Posts tagged brain cells
Articles and news from the latest research reports.
Small brain biopsies can be used to grow large numbers of patient’s own brain cells
A group of really brainy scientists have moved closer to growing “therapeutic” brain cells in the laboratory that can be re-integrated back into patients’ brains to treat a wide range of neurological conditions. According to new research published online in The FASEB Journal, brain cells from a small biopsy can be used to grow large numbers of new personalized cells that are not only “healthy,” but also possess powerful attributes to preserve and protect the brain from future injury, toxins and diseases. Scientists are hopeful that ultimately these cells could be transformed in the laboratory to yield specific cell types needed for a particular treatment, or to cross the “blood-brain barrier” by expressing specific therapeutic agents that are released directly into the brain.
"This work is an example of how integrating basic science and clinical care may reveal privileged opportunities for biomedical research," said Matthew O. Hebb, M.D., Ph.D., FRCSC, a researcher involved in the work from the Departments of Clinical Neurological Sciences (Neurosurgery), Oncology and Otolaryngology at the University of Western Ontario in Ontario, Canada. "It is our hope that the results of this study provide a footing for further advancement of personalized, cell-based treatments for currently incurable and devastating neurological disorders."
Scientists enrolled patients with Parkinson’s disease who were scheduled to have deep brain stimulation (DBS) surgery, a commonly used procedure that involves placing electrodes into the brain. Before the electrodes were implanted, small biopsies were removed near the surface of the brain and multiplied in culture to generate millions of patient-specific cells that were then subjected to genetic analysis. These cells were complex in their make-up, but exhibited regeneration and characteristics of a fundamental class of brain cells, called glia. They expressed a broad array of natural and potent protective agents, called neurotrophic factors.
"From an extremely small amount of brain tissue, we will one day be able to do very big things," said Gerald Weissmann, M.D., Editor-in-Chief of The FASEB Journal. “For centuries, treating the brain effectively and safely has been elusive. This advance opens the doors to not only new therapies for a myriad of brain diseases, but new ways of delivering therapies as well.”
(Source: eurekalert.org)
Researchers Ferret Out Function Of Autism Gene
Findings in bacteria, yeast, mice show how flawed transport gene contributes to the condition
Researchers say it’s clear that some cases of autism are hereditary, but have struggled to draw direct links between the condition and particular genes. Now a team at the Johns Hopkins University School of Medicine, Tel Aviv University and Technion-Israel Institute of Technology has devised a process for connecting a suspect gene to its function in autism.
In a report in the Sept. 25 issue of Nature Communications, the scientists say mutations in one such autism-linked gene, dubbed NHE9, which is involved in transporting substances in and out of structures within the cell, causes communication problems among brain cells that likely contribute to autism.
“Autism is considered one of the most inheritable neurological disorders, but it is also the most complex,” says Rajini Rao, Ph.D., a professor of physiology in the Institute for Basic Biomedical Sciences at the Johns Hopkins University School of Medicine. “There are hundreds of candidate genes to sort through, and a single genetic variant may have different effects even within the same family. This makes it difficult to separate the chaff from the grain, to distinguish harmless variations from disease-causing mutations. We were able to use a new process to screen variants in one candidate gene that has been linked to autism, and figure out how they might contribute to the disorder.”
An estimated one in 88 children in the United States is affected by autism spectrum disorders, a group of neurological development conditions marked by varying degrees of social, communication and behavioral problems. Scientists for years have looked for the biological roots of the problem using tools such as genome-wide association studies and gene-linkage analysis, which crunch genetic and health data from thousands of people in an effort to pinpoint disease-causing genetic variants. But while such techniques have turned up a number of gene mutations that may be linked to autism, none of them appear in more than 1 percent of people with the condition. With numbers that low, researchers need a way to screen variants in order to make a definitive link, Rao says.
For the new study, Rao and her collaborators focused on NHE9, which other researchers had flagged as a suspect in attention-deficit hyperactivity disorder, addiction and epilepsy as well as autism spectrum disorders. The gene was already known to be involved in transporting hydrogen, sodium and potassium ions in and out of cellular compartments called endosomes, and the team wondered how this function might be related to neurological conditions.
