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.

Scientists Identify ‘Clean-Up’ Snafu That Kills Brain Cells in Parkinson’s Disease

Researchers at Albert Einstein College of Medicine of Yeshiva University have discovered how the most common genetic mutations in familial Parkinson’s disease damage brain cells. The study, which published online in the journal Nature Neuroscience, could also open up treatment possibilities for both familial Parkinson’s and the more common form of Parkinson’s that is not inherited.

"Our study found that abnormal forms of LRRK2 protein disrupt an important garbage-disposal process in cells that normally digests and recycles unwanted proteins including one called alpha-synuclein - the main component of those protein aggregates that gunk up nerve cells in Parkinson’s patients," said study leader Ana Maria Cuervo, M.D., Ph.D., professor of  developmental and molecular biology, of anatomy and structural biology, and of medicine and the Robert and Renee Belfer Chair for the Study of Neurodegenerative Diseases at Einstein.

The name for the disrupted disposal process is chaperone-mediated autophagy (the word “autophagy” literally means “self-eating”). It involves specialized molecules that “guide” old and damaged proteins to enzyme-filled structures called lysosomes; there the proteins are digested into amino acids, which are then recycled within the cell.

"We showed that when LRRK2 inhibits chaperone-mediated autophagy,
alpha-synuclein doesn’t get broken down and instead accumulates to toxic levels in nerve cells,” said Dr. Cuervo.

The study involved mouse neurons in tissue culture from four different animal models, neurons from the brains of patients with Parkinson’s with  LRRK2 mutations, and neurons derived from the skin cells of Parkinson’s patients via induced pluripotent stem (iPS) cell technology. All the lines of research confirmed the researchers’ discovery.

"We’re now looking at ways to enhance the activity of this recycling system to see if we can prevent or delay neuronal death and disease," said Dr. Cuervo. "We’ve started to analyze some chemical compounds that look very promising."

Dr. Cuervo hopes that such treatments could help patients with familial as well as nonfamilial Parkinson’s - the predominant form of the disease that also involves the buildup of alpha-synuclein.

Dr. Cuervo is credited with discovering chaperone-mediated autophagy. She has published extensively on autophagy and its role in numerous diseases, such as cancer and Huntington’s disease, and its role in age-related conditions, including organ decline and weakened immunity. Dr. Cuervo is co-director of Einstein’s  Institute of Aging Research.

(Image: Shutterstock)

Parkin protects from neuronal cell death

Parkinson’s disease is the most common movement disorder and the second most common neurodegenerative disease after Alzheimer’s disease. It is characterized by the loss of dopamin-producing neurons in the substantia nigra, a region in the midbrain, which is implicated in motor control. The typical clinical signs include resting tremor, muscle rigidity, slowness of movements, and impaired balance. In about 10% of cases Parkinson’s disease is caused by mutations in specific genes, one of them is called parkin.

“Parkinson-associated genes are particularly interesting for researchers, since insights into the function and dysfunction of these genes allow conclusions on the pathomechanisms underlying Parkinson’s disease”, says Dr. Konstanze Winklhofer of the Adolf Butenandt Institute at the LMU Munich, who is also affiliated with the German Center for Neurodegenerative Diseases (DZNE). Winklhofer and her colleagues had previously observed that parkin can protect neurons from cell death under various stress conditions. In the course of this project, it became obvious that a loss of parkin function impairs the activity and integrity of mitochondria, which serve as the cellular power stations. In their latest publication, Winklhofer and coworkers uncovered the molecular mechanism that accounts for parkin’s neuroprotective action.

“We discovered a novel signaling pathway that is responsible for the neuroprotective activity of parkin,” Winklhofer reports. The central player of this pathway is a protein called NEMO, which is activated by the enzymatic attachment of a linear chain of ubiquitin molecules. This reaction is promoted by parkin, thereby enabling NEMO to activate a signal cascade, which ultimately leads to the expression of a specific set of genes. Winklhofer’s team identified one essential gene targeted by this pathway, which turned out to code for the mitochondrial protein OPA1. OPA1 maintains the integrity of mitochondria and prevents stress-induced neuronal cell death.

“These findings suggest that strategies to activate this signal pathway or to enhance the synthesis of OPA1 in cells exposed to stress could be of therapeutic benefit,” Winklhofer points out.

