Astrocytes are the most abundant central nervous system cell, yet their having a role in the progression of ALS has been discovered only recently. The star-shaped cells configure themselves into a close network, creating micro-environments where they contact neurons. These areas are critical for proper activity of neurons at synapses.

We plan to analyze the physical changes that astrocytes undergo throughout the progression of ALS, to determine how changes in this highly structured network, or syncytium, as it's called, influence motor neurons' susceptibility to cell death.

Too much glutamate - a major nerve transmitter - can kill motor neurons and is thought to be one way in which these cells may die in ALS. Normally, glutamate levels are kept low around motor neurons via transporter molecules that "mop up" any excess. The transporters exist, for the most part, on the glial cells that live in intimate contact with motor neurons.

Our grant will be used to generate laboratory cultures of human glial cells from their ancestor cells, human neural stem cells. The cultured glial cells will also be altered slightly, to allow us to visualize their transporter molecules and tell if they're active.

One great benefit of these cultures will be in screening drugs or other therapies for ALS. Those that activate or boost numbers of transporters and, thus, reduce toxic glutamate, would be the ones of choice.

Also, by adding the (G93A SOD1) gene for the mutation found in some familial ALS to the cultured glial cells, we'll have a cell model for the disease, one which should show if ALS brings abnormalities in the way transporter molecules are formed or carried to their home sites on cell membranes.

While not included directly in this grant, our other studies currently funded by the NIH will tell whether human glial cells made to express larger than usual numbers of glutamate transporters can slow down disease progression in a classic model ALS rat. Overall these studies will provide essential new data on the role of a specific glutamate transporter in human glial cells in ALS. And they'll also help test new therapeutic approaches.

The glial cells that are in intimate contact with motor neurons in the ventral spinal cord likely play an important role in ALS progression. Because little is known about how these cells develop in the disease state, and if that differs from the norm, we intend to follow the various stages, beginning with the earliest glial progenitor cells in mouse SOD1 models of ALS. We'll analyze the progenitors' cell structure in detail, observe how quickly they proliferate and examine their ability to differentiate into mature glial cell types (astrocytes, oligodendrocytes) or motor neurons. Our work should provide insight into cell interactions that influence the survival of motor neurons in ALS.

Motor neurons of ALS patients typically contain abnormal deposits of the protein TDP-43. Some researchers have suggested such deposits are important in tripping ALS or in its progress. There is, however, no concrete proof of this. We have developed a model of ALS in the fruit fly, Drosophila by introducing the human TDP-43 gene. We hope to use this model to understand the protein's role in the disease.

Genes are surely involved in ALS - both in sporadic and in the familial sorts - in the way the disease varies, for example, in what age it begins, in what body site it strikes first and in its severity. It's important to identify these genes, in part because they may clarify the overall biology of the disease. But, even more important, they may provide new targets for therapy. In our grant work, we're using easily-studied zebrafish embryos as ALS models. They carry the human mutant SOD1gene that causes some forms of the disease. By knocking out various candidate genes, we hope to learn which ones truly modify ALS.

Though we're aware of specific mutations leading to some types of familial ALS, the role genes or their mutations play in more common sporadic disease is unclear.

To identify specific genetic variations tied to increased risk of the sporadic disease, we recently completed a whole-genome survey of 276 American sporadic ALS patients and 276 normal controls using state of the art DNA chips. The study was repeated in a separate sample set of 276 Italian sporadic ALS patients. While this first work was an undeniably necessary start, such in-depth analysis of potentially telling sites on DNA in a sample this size has the down side of increasing the risk of false positive associations within the data.

So our proposed, expanded project -- which adds data from 2,304 patients and controls -- should reveal which of the 6,200 DNA sites in the original study is genuinely associated with ALS susceptibility. Later sequencing of the highlighted DNA should not only identify precisely which genetic variations underlie ALS, but could help expose biological pathways that effect motor neuron death.