Rao’s collaborators at Tel Aviv University and Technion-Israel Institute of Technology constructed a computer model of the NHE9 protein based on previous research on a distant relative in bacteria. They then used the model to predict how autism-linked variants in the NHE9 gene would affect the protein’s shape and function. Some of them were predicted to cause dramatic changes, while other changes appeared to be more subtle.
Rao’s team next tested how these variant forms of NHE9 would affect a relatively simple organism often used in genetic studies: yeast. “Using yeast to screen the function of variants was a quick, easy and inexpensive way of figuring out which were worth further study, and which we could ignore because they didn’t have any effect,” Rao says. To do that, the team engineered the yeast form of NHE9 to have the variants seen in autistic people.
For those mutations that did have a detectable effect on the yeast, the team moved on to a third and more challenging step, in mouse brains. They homed in on astrocytes, a type of brain cell that clears the signaling molecule glutamate out of the way after it has performed its job of delivering a message across a synapse between two nerve cells. Using lab-grown mouse astrocytes with variant forms of NHE9, the researchers found a change in the pH (acidity) inside cellular compartments called endosomes, which in turn altered the ability of cells to take up glutamate. Because endosomes are the vehicles that deliver cargo essential for communication between brain cells, changing their pH alters traffic to and from the cell surface, which could affect learning and memory, Rao says. “Elevated glutamate levels are known to trigger seizures, perhaps explaining why autistic patients with mutations in NHE9 and related genes also have seizures,” she notes.
Rao and her team hope that pinpointing the importance of this trafficking mechanism in autism spectrum disorders may lead to the development of new drugs for autism that alter endosomal pH. As the use of genomic data becomes increasingly commonplace in the future, the step-wise strategy devised by her team can be used to screen gene variants and identify at-risk patients, she says.
(Source: hopkinsmedicine.org)
Scientists identify brain circuitry that triggers overeating
The finding shows that certain parts of brain cells could play a critical role in anorexia, bulimia, binge eating disorder, and obesity.
Sixty years ago scientists could electrically stimulate a region of a mouse’s brain causing the mouse to eat, whether hungry or not. Now researchers from UNC School of Medicine have pinpointed the precise cellular connections responsible for triggering that behavior. The finding, published September 27 in the journal Science, lends insight into a cause for obesity and could lead to treatments for anorexia, bulimia nervosa, and binge eating disorder, the most prevalent eating disorder in the United States.
“The study underscores that obesity and other eating disorders have a neurological basis,” said senior study author Garret Stuber, PhD, assistant professor in the department of psychiatry and department of cell biology and physiology. He’s also a member of the UNC Neuroscience Center. “With further study, we could figure out how to regulate the activity of cells in a specific region of the brain and develop treatments.”
Cynthia Bulik, PhD, Distinguished Professor of Eating Disorders at UNC School of Medicine and the Gillings School of Global Public Health, said, “Stuber’s work drills down to the precise biological mechanisms that drive binge eating and will lead us away from stigmatizing explanations that invoke blame and a lack of willpower.” Bulik was not part of the research team.
Back in the 1950s, when scientists electrically stimulated a region of the brain called the lateral hypothalamus, they knew that they were stimulating many different types of brain cells. Stuber wanted to focus on one cell type — gaba neurons in the bed nucleus of the stria terminalis, or BNST. The BNST is an outcropping of the amygdala, the part of the brain associated with emotion. The BNST also forms a bridge between the amygdala and the lateral hypothalamus, the brain region that drives primal functions such as eating, sexual behavior, and aggression.
The BNST gaba neurons have a cell body and a long strand with branched synapses that transmit electrical signals into the lateral hypothalamus. Stuber and his team wanted to stimulate those synapses by using an optogenetic technique, an involved process that would let him stimulate BNST cells simply by shining light on their synapses.
Typically, brain cells don’t respond to light. So Stuber’s team used genetically engineered proteins — from algae — that are sensitive to light and used genetically engineered viruses to deliver them into the brains of mice. Those proteins then get expressed only in the BNST cells, including in the synapses that connect to the hypothalamus.
His team then implanted fiber optic cables in the brains of these specially-bred mice, and this allowed the researchers to shine light through the cables and onto BNST synapses. As soon as the light hit BNST synapses the mice began to eat voraciously even though they had already been well fed. Moreover, the mice showed a strong preference for high-fat foods.
