Current Projects
Following is information on projects we are currently funding from 2008 - 2010. This information is also available in our 2009/2010 Annual Report.
Click here for summaries of projects funded prior to 2008.
Project: Genome-wide association study of samples from the UK National Motor Neuron Disease DNA Bank
Investigator: Ammar Al-Chalabi, PhD FRCP DipStat King’s College London, United Kingdom
About 5% of people with amyotrophic lateral sclerosis (ALS), known as a motor neuron disease in the UK, have a family history of ALS. For the remainder, who have so-called sporadic ALS, we know from studies of identical and non-identical twins that genetic factors contribute to disease risk. One way to find gene variations that might increase the risk of someone developing sporadic ALS is to compare the genetic makeup of large numbers of people with sporadic ALS, with the genetic makeup of large numbers of people without ALS, to see if there is any consistent difference.
In this study, about 600 samples from a national bank of ALS DNA samples in the UK were compared with results from about 4,000 samples from the general UK population. The signal of a genetic contribution to sporadic ALS was seen on chromosome 9. These results were then combined with information from seven other countries to make the largest genetic study of ALS ever -- 4,000 people with ALS and 8,000 people without. The association signal became even stronger. This means that people who carry the risky genetic variant at this gene address have a slightly higher risk of ALS than people who do not, and it accounts for about 10% of people with sporadic ALS. At this gene address, there are only three genes called MOBKL2B, IFNK and C9orf72. Efforts are underway to find out which gene is the problem. The results have been published in the prestigious journal, Lancet Neurology.
Project: Phase III: Production Four Version III ATLIS Devices
Investigator: Patricia Andres, MS, DPT Massachusetts General Hospital (MGH), United States
In response to a critical need for improved outcome measures in ALS clinical research, to accurately and efficiently measure strength in small groups of patients, we developed a new device called Accurate Test of Limb Isometric Strength (ATLIS). ATLIS tests 12 muscle groups in the limbs using a fixed, wireless load cell. A validation study, supported by MDA, demonstrated excellent reliability and very positive user acceptance.
Last year, ATA provided funding for two new production-ready strength measurement prototypes (Version III). Funds from the Neurology Clinical Trials Unit at MGH were used to build two additional prototypes. A total of five ATLIS III prototypes are in use in clinical research sites throughout the country to collect ATLIS data from 500 healthy adults that will be used to allow raw ATLIS data to be converted to a percent of predicted normal values. Use of percent of normal will mitigate differences between subjects and enable disease progression rates to be calculated based on a 100 point scale. These five ATLIS prototypes will also be used in the tamoxifen/creatine trial to directly compare ATLIS with HHD in this longitudinal study.
We are extremely grateful to the support that ATA has provided through various Reasearching a cure for Amyotrophic Lateral Sclerosis stages of ATLIS prototype development. We feel that ATLIS will prove to be an efficient and practical method to measure disease progression in ALS and enable candidate drugs to be screened quicker and with less expense.
Project: Multicenter Study for Validation of ALS Biomarkers
Investigator: Merit E. Cudkowicz, MD, MSc. Massachusetts General Hospital (MGH), United States
Twenty-five Northeast ALS Consortium (NEALS) Centers are currently participating in a Multicenter Study for the Validation of ALS Biomarkers with the primary objective of identifying factors that contribute to the pathogenesis of ALS. Development of disease biomarkers and diagnostic laboratory tests would facilitate earlier treatment intervention, help monitor treatment efficacy, and, ultimately, lead to the identification of targets that could be used in therapy development. Sponsored by ALSA, ATA and the NIH, and working in conjunction with Metabolon, Inc., blood and cerebrospinal fluid (CSF) is collected for metabolomic testing. Metabolomics provides a new approach to evaluate global biochemical defects in ALS and to establish characteristic and unique metabolic patterns for ALS and its different phenotypic forms. It is believed that these signatures and knowledge of the corresponding molecular structures will provide diagnostic markers for the disease and provide insights into disease mechanisms. A total of 650 blood and 300 CSF samples will be collected from healthy individuals, ALS disease mimics and people with ALS. In addition, up to 600 blood samples from all groups will be collected for a sub-study for DNA analysis.
Currently, there are 445 total volunteers enrolled. A total of 411 blood samples have been collected along with 226 longitudinal blood samples, 190 CSF samples and 246 DNA samples.