We have developed new techniques to investigate pathology of the peripheral nervous system with greater accuracy and efficiency. Now we're using them successfully to study growth factor and other types of cell signaling, nerve regeneration, and the effects of nerve demyelination. Applying these approaches to ALS models is especially timely; we simply don't know the extent and pace of neuronal changes in these disorders. A secondary but important goal is to find a simple, rapid indicator of the state of health of the peripheral nervous system, one that can be used by any investigator to any rodent model of motor neuron or peripheral nerve disease.

Our work is aimed at the cellular repair and protection of the so-called corticospinal motor neurons that degenerate specifically in ALS and some other diseases. These neurons connect the brain to the spinal cord to control and effect voluntary movement. We know, in mammals, that CSMNs controlling the arms develop differently from CSMNs that control the legs. What molecular differences underlie this fact? Do they play a part in ALS's different onset in patients? In answering these questions, we hope to develop techniques to replenish degenerating CSMNs, and prevent their degeneration during ALS in the first place.

Neurotrophins are natural molecular attractants that also appear to protect neurons after traumatic injury. They show the same effect in nerve cultures from animal models of neurodegenerative diseases. Neurotrophins' diverse activities typically occur through their binding with tyrosine kinase receptors (Trk), molecules key to a cell's internal signaling. While studies show that bathing spinal motor neurons in neurotrophins may slow ALS, delivering therapeutic doses into the spinal cord is difficult. The nervous system's blood-brain barrier is hard to broach.

By understanding neurotrophins' behavior, we propose a way to duplicate their useful effect within the spinal cord without their having to be present. It involves activating Trk receptors via small molecules able to penetrate the blood brain barrier. We know this transactivation rescues animal facial motor neurons from cell death following injury. Now we propose to see if it will improve the survival of motor neurons in a transgenic mouse model of ALS.

The molecule retinoic acid (RA) acts as a signaling agent that’s essential for spinal motor neuron development. By identifying two genes that RA regulates – genes turned on at different times in motor neuron development – Sockanathan hopes to learn critical information about how spinal motor neurons develop and how their early experiences in cells determine what they do. Such finds are invaluable in the search for ways to repair damaged nervous systems or even, perhaps, to prevent their injury.

Weichun Lin’s team has created a novel transgenic mouse—based, in part, on a fairly newly-discovered, inheritable ALS mutation—that develops motor neuron disease with key features of the human disease. The mice become paralyzed at 5 months of age, and the paralysis progresses to the rest of the body within ten months. The specific cell error the model mice show should shed light on poorly-explored aspects of the ALS process, namely, events at the motor neuron synapse and those involving the cell’s system for removing damaged proteins. Lin’s grant supports his month by month study of motor neurons and synapses in this new model. He hopes to see how synaptic and conduction changes relate to outward signs of the disease.

The fruit fly, Drosophila melanogaster, provides one of the most powerful gene-based systems for studying development and biology of the neuromuscular system. Because of the ability to express mutant human genes in Drosophila, the flies have recently been used as models of many neurodegenerative illnesses such as Alzheimer’s, Parkinson’s and Huntington’s diseases. Such models allow rapid screening of all other genes, to search for those that modify the disease process in some way. They also reveal potential drug targets.

We’re exploring mutations in genes involved in internal transport of materials within a motor neuron’s axons, specifically those associated with motor neuron degerneration in humans. Since these genes are highly conserved from flies to humans, we expect that similar mutations in the fly will reproduce characteristics of the human disease. Our goal is to create a genetic model of motor neuron disease in Drosophila and then use this model to search for new drug targets for ALS.

Mutations in the protein dynactin, part of a neuron’s internal transport system, are suspect as a cause of amyotrophic lateral sclerosis. However, since the genetic findings in more than 2,000 patients and controls cannot reveal a cause-effect relationship for each of the mutations, we propose to investigate the detected sequence changes in transgenic mice.