“They would essentially eat up to half their daily caloric intake in about 20 minutes,” Stuber said. “This suggests that this BNST pathway could play a role in food consumption and pathological conditions such as binge eating.”
Stimulating the BNST also led the mice to exhibit behaviors associated with reward, suggesting that shining light on BNST cells enhanced the pleasure of eating. On the flip side, shutting down the BNST pathway caused mice to show little interest in eating, even if they had been deprived of food.
“We were able to really home in on the precise neural circuit connection that was causing this phenomenon that’s been observed for more than 50 years,” Stuber said.
The study, which uses technologies highlighted in the new National Institutes of Health Brain Initiative, suggests that faulty wiring in BNST cells could interfere with hunger or satiety cues and contribute to human eating disorders, leading people to eat even when they are full or to avoid food when they are hungry. Further research is needed to determine whether it would be possible to develop drugs that correct a malfunctioning BNST circuit.
“We want to actually observe the normal function of these cell types and how they fire electrical signals when the animals are feeding or hungry,” Stuber said. “We want to understand their genetic characteristics – what genes are expressed. For example, if we find cells that become really activated after binge eating, can we look at the gene expression profile to find out what makes those cells unique from other neurons.”
And that, Stuber said, could lead to potential targets for drugs to treat certain populations of patients with eating disorders.
Propofol Discovery May Aid Development of New Anesthetics
Researchers at Washington University School of Medicine in St. Louis and Imperial College London have identified the site where the widely used anesthetic drug propofol binds to receptors in the brain to sedate patients during surgery.
Until now, it hasn’t been clear how propofol connects with brain cells to induce anesthesia. The researchers believe the findings, reported online in the journal Nature Chemical Biology, eventually will lead to the development of more effective anesthetics with fewer side effects.
“For many years, the mechanisms by which anesthetics act have remained elusive,” explained co-principal investigator Alex S. Evers, MD, the Henry E. Mallinckrodt Professor and head of the Department of Anesthesiology at Washington University. “We knew that intravenous anesthetics, like propofol, act on an important receptor on brain cells called the GABA-A receptor, but we didn’t really know exactly where they bound to that receptor.”
Propofol is a short-acting anesthetic often used in patients having surgery. It wears off quickly and is less likely to cause nausea than many other anesthetics. But the drug isn’t risk-free. Its potentially dangerous side effects include lowering blood pressure and interfering with breathing.
In an attempt to understand how propofol induces anesthesia during surgery, scientists have tried to identify its binding site within the gamma-aminobutyric acid type A (GABA-A) receptor on brain cells. Activating these receptors — with propofol, for example — depresses a cell’s activity.
Researchers have altered the amino acids that make up the GABA-A receptor in attempts to find propofol’s binding site, but Evers said those methods couldn’t identify the precise site with certainty.
“In previous work to directly identify anesthetic binding sites, GABA-A receptors had to be extracted from membranes and purified prior to performing the binding studies,” he said. “Our method allowed us to study propofol binding to the intact receptor in its native membrane environment.”
Having developed the techniques to analyze the interactions between anesthetics and GABA-A receptors in their native environment, Evers’ laboratory teamed up with a group at Imperial College that had been taking the same approach. Led by Nicholas P. Franks, PhD, professor of biophysics and anaesthetics, the group has spent years creating a photoanalogue of propofol that both behaves in precisely the same way as propofol and contains a labeling group that permanently attaches to its binding site on the GABA-A receptor when exposed to a specific wavelength of light.
In creating the analogue of propofol, it’s as if the researchers put a tiny hook onto the molecule so that when it binds to the GABA-A receptor, it grabs onto the receptor and won’t let go.
“Normally, an anesthetic drug binds to the GABA-A receptor transiently,” Franks explained. “But for the purposes of this research, we wanted to create an analogue that behaved exactly like propofol except that we could activate this chemical hook to permanently bind the drug to the receptor. The next step was then to extract the receptor, cut it into pieces and identify the precise piece of the protein where the propofol analogue had attached to the receptor. This was the tricky step that the Evers group at Washington University had perfected.”
Evers and Franks believe this technique has implications beyond propofol and other anesthetics.