Specific biomarkers for ALS would be valuable because they may point to new hypotheses in the pathogenesis, allow earlier and more accurate disease diagnosis, and may serve as surrogate indices of disease activity that enhances monitoring for therapeutic effect in treatment trials.
Project: Analysis of mTOR and Synaptic Activity in Amytotrophic Lateral Sclerosis
Investigator: Eric Frank, Tufts University, United States
In this project, we characterized the synaptic connectivity between various types of sensory neurons and the motoneurons that they innervate in hSOD1 mutant mice during early postnatal stages. Our goal was to determine whether this mutation altered neural circuits in the spinal cord at early postnatal stages, well before the loss of motoneurons that is a hallmark of the ALS phenotype.
Synaptic connections were assessed by making electrical recordings from motoneurons. This was in response to stimulation of sensory nerves in isolated spinal cord preparations from nine day old mice pups expressing the hSOD1G93A or hSODwt genes. We found that the specificity of synaptic inputs from sensory neurons was abrogated in mutant mice. Normally, inputs from a motoneuron’s own muscle afferents are more than ten times as large as those from unrelated muscles.
In contrast, these connections are much less specific in hSOD- 1G93A mice; inputs from unrelated muscles were more than twice their normal amplitude. There was also an increase in the excitation of motoneurons caused by stimulation of cutaneous nerves. Stimulation of the saphenous nerve elicited synaptic potentials that were twice as large as in hSODwt mice. Intracellular recordings suggested that cutaneous axons were often projecting to inappropriate motoneurons, again suggesting a loss of synaptic specificity.
Taken together, our results show that although many of the direct synaptic pathways are intact in neonatal hSOD1 mutant mice, the selectivity of these pathways is appreciably reduced. This reduction in specificity may contribute to the loss of motor coordination noted in earlier investigations at times prior to the outright loss of motoneurons.
Project: Transgenic Mouse Models of FUS/TLS-mediated ALS
Investigator: Lawrence J. Hayward, M.D., Ph.D., University of Massachusetts Medical School, United States
Transgenic Mouse Models of FUS/TLS-mediated ALS Genetic defects that affect proteins involved in the regulation of RNA processing or transport have been linked to both familial and sporadic forms of ALS. Mutant variants of one of these proteins, FUS, have been estimated to cause ~5% of familial ALS. Mutations near the C-terminus of the FUS gene cause motor neuron loss, but the mechanism(s) of toxicity have not yet been identified.
In this project, we are establishing transgenic mouse models that express either normal or mutant human FUS protein in the brain and spinal cord. We are characterizing 15 independent transgenic lines to detect altered FUS sub-cellular localization and other neuropathological features and to assess quantitative motor function and survival. These results will indicate whether the FUS mutations trigger dominant gain-of-function, loss-of-function, or dominant-negative mechanism(s) affecting motor neurons. Moreover, these insights will enable the identification of novel targets for slowing the neuronal degeneration and will accelerate rapid preclinical testing of treatment strategies for ALS.
Project: Zebrafish Models of FUS-ALS
Investigator: Lawrence J. Hayward, M.D., Ph.D., University of Massachusetts Medical School, United States
Over the past four years, gene defects that may alter the function of several proteins involved in the processing of RNA have been linked to both familial and sporadic forms of ALS. Mutant variants of one of these proteins, FUS, have been estimated to cause ~5% of familial ALS. FUS normally helps to coordinate important cellular reactions such as splicing of nascent RNA in the nucleus and translation of the RNA message into protein in the cytoplasm. Initial studies suggest that mutant FUS may be localized abnormally in the cytoplasm of affected motor neurons in ALS, but the mechanism(s) by which dominant expression of the mutants injures motor neurons is not known.
The goal of this project is to establish and characterize zebrafish models of FUS-mediated ALS based on transgenic, knock-in, and knock-out manipulation of the zebrafish FUS gene. Zebrafish are increasingly being used to study mechanisms related to neurodegenerative diseases because of their small size and the ability to produce hundreds of eggs per day. Zebrafish embryos develop a functional motor system within three days after fertilization, and because zebrafish embryos are optically transparent, their morphology and physiology can be directly visualized. Experiments using fluorescent molecules to report back on the health of the nervous system can be designed, and hundreds of drugs can be tested in large numbers of individual animals over a few days. Our long-term goal is to use these genetic zebrafish models to identify novel drug targets and screen for their effectiveness in treating ALS.