The newly identified signal pathway may also be relevant in the context of other neurological conditions that are characterized by the loss of specific neurons. Konstanze Winklhofer and her group are already engaged in further projects designed to determine whether other molecules regulated by this pathway might provide targets for therapeutic interventions.

New chemical probe provides tool to investigate role of malignant brain tumor domains

In an article published as the cover story of the March 2013 issue of Nature Chemical Biology, Lindsey James, PhD, research assistant professor in the lab of Stephen Frye, Fred Eshelman Distinguished Professor in the UNC School of Pharmacy and member of the UNC Lineberger Comprehensive Cancer Center, announced the discovery of a chemical probe that can be used to investigate the L3MBTL3 methyl-lysine reader domain. The probe, named UNC1215, will provide researchers with a powerful tool to investigate the function of malignant brain tumor (MBT) domain proteins in biology and disease.

“Before this there were no known chemical probes for the more than 200 domains in the human genome that recognize methyl lysine. In that regard, it is a first in class compound. The goal is to use the chemical probe to understand the biology of the proteins that it targets,” said Dr. James.

Chromatin regulatory pathways play a fundamental role in gene expression and disease development, especially in the case of cancer. While many chemical probes work through the inhibition of enzyme activity, L3MBTL3 functions as a mediator of protein-to-protein interactions, which have been historically difficult to target with small, drug-like molecules.The researchers found three to four further disease subtypes within TN tumors, with more than 75 percent of the tumors falling into the basal-like subtype. Further research is needed to identify the distinct biomarkers shared by the expanded subtypes of TN cancers. The ultimate goal will be to target the individual biomarkers of these subtypes and create therapies that target their individual biology, according to Dr. Perou.

“Many people believe that protein-protein interactions are difficult to target. Often they have a large surface area, so it is hard for small molecules to go in and intervene,” said Dr. James.

Almost 40 percent of the genes that drive cancer can be mapped to dysfunction within signaling pathways. In the last five years, chemical probe development has allowed researchers to make fundamental observations of the role of these pathways in cancer development, as well as pointing to potential targets for new therapies. Each of the complex interactions within the signaling pathways represents a potential point where a therapy can be applied, and the probes allow researchers to interact with these processes at the molecular level and observe the overall effect of their perturbation on the disease state.

In a 2008 Nature Chemical Biology commentary, Dr. Frye outlined the qualities that make a good chemical probe. To Frye, a good chemical probe must be highly selective to enable specific questions to be asked and it must function as well in a cell as in the test tube, providing clear quantitative data with a well understood mechanism of action in either situation. It also must be available to all academic researchers without restrictions on its use, a criteria that the L3MBTL3 probe fulfills through the Frye lab’s commitment to provide researchers with the probe free of charge on request and UNC1215 is already available through commercial vendors as well.

Research reveals Huntington’s hope

Researchers in Scotland and Germany have discovered a molecular mechanism that shows promise for developing a cure for Huntington’s Disease (HD).

Scientists from the University of Dundee, the German Center for Neurodegenerative Diseases (DZNE) in Bonn, the Max-Planck Institute for Molecular Genetics in Berlin and the Johannes Gutenberg-Universität Mainz have found a mechanism that specifically stirs and induces the synthesis of disease-making protein in HD patients.

Their data lead to the conclusion that a selective overproduction of aberrant Huntington protein in patients is a key step in the establishment of the disease, which affects 1 in 10,000 people in Western countries and is so far incurable.

"This is a very promising strategy to develop a small molecule drug therapy that is able to inhibit the production of disease-making protein," said Professor Susann Schweiger of the University of Dundee and Johannes Gutenberg-Universität Mainz.

"Theoretically, if you don’t have the disease-making protein then you don’t have the disease. Obviously we still have work to do to develop a drug to target these mechanisms and inhibit the production of this protein but we think this research is attractive to drug discovery and ongoing work in this area is being carried out."

The gene responsible for causing Huntington’s Disease was first identified in 1993, leading to hopes that a specific therapy for HD would soon be on the market. However, cell biology and brain pathology of HD showed it to be more complicated than originally anticipated and only symptomatic treatments to slightly relieve the distress of single components of the disease are currently available.