“Anesthetics have desirable effects — they induce anesthesia, for example — but they also have undesirable effects,” Evers said. “Propofol can lower blood pressure or interfere with breathing, for example. By understanding precisely what the binding sites look like on the proteins that induce those potential problems, we eventually hope to design and select for drugs that have the benefits we want without dangerous side effects.”
Using the techniques they have developed, Evers and Franks now plan to identify binding sites of other anesthetic agents. They believe their approach also can be used to study other types of drugs, such as psychiatric agents and anti-seizure drugs.
Ground breaking research identifies promising drugs for treating Parkinson’s
New drugs which may have the potential to stop faulty brain cells dying and slow down the progression of Parkinson’s, have been identified by scientists in a pioneering study which is the first of its kind.
Experts from the world leading Sheffield Institute for Translational Neuroscience (SITraN) conducted a large scale drugs trial in the lab using skin cells from people with this progressive neurological condition which affects one in every 500 people in the UK.
The researchers tested over 2,000 compounds to find out which ones could make faulty mitochondria work normally again.
Mitochondria act as the power generators in all cells of our body, including the brain. Malfunctioning mitochondria are one of the main reasons why brain cells die in Parkinson’s.
One of the promising medications identified though the research is a synthetic drug called ursodeoxycholic acid (UDCA).
This licenced drug has been in clinical use for several decades to treat certain forms of liver disease which means that researchers will be able to immediately start a clinical trial to test its safety and tolerability in people with Parkinson’s.
This will discover the optimum dose to ensure that enough of the drug reaches the part of the brain where Parkinson’s develops.
Based on this information, larger randomized controlled trials can be carried out to assess the potential of UDCA to treat Parkinson’s.
The extensive drug screen, which took over five years to complete, was funded by leading research charity Parkinson’s UK, and was carried out in collaboration with the University of Trondheim, Norway.
Dr Oliver Bandmann, Reader in Neurology at SITraN, said: “Parkinson’s is so much more than just a movement disorder.
It can also lead to depression and anxiety, and a host of distressing day to day problems like bladder and bowel dysfunction.
"The best treatments currently available only improve some of the symptoms, rather than tackle the reason why Parkinson’s develops in the first place, so there is a desperate need for new drug treatments which could actually slow down the disease progression”.
"We are hopeful that this group of drugs can one day make a real difference to the lives of people with Parkinson’s”.
The results of the ground breaking study are published in the leading Neuroscience journal BRAIN.
Dr Kieran Breen, Director of Research and Innovation at Parkinson’s UK commented: “This is a really exciting time for Parkinson’s research. For the first time, we are starting to identify drugs that will treat the Parkinson’s – possibly slow down or halt its progression – rather than just the symptoms.
“This will bring us closer to our ultimate goal of a cure for Parkinson’s. We look forward to working closely with Dr Bandmann to develop this treatment”.
Scientists watch live brain cell circuits spark and fire
NIH-funded scientists show new genetically engineered proteins may be important tool for the President’s BRAIN Initiative
Scientists used fruit flies to show for the first time that a new class of genetically engineered proteins can be used to watch electrical activity in individual brain cells in live brains. The results, published in Cell, suggest these proteins may be a promising new tool for mapping brain cell activity in multiple animals and for studying how neurological disorders disrupt normal nerve cell signaling. Understanding brain cell activity is a high priority of the President’s Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative.
Brain cells use electricity to control thoughts, movements and senses. Ever since the late nineteenth century, when Dr. Luigi Galvani induced frog legs to move with electric shocks, scientists have been trying to watch nerve cell electricity to understand how it is involved in these actions. Usually they directly monitor electricity with cumbersome electrodes or toxic voltage-sensitive dyes, or indirectly with calcium detectors. This study, led by Michael Nitabach, Ph.D., J.D., and Vincent Pieribone, Ph.D., at the Yale School of Medicine, New Haven, CT, shows that a class of proteins, called genetically encoded fluorescent voltage indicators (GEVIs), may allow researchers to watch nerve cell electricity in a live animal.