Skeletal muscles provide signals to the motor neurons that stimulate them, signals that promote the neurons’ differentiation. Without these muscle-derived “retrograde” signals, motor neurons develop and function abnormally.

Although generating retrograde signals stems, in part, on the operation of a chemical receptor on muscle - specifically on a the tyrosine kinase (or MuSK) portion of the receptor, the exact nature of the signals themselves isn’t known.

We propose to take advantage of mice, genetically altered with respect to MuSK expression, to identify the critical muscle-derived retrograde signals that regulate motor neuron differentiation.

We have found in earlier experiments that semaphorin3A, a protein that helps guide developing axons by selectively repelling them from places they shouldn’t be, is released by Schwann cells—a type of non-neural typically in close contact with neurons.

In the mouse model of ALS we studied, the expressed semaphorin3A reaches the neuromuscular junction, the site where motor nerves signal muscle targets. We plan to test the hypothesis that semaphorin3A causes the gradual breakdown of specific neuromuscular synapses in ALS mice. If correct, this would show for the first time that release of this chemorepulsive protein in error is part of a cascade of molecular events that can lead to motor neuron degeneration in a mouse model for ALS.

With their grant, Scott Banta’s team will engineer novel peptides—short segments of proteins—able to cross the brain’s natural barricade against “foreign” molecules, the blood brain barrier (BBB). Further, the scientists intend to attach therapeutic substances to the peptides and insure ability of the complex to target specific brain cells. Recently, Banta identified a family of natural peptides that can traverse cell membranes while carrying cargoes. Using sophisticated molecular biology techniques and elaborate selection methods, he hopes to identify the right peptides to reach a given target. The new techniques may be valuable, ultimately, in treating devastating disorders of the central nervous system.

ALS4 is a rather recently-discovered, inherited form of ALS. Because it’s caused by a dominant gene, only one copy need be inherited to have the disease. This type of ALS is characterized by early onset and by slow disease progression. Life span in those with ALS4 is typically normal.

Patients have mutations in what’s called the senetaxin gene. The protein product of that gene in the healthy form is thought to have ability to alter the molecular shape of DNA or RNA. That’s of interest because research has found that defects in such proteins or protein complexes are somehow tied to motor neuron degeneration.

Our research aims to see if mutant senetaxin can cause ALS4 in mice. The result would be an animal model of an early onset, slowly progressing form of ALS, one that might reveal new information about molecular mechanisms of ALS or other motor neuron diseases.

For the past two years, Packard scientists Don Cleveland and Larry Goldstein have used an unusual way to see if ALS’s toxic process originates exclusively in motor neuron cells. Their strategy was to construct chimeric mice — those containing both normal cells and ones with ALS-linked mutations in the SOD1 gene tied to inherited forms of the disease.

This work, now published in Science, led them to conclude that ALS is, most definitely, “non cell autonomous,” that is, toxicity does not begin solely from damage only in motor neurons. Normal motor neurons, they found, begin to show cell characteristics of ALS when surrounding cells that aren’t even neurons carry the mutation.

More important, they discovered that SOD1 mutation-carrying motor neurons live longer when surrounded by healthy non-neural cells. In fact, it’s possible none of the crucial toxicity develops directly within motor neurons. This outcome has bearing in many directions including potential stem cell therapies: replacing non-neural cells, for example, is much easier than adding new motor neurons. Such an approach might become an effective ALS therapy.

The main question to answer, the scientists say, is which cells are damaged directly by SOD1 mutations? Knowing that will shed light on the mechanism behind the selective killing of motor neurons. It should also give rise to a therapeutic approach — either stem-cell or drug-based — based on understanding. That’s the sort with a chance of success.