Project: Analysis of mTOR & Synaptic Activity in ALS
Investigator: Zhigang He, Children’s Hospital Boston, United States
In ALS, available evidence suggests that motoneurons become dysfunctional very early in this disease. Electrophysiological studies reveal substantial increases in neuronal excitability and synaptic activity as early as the first postnatal week. These changes may be paralleled by alterations in synaptic connectivity with motoneurons, a possibility that has not been explored previously. These early changes are apparent well before the onset of neuromuscular degeneration and motoneuron loss, suggesting that it may be critical to understand the functional alterations in motoneurons during postnatal development.
In addition, the molecular mechanisms for these and other abnormalities in the motoneurons of ALS models and patients remain unclear. Our preliminary results suggest that the mammalian target of rapamycin (mTOR) activity is prematurely downregulated in both upper and lower motoneurons of ALS mice at 10 weeks. This pathway is known to be a central regulator of cell survival and growth and remodeling of axons and dendrites as well as synaptic structures. We thus hypothesize that alterations of mTOR activity in motoneurons may be an important underlying mechanism in the development of motoneuron dysfunction and death in ALS models and perhaps in human patients.
In this application, we propose to utilize a combination of electrophysiological, behavioral, genetic, and pharmacological means to test this hypothesis. We are in the process of determining the time course of the down-regulation of mTOR in ALS mice and its correlation with the development of ALS pathology, testing the hypothesis that synaptic inputs to motoneurons are affected in ALS mice at early postnatal stages, and assessing the potential therapeutic effects of modifications in the mTOR pathway on slowing or halting the progression of ALS.
Project: Characterization of Three Novel ALS Genes
Investigator: John Landers, University of Massachusetts Medical School, United States
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease caused by the selective loss of motor neurons. It is ultimately lethal with a typical patient survival of two to five years. Although most ALS cases are sporadic in nature, ~10% are familial.
In recent years, the identification of genetic causes of familial amyotrophic lateral sclerosis (FALS) has greatly contributed to understanding the pathogenesis of ALS in general. Unfortunately, only one-third of the underlying genetic causes of FALS have been explained to date. Through our efforts, we have identified novel genes, which contribute to the development and progression of ALS.
Using a candidate gene approach, we have discovered that mutations in the Paraoxonase gene cluster contribute to familial and, to a lesser extent, sporadic ALS. Through a whole genome association study, we have identified a variant in the Kinesin- Associated Protein 3 (KIFAP3) gene that acts as a modifier of survival in sporadic ALS.
Lastly, we are focused on the identification of a gene causal for FALS within a genomic region determined by linkage analysis. This project is focused on understanding how each of these contributes to ALS and other neurodegenerative diseases. Understanding how these genes influence the process of neurodegeneration will aid in the development of novel strategies to interfere or prevent this process.
Project: Regulation of Alternative Splicing by TDP43
Investigator: Tom Maniatis PhD (and Brad Friedman), College of Physicians and Surgeons, Columbia University, United States
TDP43 is an RNA processing factor that is mislocalized and abnormally processed in neurons and glia of a majority of SOD1- unrelated ALS patients. Rare mutations in TDP43 segregate with the disease in certain ALS families, suggesting that it plays a causative role. However, neither the pathway by which TDP43 abnormalities can lead to neuron death nor the normal function of TDP43 is understood. Two new genomic technologies If successful the proposed studies will shed light on a previously unexplored aspect of the ALS disease mechanisms, and in the process identify novel therapeutic strategies.
First, RNA-Seq will be performed to analyze RNA from TDP43- depleted and control embryonic spinal cord cultures in order to detect TDP43-regulated RNA processing. This technique can uncover differential nucleic acid processing at all levels -- transcription, splicing, poly-adenylation and RNA editing. In addition this method can be used to detect DNA mutations.
Second, CLIP-Seq will be used to identify the RNA binding sites of TDP43 in the same cells. CLIP-Seq is a method by which total cellular RNA directly bound to a specific protein can be purified and massively sequenced. Analysis of the binding sites determined by CLIP-Seq and the regulated exons determined by RNA-Seq will provide important insights into the role of TDP43 in RNA processing. This comparison may also identify genes or pathways altered by the loss of function of TDP43 that lead to motor neuron death.
If successful the proposed studies will shed light on a previously unexplored aspect of the ALS disease mechanisms, and in the process identify novel therapeutic strategies.
Project: Blockade of Integrin-mediated Peripheral Nervous System inflammation in ALS.