Dr. Pieribone and his colleagues helped develop ArcLight, the protein used in this study. ArcLight fluoresces, or glows, as a nerve cell’s voltage changes and enables researchers to watch, in real time, the cell’s electrical activity. In this study, Dr. Nitabach and his colleagues engineered fruit flies to express ArcLight in brain cells that control the fly’s sleeping cycle or sense of smell. Initial experiments in which the researchers simultaneously watched brain cell electricity with a microscope and recorded voltage with electrodes showed that ArcLight can accurately monitor electricity in a living brain. Further experiments showed that ArcLight illuminated electricity in parts of the brain that were previously inaccessible using other techniques. Finally, ArcLight allowed the researchers to watch brain cells spark and fire while the flies were awakening and smelling. These results suggest that in the future neuroscientists may be able to use ArcLight and similar GEVIs in a variety of ways to map brain cell circuit activity during normal and disease states.
(Source: ninds.nih.gov)
The flexible tail of the prion protein poisons brain cells
For decades, there has been no answer to the question of why the altered prion protein is poisonous to brain cells. Neuropathologists from the University of Zurich and University Hospital Zurich have now shown that it is the flexible tail of the prion protein that triggers cell death. These findings have far-reaching consequences: only those antibodies that target the tail of the prion protein are suitable as potential drugs for combating prion diseases.
Prion proteins are the infectious pathogens that cause Mad Cow Disease and Creutzfeldt-Jakob disease. They occur when a normal prion protein becomes deformed and clumped. The naturally occurring prion protein is harmless and can be found in most organisms. In humans, it is found in our brain cell membrane. By contrast, the abnormally deformed prion protein is poisonous for the brain cells. Adriano Aguzzi, Professor of Neuropathology at the University of Zurich and University Hospital Zurich, has spent many years exploring why this deformation is poisonous. Aguzzi’s team has now discovered that the prion protein has a kind of «switch» that controls its toxicity. This switch covers a tiny area on the surface of the protein. If another molecule, for example an antibody, touches this switch, a lethal mechanism is triggered that can lead to very fast cell death.
Flexible tail induces cell death
In the current edition of «Nature», the scientists demonstrate that the prion protein molecule comprises two functionally distinct parts: a globular domain, which is tethered to the cell membrane, and a long and unstructured tail. Under normal conditions, this tail is very important in order to maintain the functioning of nerve cells. By contrast, in the case of a prion infection the pathogenic prion protein interacts with the globular part and the tail causes cell death – this is the hypothesis put forward by the researchers.
Aguzzi and his team tested this by generating mimetic antibodies in tissue sections from the cerebellum of mice which have a similar toxicity to that of a prion infection. The researchers found that these antibodies tripped the switch of the prion protein. «Prion proteins with a trimmed version of the flexible tail can, however, no longer damage the brain cells, even if their switch has been recognized by antibodies», explains Adriano Aguzzi. «This flexible tail is responsible for causing cell death.» If the tail is bound and made inaccessible using a further antibody, activation of the switch can likewise no longer trigger cell death.
«Our discovery has far-reaching consequences for understanding prion diseases», says Aguzzi. The findings reveal that only those antibodies that target the prion protein tail are suitable for use as potential drugs. By contrast, antibodies that trip the switch of the prion are very harmful and dangerous.
Researchers discover how brain cells change their tune
Brain cells talk to each other in a variety of tones. Sometimes they speak loudly but other times struggle to be heard. For many years scientists have asked why and how brain cells change tones so frequently. Today National Institutes of Health researchers showed that brief bursts of chemical energy coming from rapidly moving power plants, called mitochondria, may tune brain cell communication.
"We are very excited about the findings," said Zu-Hang Sheng, Ph.D., a senior principal investigator and the chief of the Synaptic Functions Section at the NIH’s National Institute of Neurological Disorders and Stroke (NINDS). "We may have answered a long-standing, fundamental question about how brain cells communicate with each other in a variety of voice tones."
The network of nerve cells throughout the body typically controls thoughts, movements and senses by sending thousands of neurotransmitters, or brain chemicals, at communication points made between the cells called synapses. Neurotransmitters are sent from tiny protrusions found on nerve cells, called presynaptic boutons. Boutons are aligned, like beads on a string, on long, thin structures called axons. They help control the strength of the signals sent by regulating the amount and manner that nerve cells release transmitters.