The new discovery once again raises hopes that a curative therapy can be established. The scientists found that it was mainly three proteins - the mammalian target of rapamycin (mTOR), protein phosphatase 2A (PP2A) and Midline 1 (MID1) - that specifically drive the production of disease-making protein in HD patients.

As a result, more and more aberrant protein is produced with time, which leads to a protein overload in the cell. By interfering with the function of the three proteins it is possible to disrupt this circle and prevent the synthesis of aberrant protein in HD patients.

The Dundee-Germany research is published in the latest edition of the Nature Communications journal.

Blood marrow derived cells regulate appetite

Bone marrow cells that produce brain-derived eurotrophic factor (BDNF), known to affect regulation of food intake, travel to part of the hypothalamus in the brain where they “fine-tune” appetite, said researchers from Baylor College of Medicine and Shiga University of Medical Science in Otsu, Shiga, Japan, in a report that appears online in the journal Nature Communications.

"We knew that blood cells produced BDNF," said Dr. Lawrence Chan, professor of molecular and cellular biology and professor and chief of the division of diabetes, endocrinology & metabolism in the department of medicine and director of the federally funded Diabetes Research Center, all at BCM. The factor is produced in the brain and in nerve cells as well. "We didn’t know why it was produced in blood cells."

Fluorescent marker reveals surprise

Dr. Hiroshi Urabe and Dr. Hideto Kojima, current and former postdoctoral fellows in Chan’s laboratory respectively, looked for BDNF in the brains of mice who had not been fed for about 24 hours. The bone marrow-derived cells had been marked with a fluorescent protein that showed up on microscopy. To their surprise, they found cells producing BDNF in a part of the brain’s hypothalamus called the paraventricular nucleus.

"We knew that in embryonic development, some blood cells do go to the brain and become microglial cells," said Chan. (Microglial cells form part of the supporting structure of the central nervous system. They are characterized by a nucleus from which "branches" expand in all directions.) "This is the first time we have shown that this happens in adulthood. Blood cells can go to one part of the brain and become physically changed to become microglial-like cells."

However, these bone marrow cells produce a bone marrow-specific variant of BDNF, one that is different from that produced by the regular microglial cells already in the hypothalamus.

Only a few of these blood-derived cells actually reach the hypothalamus, said Chan.

"It’s not very impressive if you look casually under the microscope," he said. However, a careful scrutiny showed that the branching nature of these cells allow them to come into contact with a whole host of brain cells.

"Their effects are amplified," said Chan.

Curbing the urge

Mice that are born lacking the ability to produce blood cells that make BDNF overeat, become obese and develop insulin resistance (a lack of response to insulin that affects the ability to metabolize glucose). A bone marrow transplant that restores the gene for making the cells that produce BDNF can normalize appetite, said Chan. However, a transplant of bone marrow that does not contain this gene does not reverse overeating, obesity or insulin resistance.

When normal bone marrow cells that produce BDNF are injected into the third ventricle (a fluid-filled cavity in the brain) of mice that lack BDNF, they no longer have the urge to overeat, said Chan.

All in all, the studies represent a new mechanism by which these bone-marrow derived cells control feeding through BDNF and could provide a new avenue to attack obesity, said Chan.

He and his colleagues hypothesize that the bone marrow cells that produce BDNF fine tune the appetite response, although a host of different appetite-controlling hormones produced by the regular nerve cells in the hypothalamus do the lion’s share of the work.

"Bone marrow cells are so accessible," said Chan. “If these cells play a regulatory role, we could draw some blood, modify something in it or add something that binds to blood cells and give it back. We may even be able to deliver medication that goes to the brain," crossing the blood-brain barrier. Even a few of these cells can have an effect because their geometry means that they have contact with many different neurons or nerve cells.

Researchers find controlling element of Huntington’s disease: Molecular troika regulates production of harmful protein

A three molecule complex may be a target for treating Huntington’s disease, a genetic disorder affecting the brain. This finding by an international research team including scientists from the German Center for Neurodegenerative Diseases (DZNE) in Bonn and the University of Mainz was published in the online journal “Nature Communications”. The report states that the so-called MID1 complex controls the production of a protein which damages nerve cells.

