toxic fragments of amyloid beta protein that builds up in the brains of Alzheimer’s disease patients.

What’s more, he developed a method to clear this amyloid beta when it begins to build up in neurons — a gene therapy strategy he has shown works in rodents. Work continues on this potential therapy.

In this study, Moussa found that parkin “tags” TDP-43 protein in the nucleus with a molecule that takes it from the nucleus and into the cytoplasm of the cell. “This is good. If TDP-43 is in the cytoplasm, it will prevent further nuclear damage and deregulation of genetic materials that determine protein identity,” he says.

"This discovery challenges the dogma that accumulation of TDP-43 in the cytoplasm is," Moussa says. "We think parkin is tagging proteins in the nucleus for destruction, but there just isn’t enough parkin around — compared with over-production of TDP-43 — to do the job."

Moussa says his next research steps will be to inject a drug that activates parkin to see whether that can prolong the lifespan and reduce motor defects in mice with ALS.

(Image: iStock)

Filed under nerve cells neurodegenerative diseases parkin brain cells ALS protein neuroscience science

46 notes

Transplanted neural stem cells treat ALS in mouse model Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is untreatable and fatal. Nerve cells in the spinal cord die, eventually taking away a person’s ability to move or even breathe. A consortium of ALS researchers at multiple institutions, including Sanford-Burnham Medical Research Institute, Brigham and Women’s Hospital, and the University of Massachusetts Medical School, tested transplanted neural stem cells as a treatment for the disease. In 11 independent studies, they found that transplanting neural stem cells into the spinal cord of a mouse model of ALS slows disease onset and progression. This treatment also improves host motor function and significantly prolongs survival. Surprisingly, the transplanted neural stem cells did not benefit ALS mice by replacing deteriorating nerve cells. Instead, neural stem cells help by producing factors that preserve the health and function of the host’s remaining nerve cells. They also reduce inflammation and suppress the number of disease-causing cells in the host’s spinal cord. These findings, published December 19 in Science Translational Medicine, demonstrate the potential neural stem cells hold for treating ALS and other nervous system disorders. “While not a cure for human ALS, we believe that the careful transplantation of neural stem cells, particularly into areas that can best sustain life—respiratory control centers, for example—may be ready for clinical trials,” Evan Y. Snyder, M.D., Ph.D., director of Sanford-Burnham’s Stem Cell and Regenerative Biology Program and senior author of the study. Neural stem cells In this study, researchers at multiple institutions conducted 11 independent studies to test neural stem cell transplantation in a well-established mouse model of ALS. They all found that this cell therapy reduced the symptoms and course of the ALS-like disease. They observed improved motor performance and respiratory function in treated mice. Neural stem cell transplant also slowed the disease’s progression. What’s more, 25 percent of the treated ALS mice in this study survived for one year or more—roughly three to four times longer than untreated mice. Neural stem cells are the precursors of all brain cells. They can self-renew, making more neural stem cells, and differentiate, becoming nerve cells or other brain cells. These cells can also rescue malfunctioning nerve cells and help preserve and regenerate host brain tissue. But they’ve never before been studied extensively in a good model of adult ALS. How neural stem cells benefit ALS mice Transplanted neural stem cells helped the ALS mice, but not for the obvious reason—not because they became nerve cells, replacing those missing in the ALS spinal cord. The biggest impact actually came from a series of other beneficial neural stem cell activities. It turns out neural stem cells produce protective molecules. They also trigger host cells to produce their own protective molecules. In turn, these factors help spare host nerve cells from further destruction. Then a number of other positive events take place in treated mice. The transplanted normal neural stem cells change the fate of the host’s own diseased neural stem cells—for the better. This change decreases the number of toxin-producing, disease-promoting cells in the host’s spinal cord. Transplanted neural stem cells also reduce inflammation. “We discovered that cell replacement plays a surprisingly small role in these impressive clinical benefits. Rather, the stem cells change the host environment for the better and protect the endangered nerve cells,” said Snyder. “This realization is important because most diseases are now being recognized as multifaceted in their cause and their symptoms—they don’t involve just one cell type or one malfunctioning process. We are coming to recognize that the multifaceted actions of the stem cell may address a number of these disease processes.”

Transplanted neural stem cells treat ALS in mouse model

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is untreatable and fatal. Nerve cells in the spinal cord die, eventually taking away a person’s ability to move or even breathe. A consortium of ALS researchers at multiple institutions, including Sanford-Burnham Medical Research Institute, Brigham and Women’s Hospital, and the University of Massachusetts Medical School, tested transplanted neural stem cells as a treatment for the disease. In 11 independent studies, they found that transplanting neural stem cells into the spinal cord of a mouse model of ALS slows disease onset and progression. This treatment also improves host motor function and significantly prolongs survival.

Surprisingly, the transplanted neural stem cells did not benefit ALS mice by replacing deteriorating nerve cells. Instead, neural stem cells help by producing factors that preserve the health and function of the host’s remaining nerve cells. They also reduce inflammation and suppress the number of disease-causing cells in the host’s spinal cord. These findings, published December 19 in Science Translational Medicine, demonstrate the potential neural stem cells hold for treating ALS and other nervous system disorders.

“While not a cure for human ALS, we believe that the careful transplantation of neural stem cells, particularly into areas that can best sustain life—respiratory control centers, for example—may be ready for clinical trials,” Evan Y. Snyder, M.D., Ph.D., director of Sanford-Burnham’s Stem Cell and Regenerative Biology Program and senior author of the study.

Neural stem cells

In this study, researchers at multiple institutions conducted 11 independent studies to test neural stem cell transplantation in a well-established mouse model of ALS. They all found that this cell therapy reduced the symptoms and course of the ALS-like disease. They observed improved motor performance and respiratory function in treated mice. Neural stem cell transplant also slowed the disease’s progression. What’s more, 25 percent of the treated ALS mice in this study survived for one year or more—roughly three to four times longer than untreated mice.

Neural stem cells are the precursors of all brain cells. They can self-renew, making more neural stem cells, and differentiate, becoming nerve cells or other brain cells. These cells can also rescue malfunctioning nerve cells and help preserve and regenerate host brain tissue. But they’ve never before been studied extensively in a good model of adult ALS.

How neural stem cells benefit ALS mice

Transplanted neural stem cells helped the ALS mice, but not for the obvious reason—not because they became nerve cells, replacing those missing in the ALS spinal cord. The biggest impact actually came from a series of other beneficial neural stem cell activities. It turns out neural stem cells produce protective molecules. They also trigger host cells to produce their own protective molecules. In turn, these factors help spare host nerve cells from further destruction.

Then a number of other positive events take place in treated mice. The transplanted normal neural stem cells change the fate of the host’s own diseased neural stem cells—for the better. This change decreases the number of toxin-producing, disease-promoting cells in the host’s spinal cord. Transplanted neural stem cells also reduce inflammation.

“We discovered that cell replacement plays a surprisingly small role in these impressive clinical benefits. Rather, the stem cells change the host environment for the better and protect the endangered nerve cells,” said Snyder. “This realization is important because most diseases are now being recognized as multifaceted in their cause and their symptoms—they don’t involve just one cell type or one malfunctioning process. We are coming to recognize that the multifaceted actions of the stem cell may address a number of these disease processes.”

Filed under stem cells stem cell transplantation ALS nervous system nerve cells science

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