Mitochondria are known as the cell’s power plant because they use oxygen to convert many of the chemicals cells use as food into adenosine triphosphate (ATP), the main energy that powers cells. This energy is essential for nerve cell survival and communication. Previous studies showed that mitochondria can rapidly move along axons, dancing from one bouton to another.
In this study, published in Cell Reports, Dr. Sheng and his colleagues show that these moving power plants may control the strength of the signals sent from boutons.
"This is the first demonstration that links the movement of mitochondria along axons to a wide variety of nerve cell signals sent during synaptic transmission," said Dr. Sheng.
The researchers used advanced microscopic techniques to watch mitochondria move among boutons while they released neurotransmitters. They found that boutons sent consistent signals when mitochondria were nearby.
"It’s as if the presence of mitochondria causes a bouton to talk in a monotone voice," said Tao Sun, Ph.D., a researcher in Dr. Sheng’s laboratory and the first author of the study.
Surprisingly, when the mitochondria were missing or moving away from boutons, the signal strength fluctuated. The results suggested that the presence of stationary power plants at synapses controls the stability of the nerve signal strength.
To test this idea further, the researchers manipulated mitochondrial movement in axons by changing levels of syntaphilin, a protein that helps anchor mitochondria to the nerve cell’s skeleton found inside axons. Removal of syntaphilin resulted in faster moving mitochondria and electrical recordings from these neurons showed that the signals they sent fluctuated greatly. Conversely, elevating syntaphilin levels in nerve cells arrested mitochondrial movement and resulted in boutons that spoke in monotones by sending signals with the same strength.
"It’s known that about one third of all mitochondria in axons move. Our results show that brain cell communication is tightly controlled by highly dynamic events occurring at numerous tiny cell-to-cell connection points," said Dr. Sheng.
In separate experiments the researchers watched ATP energy levels in these tiny boutons as they sent nerve messages.
"The levels fluctuated more in boutons that did not have mitochondria nearby," said Dr. Sun.
The researchers also found that blocking ATP production in mitochondria with the drug oligomycin reduced the size of the signals boutons sent even if a mitochondrial power plant was nearby.
"Our results suggest that local ATP production by nearby mitochondria is critical for consistent neurotransmitter release," said Dr. Sheng. "It appears that variability in synaptic transmission is controlled by rapidly moving mitochondria which provide brief bursts of energy to the boutons they pass through."
Problems with mitochondrial energy production and movement throughout nerve cells have been implicated in Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and other major neurodegenerative disorders. Dr. Sheng thinks these results will ultimately help scientists understand how these problems can lead to disorders in brain cell communication.
"Our findings reveal the cellular mechanisms that tune brain communication by regulating mitochondrial mobility, thus advancing our understanding of human neurological disorders," said Dr. Sheng.
For a healthy brain, don’t let the trash pile up
Recycling is not only good for the environment, it’s good for the brain. A study using rat cells indicates that quickly clearing out defective proteins in the brain may prevent loss of brain cells.
Results of a study in Nature Chemical Biology suggest that the speed at which damaged proteins are cleared from neurons may affect cell survival and may explain why some cells are targeted for death in neurodegenerative disorders. The research was supported by the National Institute of Neurological Disorders and Stroke (NINDS), part of the National Institutes of Health.
One of the mysteries surrounding neurodegenerative diseases is why some nerve cells are marked for destruction whereas their neighbors are spared. It is especially puzzling because the protein thought to be responsible for cell death is found throughout the brain in many of these diseases, yet only certain brain areas or cell types are affected.
In Huntington’s disease and many other neurodegenerative disorders, proteins that are misfolded (have abnormal shapes), accumulate inside and around neurons and are thought to damage and kill nearby brain cells. Normally, cells sense the presence of malformed proteins and clear them away before they do any damage. This is regulated by a process called proteostasis, which the cell uses to control protein levels and quality.
In the study, Andrey S. Tsvetkov and his colleagues from the University of California, San Francisco (UCSF) and Duke University, Durham, N.C., showed that differences in the rate of proteostasis may be the clue to understanding why certain nerve cells die in Huntington’s, a genetic brain disorder that leads to uncontrolled movements and death.