The long DNA sequences in Huntington’s disease lead to changes in a certain protein called “Huntingtin”. The DNA is like an archive of blueprints for proteins. Errors in the DNA therefore result in defective proteins. “Huntingtin is essential for the organism’s survival. It is a multi-talent which is important for many processes,” emphasises Krauss. “If the protein is defective, brain cells may die.“

In the spotlight: protein synthesis
In the current study, the scientists around Sybille Krauss and the Mainz-based human geneticist Susann Schweiger took a closer look at a critical stage of protein production – translation. At this step, a copy of the DNA, the so-called messenger RNA, is processed by the cell’s protein factories. In patients with Huntington’s disease, the messenger RNA contains an unusually high number of consecutive CAG sequences – CAG representing the building plan for the amino acid glutamine.

These repetitive sequences have a direct consequence: more glutamine than normal is built into Huntingtin, which is therefore defective. Sybille Krauss and her colleagues have now identified a group of three molecules, which regulate the production of this protein. “We were able to show that this complex binds to the messenger RNA and controls the synthesis of defective Huntingtin,” says Krauss. When the scientists reduced the concentration of this so-called MID1 complex in the cell, production of the defective protein declined.

“If we could find a way of influencing this complex, for example with pharmaceuticals, it is quite possible that we could directly affect the production of defective Huntingtin. This kind of treatment would not just treat the symptoms but also the causes of Huntington’s disease,” says Krauss.

Background:Three molecules come together
The complex consists of MID1, from which it gets its name, and the proteins PP2Ac and S6K. “Every single one of these proteins is known to be important for translation. We have discovered that in the specific case of Huntington’s disease, they together bind to the CAG sequences. This was previously unknown. We also found that binding increases with repeat lengths,” says Krauss. “In sequences of normal length, we found only weak binding or none at all.”

The Bonn-based molecular biologist and her colleagues investigated the effect of the MID1 complex and the interaction between its components in a series of elaborate laboratory experiments. “This project took several years of research work,” says Krauss. Along with biochemical procedures, the scientists used cell cultures and analysed proteins from the brains of mice. The mice’s genetic code had been modified in such a way that it contained elongated CAG-repeats as it is typical for Huntington’s disease.

From previous studies it was already known that the protein MID1 tends to bind messenger RNAs. The scientists were now able to show that MID1 also attaches to messenger RNAs with excessively long CAG sequences. Furthermore, experiments showed that PP2Ac and S6K also bound the RNA in the presence of MID1. However, if the MID1 was depleted, this binding did not occur. “From this, we can conclude that these three proteins form a molecular complex, which binds to the RNA. MID1 is a key component. It actually seems to keep together its binding partners,” Krauss comments on the results of the experiments.

Complex controls protein production
The researchers were also able to prove that the MID1 complex controls the translation of RNA with excessively long CAG sequences. For this, they investigated various cell cultures. The cells produced either normal Huntingtin or – due to excessively long sequences in their DNA – a defective version of this protein. The scientists reduced the occurrence of MID1 inside the cells using a procedure known as “knock-down”. The elimination of this protein, which is a major part of the MID1 complex, had direct consequences: the production of defective Huntingtin declined. “However, it did not affect the production of normal Huntingtin,” emphazises Krauss. “This further proves that the MID1 complex specifically targets RNAs with excessively long CAG sequences.”

Highly specific
The Bonn-based molecular biologist sees this specific influence as a chance to treat Huntington’s disease: “The MID1 complex is a promising target for therapy. It indicates a possibility to suppress the production of defective Huntingtin only, while not affecting the production of normal Huntingtin. This is of particular significance, because the normal protein is also being produced in the patients’ bodies and it is important for the organism.”

A suitable active substance has yet to be found, says Krauss. However, the next developments are in sight: “We now want to test potential substances in the laboratory,” she says.

Clues to Fetal Alcohol Risk: Molecular switch promises new targets for diagnosis and therapy

Fetal alcohol syndrome is the leading preventable cause of developmental disorders in developed countries. And fetal alcohol spectrum disorder (FASD), a range of alcohol-related birth defects that includes fetal alcohol syndrome, is thought to affect as many as 1 in 100 children born in the United States.