Much has been made about using neural stem cells — those in the adult central nervous system of humans and other mammals — as possible repair agents once ALS is slowed or cured. But not many people realize adult neural stem cells also provide a powerful way to study cell behavior. And with Packard scientists recently showing that interactions between motor neurons and their surrounding cells are crucial to the course of the disease, the time is ripe for a good way to study them.

New Packard researcher, Hongjun Song, is preparing laboratory cultures of stem cells to model normal cell interactions and contrast them with what occurs in ALS. He chooses these specific stem cells because they’re endowed with nervous system characteristics but, unlike adult nerve cells taken from the body, they reproduce and survive in lab dishes. Also, as stem cells, they can morph into the various types of cells in the nervous system.

Song will work with both normal stem cells and those carrying the mutant SOD1 genes that can trigger ALS characteristics. He’ll also use both mice and human cells in his cultures. His system should let him follow the course of disease at a cell level. It should also show how crucial each type of nervous system cell is to the process.

Defects in transporting molecules throughout nerve cells have been proposed as a pathogenic mechanism in a number of neurodegenerative diseases. More recently, a mutation in a “motor” protein (p150) that enables transport was found in a family with amyotrophic lateral sclerosis (ALS). In this proposal, we intend to investigate whether the mutant motor protein is sufficient to cause ALS in mice, and, if so, to provide a model that will clarify molecular mechanisms involved in this disease.

There is a suspicion among ALS researchers that the genetics of ALS may not be as simple as it seems. We now know that about 10% of cases of ALS are inherited from parent to child in a way where both develop the disease. However, lessons learned from other neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, suggest that inheritance patterns may be more complex. In these diseases parents
may pass a gene or a group of genes to a child that increase susceptibility to disease as opposed to causing the disease directly.

Researchers at Emory University are teaming with deCode Genetics in Iceland, in a project to search for such susceptibility genes. Blood samples from ALS patients and their unaffected parents and siblings will be screened for patterns of inheritance of DNA that may suggest susceptibility to ALS. DNA from hundreds of patients and their living parents will be needed to complete this study. Since most ALS patients are over 50 years old, finding patients with living parents will require a nationwide campaign to collect these blood samples. Emory is developing the necessary tools to handle this task and will be encouraging patients and practitioners around the country to participate.

More than 90 different mutations in the SOD1 gene have been identified that cause ALS. By expressing this disease causing gene in different systems, many models—both live animal and organ or cell cultures—have overwhelmingly demonstrated that mutant SOD1 causes motor neuron degeneration through some toxic property of the protein it codes for (Cleveland and Rothstein, 2001). These findings explain why offspring of one parent with mutant SOD1 and another with a normal gene nevertheless develop ALS.
An effective therapy for ALS caused by toxic SOD1 gene mutations would be to eliminate the defective protein the mutant gene produces. This project explores the possibility of using a new technique, RNA interference (RNAi), to disrupt cell production of mutant SOD1 protein while letting the cell continue to make the normal version.

In ALS, the presumed cause of muscle movement loss has always been the death of motor neurons. New research is showing, however, that while motor neuron death does occur, it's the pulling away of neuron from muscle that appears to precipitate disease symptoms. Our work will study early changes in biology that occur at the motor neuron/muscle junction and relate them to earlier or subsequent changes in the motor neuron proper. We use a multidisciplinary approach to discover which portion of the nervous system shows ALS's earliest changes. Our intent is to find a biomarker for the disease - one that will report on the usefulness of new therapies.

Because ALS becomes fatal when motor neurons controlling breathing are affected, a treatment that replaces injured or dead neural cells makes great sense. Various approaches to this have been tried so far, without good effect. We propose using replacement motor neurons derived from human embryonic stem cells. By transplanting them into the spinal cord portion that affects breathing muscles, and then tracking these cells in rat models of ALS, we'll assay how well they function. This work will help create a strategy for eventual trials of such therapy in patients.