To measure how quickly proteins are cleared away from cells, the researchers developed a new technique called optical pulse-labeling, allowing them to follow specific proteins in individual living cells. To test the technique, they grew brain cells in a dish and turned on Dendra2, a photoswitchable protein that glows from green to red after being hit by a specific type of light. Both the red and green glow can be followed until the protein is cleared from the cell. In this way, the researchers could track the lifetime of newly produced Dendra2 (which glows green) and older, photoswitched Dendra2 (which glows red) until the protein was cleared away from the cell.
"Before this new technique, there was no way to look at individual neurons and their capacity to handle proteins. This method provides a real-time readout of how fast proteins are turned over in neurons and gives us a look at some of the mechanisms involved," said Margaret Sutherland, Ph.D., program director at NINDS.
The researchers followed Dendra2 in a set of striatal neurons, which they obtained from rats. The striatum (where striatal neurons are located) is a brain region involved in a number of brain functions including planning movements and is most heavily affected in Huntington’s disease. They discovered that the mean lifetime of the protein (how long it remained in the cell) varied three- to fourfold, suggesting that rates of proteostasis were different among individual neurons. In other words, some cells may process an identical protein much slower than others.
Then, the researchers investigated how cells deal with different forms of huntingtin, the protein involved in Huntington’s. They fused Dendra2 on the end of a normal or mutant version of huntingtin to track how long the protein remained in cells. The mutant version of huntingtin is longer, and contains three building blocks of the protein repeated an abnormal number of times. These repeats in huntingtin are what cause it to misfold, eventually leading to neuron death and the symptoms of the disease. As predicted, in their experiments, the mutant form of huntingtin caused more rat cells to die than did the normal form of the protein.
The researchers found that the amount of time the mutant protein remained in the cell predicted neuronal survival: shorter mean lifetimes of mutant huntingtin were associated with longer neuronal survival. A shorter mean lifetime indicates that a protein does not remain in the cell for a long time, and that proteostasis is working effectively to clear it away. This suggests that improving proteostasis in Huntington’s brains may improve neuronal survival.
To test this idea, the researchers activated Nrf2, a protein known to regulate protein processing. When Nrf2 was turned on, the mean lifetime of huntingtin was shortened, and the neuron lived longer.
"Nrf2 seems like a potentially exciting therapeutic target. It is profoundly neuroprotective in our Huntington’s model and it accelerates the clearance of mutant huntingtin," said Dr. Steven Finkbeiner, senior author of the paper.
Although both striatal and cortical neurons are affected by mutant huntingtin, striatal neurons are more susceptible to cell death. The investigators found that striatal neurons were not as effective as cortical neurons in recognizing and clearing away the mutant protein.
"One surprising finding from these experiments was the significance of single cells’ ability to clear mutant huntingtin. It turned out that this ability largely predicted their susceptibility, whether that neuron came from the most vulnerable region of the brain – the striatum, or the cortex, which is less vulnerable," said Dr. Finkbeiner. The findings indicate that the toxicity of the damaged proteins may cause neurodegeneration by interfering with the proteostasis system, affecting how quickly they are cleared from neurons.
"The results should remind us that focusing on the disease-causing proteins is only one side of the coin. To understand why some cells die and others are spared, we may need to recognize that there are major, largely unrecognized cell-specific differences in the ways that various types of neurons recognize and dispose of disease-causing proteins," continued Dr. Finkbeiner.
The researchers explored potential mechanisms behind differences in proteostasis. One way that cells normally get rid of proteins is through autophagy — a process in which proteins are packed up into spheres and then broken down. Results in this paper suggested that neurons increased the rate of autophagy when they sensed that the mutant form of huntingtin was accumulating, indicating the autophagy system may be a drug target.
"These findings provide evidence that our brains have powerful coping mechanisms to deal with disease-causing proteins. The fact that some of these diseases don’t cause symptoms we can detect until the fourth or fifth decade of life, even when the gene has been present since birth, suggests that those mechanisms are pretty good," said Dr. Finkbeiner.
Future research is needed to determine why coping mechanisms fail as brain cells age and how neurons in the healthy brain keep the proteostasis system functioning.
"New research methods that help us understand how individual neurons function will increase our understanding of central nervous system disorders and help identify new treatments. It is critical to continue working on the methods such as those described in this paper," said Dr. Sutherland.