Any amount of alcohol consumed by the mother during pregnancy poses a risk of FASD, a condition that can include the distinct pattern of facial features and growth retardation associated with fetal alcohol syndrome as well as intellectual disabilities, speech and language delays, and poor social skills. But drinking can have radically different outcomes for different women and their babies. While twin studies have suggested a genetic component to susceptibility to FASD, researchers have had little success identifying who is at greatest risk or what genes are at play.

Research from Harvard Medical School and Veterans Affairs Boston Healthcare System sheds new light on this question, identifying for the first time a signaling pathway that might determine genetic susceptibility for the development of FASD. The study was published online Feb. 19 in the journal Proceedings of the National Academy of Sciences.

“Our work points to candidate genes for FASD susceptibility and identifies a path for the rational development of drugs that prevent ethanol neurotoxicity,” said Michael Charness, chief of staff at VA Boston Healthcare System and HMS professor of neurology. “And importantly, identifying those mothers whose fetuses are most at risk could help providers better target intensive efforts at reducing drinking during pregnancy.”

The discovery also solves a riddle that had intrigued Charness and other researchers for nearly two decades. In 1996, Charness and colleagues discovered that alcohol disrupted the work of a human protein critical to fetal neural development—a major clue to the biological processes of FASD. The protein, L1, projects through the surface of a cell to help it adhere to its neighbors. When Charness and his team introduced the protein to a culture of mouse fibroblasts cells, L1 increased cell adhesion. Tellingly, the effect was erased in the presence of ethanol (beverage alcohol).

Charness and his team went on to develop multiple cell lines from that first culture, and that’s where they encountered the riddle: In some of those lines, alcohol disrupted L1’s adhesive effect, while in others it did not.

“How could it be possible that a cell that expresses L1 is completely sensitive to alcohol, and others that express it are completely insensitive?” asked Charness, who is also faculty associate dean for veterans hospital programs at HMS and assistant dean at Boston University School of Medicine.

Clearly, something else was affecting the protein’s sensitivity to alcohol — but what? Studies of twins provided one clue: Identical twins are more likely than fraternal twins to have the same diagnosis, positive or negative, for FASD. “That concordance suggests that there are modifying genes, susceptibility genes, that predispose to this condition,” Charness said.

Stopping cold: USC scientists turn off the ability to feel cold

USC neuroscientists have isolated chills at a cellular level, identifying the sensory network of neurons in the skin that relays the sensation of cold.

David McKemy, associate professor of neurobiology in the USC Dornsife College of Letters, Arts and Sciences, and his team managed to selectively shut off the ability to sense cold in mice while still leaving them able to sense heat and touch.

In prior work, McKemy discovered a link between the experience of cold and a protein known as TRPM8 (pronounced trip-em-ate), which a sensor of cold temperatures in neurons in the skin, as well as a receptor for menthol, the cooling component of mint. Now, in a paper appearing in the Journal of Neuroscience on February 13, McKemy and his co-investigators have isolated and ablated the neurons that express TRPM8, giving them the ability to test the function of these cells specifically.

Using mouse-tracking software program developed by one of McKemy’s students, the researchers tested control mice and mice without TRPM8 neurons on a multi-temperature surface. The surface temperature ranged from 0 degrees to 50 degrees Celsius (32 to 122 degrees Farenheit), and mice were allowed to move freely among the regions.

The researchers found that mice depleted of TRPM8 neurons could not feel cold, but still responded to heat. Control mice tended to stick to an area around 30 degrees Celsius (86 degrees Fahrenheit) and avoided both colder and hotter areas. But mice without TRPM8 neurons avoided only hotter plates and not cold — even when the cold should have been painful or was potentially dangerous.

In tests of grip strength, responses to touch, or coordinated movements, such as balancing onto a rod while it rotated, there was no difference between the control mice and the mice without TRPM8-expressing neurons.

By better understanding the specific ways in which we feel sensations, scientists hope to one day develop better pain treatments without knocking out all ability to feel for suffering patients.

"The problem with pain drugs now is that they typically just reduce inflammation, which is just one potential cause of pain, or they knock out all sensation, which often is not desirable," McKemy said. "One of our goals is to pave the way for medications that address the pain directly, in a way that does not leave patients completely numb."