Most ALS patients die from respiratory failure as the muscles that control breathing become weak. While many cell transplantation strategies have focused on replacing the motor neurons that service those muscles, our work focuses on use of human glial stem cells to protect the motor neurons in the first place. We're exploring this first by transplanting glial stem cells into a rat model of ALS. The cells are targeted to the neck region of the spinal cord - the area containing motor nerves that innervate breathing muscles. Measurement of various signs will show if disease course has slowed. Our hope is that that targeting these specific regions will ultimately lead to human glial stem cell trials aimed at significantly prolonging patients' lives.

The EAAT2 molecule is a cell membrane protein that removes excesses of the neurotransmitter glutamate. Abnormalities in EAAT2 quantity or in its behavior have long been tied to ALS. That could make the molecule a good indicator, or biomarker, of nervous system health. At some point, it could provide a way to measure if treatment is effective.

In our supported research, we hope to create a radioligand (tracer) molecule targeted to the EAAT2 protein. The tracer will enable us to detect the EAAT2 biomarker noninvasively within the brain and spinal cord, using positron emission tomography (PET) imaging. Our challenge comes in designing a tracer with a unique chemical structure — a molecule with a high affinity for the EAAT2 target protein, one that can penetrate the brain’s natural barriers and that reaches specific central nervous system tissues. Ultimately, data from our studies may lead to a noninvasive way to monitor ALS in the clinic.

Regenerating motor neurons can distinguish between nerve pathways that lead to skin and those that lead to muscle. We have recently shown that these pathways differ in the types or patterns of release of the growth factors that they produce. However, we don’t yet understand how that plays a part in motor neuron growth toward muscle targets.

My work for the Packard Center aims to shed light on the process, largely by creating a small, controlled system to study. Our cultures will consist of sections of spinal cord containing motor neurons placed close enough to living nerve to enable the motor neurons to regenerate into the nerve.

In this situation, we can explore the role of different growth factors and even cell structure that affects motor neuron outgrowth. We hope to apply this knowledge to patients, ultimately, to develop ways to overcome the effects of injury or disease in the nervous system.

Should it become possible to stop the progress of ALS, problems still exist. The inhibitory environment of the adult spinal cord blocks regrowth of surviving axons. Further, the spinal cord environment, in model systems at least, halts growth of potentially helpful transplanted motor neurons arising from differentiated embryonic stem cells.

New Packard Center scientist Zhigang He explains that a single axonal surface protein called the Nogo receptor (NgR) helps carry out this inhibitory activity by interacting with three major myelin components. Myelin is the insulating material that surrounds some neurons. To counter this effect, he has developed a mutant receptor that, once expressed on axons, lets mature neurons resist myelin inhibition. He hopes to use mutant Nogo receptors to tie up myelin and thus promote axon regeneration in ALS models.

The primary focus of this project is on the mechanisms of axonal degeneration in models of peripheral nerve disease. Over the past decade major roles for calcium and calcium-activated proteases have been demonstrated in both acute and chronic axonal degeneration. This project assumes the same or similar mechanisms and molecules may be involved in the motor neuron degeneration seen in ALS. The two basic aims of this research are: (1) To test whether calpain inhibition will alter pathological changes and/or prolong survival in the FALS mouse; (2) To test whether the gene for "slow Wallerian degeneration" (Wlds) will alter pathological changes and/or prolong survival in the FALS mouse.

In ALS, current attempts at treating the illness are directed at promoting the growth of motor neurons without addressing the issue of changes that occur in the distal portions of the peripheral nerves. Local factors within the distal portions of the nerve may play an important role in degree of success during regeneration of motor neurons into the target tissue. Glial cell-line Derived Neurotrophic Factor (GDNF) is very important in motor neuron regeneration and survival. This project has two goals: (1) To transplant C17.2 stem cells in a model of regeneration after prolonged denervation in order to improve degree of motor regeneration; (2) To develop robust and reliable measures of motor regeneration that we can measure at the target tissue using electrophysiological and histological methods.