(Source: eurekalert.org)
Stem cell research reveals clues to brain disease
The development of new drugs for improving treatment of Alzheimer’s and Parkinson’s disease is a step closer after recent research into how stem cells migrate and form circuits in the brain.
The results from a study by researchers at The University of Auckland’s Centre for Brain Research may hold important clues into why there is less plasticity in brains affected by Parkinson’s and Alzheimer’s disease, and links to insulin resistance and diabetes.
The major five-year project to understand how stem cells start and stop migrating in the brain has also helped to unlock the secrets of how stem cells migrate during development and in adulthood.
The study revealed new information on how connectivity between brain cells is improved or worsened, says senior study author, Dr Maurice Curtis who conceived and directed the research. The experiments were carried out at the Centre for Brain Research laboratories by Dr Hector Monzo. Collaborators included a director of the CBR, Distinguished Professor Richard Faull, Dr Thomas Park, Dr Birger Dieriks, Deidre Jansson and Professor Mike Dragunow.
“We have begun testing new novel drug compounds that target how polysialic acid is removed from the cell in the hope of improving neuron connectivity,” says Dr Curtis.
He explains that stem cells in the brain are immature brain cells that must migrate from their birthplace to a position in the brain where they will connect with other brain cells, turn into adult brain cells (neurons) and become part of the brain’s circuitry.
“Even once the neuron has found its location, the neuron’s tentacles (or dendrites) need to forage to find other neurons to connect with to form circuits. This would be easy except that in the adult brain the cells are surrounded by a fairly rigid matrix (extracellular matrix) and so migration or foraging becomes almost impossible in this high friction environment.”
“The way the cell overcomes this ‘friction’ is by placing large amounts of a special slippery molecule called ‘polysialic acid-neural cell adhesion molecule’ onto the cell surface,” says Dr Curtis. “This allows the cell to migrate or forage with only a fraction of the friction it once had and this also reduces the energy requirements of the cell.”
Once the cell has migrated to its destination, the slippery coating is removed and the cell becomes locked in place ready to connect with other cells. In the case of the dendritic foraging, the polysialic acid must be removed in order for the dendrite to connect with another cell (synapse formation).
“We have known for at least 20 years that this process occurs but despite extensive studies by a number of groups internationally we have been in the dark about what controls this process,” he says. “Studies in my laboratory have demonstrated what happens to the slippery molecules once the cell no longer needs them.”
There were three possibilities for this process:
- that enzymes cut them off the outside of the cell
- that the friction wears it off the cell or
- the cell internalises the slippery substance and recycles it ready for future use.
“For the past five years, we have systematically studied how this process is controlled,” says Dr Curtis. “Our findings have demonstrated that cells internalise the slippery molecule after receiving two specific cues.”
One of these cues is from collagen which makes up part of the rigid structure outside of the cell and the other is from a gaseous molecule called nitric oxide which triggers the outer membrane of the cell to internalise the slippery molecules.
“What we also discovered is that when there is an increased amount of insulin and insulin-like growth factor 1 (which has some similar functions to insulin) present in the culture, the cell cannot internalise the slippery molecules and instead they remain on the cell surface.”
“The key to the breakthrough was in determining that the process by which the polysialic acid is added to the cell surface was so persistent that it needed to be stopped in order to study how the polysialic acid was removed,” says Dr Curtis. “This required extensive trialling of many different cell growth conditions, enzyme concentrations and growing the cells in many different extracellular matrices.”
This is interesting because it is well known that in Parkinson’s disease and Alzheimer’s disease the brain is less sensitive to insulin, he says.
“In our studies in cells the insulin blocks the removal of polysialic acid and therefore the cell cannot connect properly and form synapses with other nearby cells.”
“This may hold major clues to why there is less plasticity in brains affected by Parkinson’s and Alzheimer’s disease in adults as well as helping to unlock the secrets of how stem cells migrate during development of the brain”, says Dr Curtis.
The Gus Fisher Postdoctoral Fellowship, the Auckland Medical Research Foundation and the Manchester Trust were the main sponsors of this research work.
The study results were published online this month in an ‘ahead of print’ version of The Journal of Neurochemistry.
(Source: auckland.ac.nz)