Investigator: Tom Maniatis PhD (and Isaac Chiu), College of Physicians and Surgeons, Columbia University, United States
Amyotrophic lateral sclerosis (ALS) is a fatal, neuro-degenerative disease characterized by the selective death of motor neurons in the brain and spinal cord. Denervation of the neuromuscular junction and pathology at the axon is an early and significant feature of ALS disease progression. However, the mechanisms governing motor axon degeneration are not well defined.
We have characterized the prominent infiltration of activated macrophages within peripheral nerves and axons of transgenic mouse models of ALS. These cells accumulated over time within ventral roots, sciatic nerves, and muscles of SOD1G37R and SOD1G93A transgenic mice, but not in SOD1WT or non-transgenic controls.
We hypothesize that interactions between peripheral macrophages and distal axons of motor neurons may aberrantly alter neuronal function and survival. During inflammation, immune cells utilize a specific set of surface integrin receptors for recruitment into affected tissues. Antibody mediated blockade of integrins has been utilized in acute and chronic disease models to inhibit macrophage and lymphocyte influx into inflamed tissues in vivo.
In this project, we propose to use anti-integrin antibodies to chronically inhibit macrophage entry into the peripheral nervous system of SOD1G93A transgenic mice during motor neuron degeneration. These experiments will potentially lead to insights on the molecular mechanisms of macrophage entry and activation, immune-axonal interactions, and direct therapeutic applications in ALS.
Project: Modulating Disease Progression in ALS Mice Using Conditional RNAi
Investigator: Michele M. Maxwell, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital and Harvard Medical School, United States
The primary goals of this research project are to develop and test a flexible system for regulatable RNAi-mediated gene silencing in vivo and to use this system to evaluate the therapeutic potential of gene knockdown approaches for the treatment of ALS. For dominantly inherited neurodegenerative diseases such as mutant SOD1-mediated familial ALS, the disease-causing mutant protein itself is an important therapeutic target. For this reason, efforts aimed at reducing the cellular load of mutant proteins via gene knockdown have become a major focus of therapy development for ALS. This approach has shown considerable promise, and previous studies have shown that RNAi-based silencing of mutant SOD1, if achieved early enough in life and in a widespread manner, can ameliorate disease in ALS mice. In most cases, however, treatments were administered to young animals long before they exhibit symptoms of disease, and these studies could not address whether knockdown of mutant SOD1 could be beneficial at later stages of disease.
In order to evaluate mutant SOD1 as a therapeutic target in the both pre- and early-symptomatic stages of disease, we devised a flexible transgenic expression system for RNAi that permits ubiquitous, but temporally regulatable, knockdown of SOD1. When introduced into existing ALS mice via genetic crosses, these regulatable RNAi transgenes permit knockdown of mutant SOD1 upon administration of a small molecule inducer. This conditional system for RNAi may aid in identifying an appropriate window for treatments designed to reduce mutant SOD1 levels in familial ALS. In addition, the transgenic expression system was designed to be readily adaptable for silencing of any desired target gene in any mouse model of motor neuron degeneration. This system will therefore provide a valuable resource for future studies designed to identify or confirm additional putative therapeutic targets for ALS.
Project: Misregulation of TDP43 and FUS mRNA in ALS
Investigator: Melissa J. Moore, HHMI/University of Massachusetts Medical School, United States
Missense mutations in the RNA/DNA binding proteins TDP-43 and FUS/TLS have been shown to be causative of familial amyotrophic lateral sclerosis (FALS). The mutant proteins accumulate in the cytoplasm and exhibit decreased in abundance in the nucleus of affected neurons. Similar cytoplasmic accumulation and nuclear clearance of TDP-43 and FUS/TLS is also observed in sporadic ALS (SALS). Thus ALS could result in part from a loss of nuclear function. We are investigating the possibility that homeostatic levels of both TDP-43 and FUS/TLS are maintained by a negative feedback loop in which alternative splicing of the mRNAs encoding these proteins is linked to nonsense mediated mRNA decay (AS-NMD). NMD is a translation-dependent degradation pathway that eliminates mRNAs whose open reading frames terminate upstream of one or more exon-exon junctions (the sites where introns were removed). Many RNA binding proteins keep their expression in check by targeting their own mRNAs to NMD via alternative splicing when intracellular protein concentrations rise. Since such a feedback loop critically depends on protein function in the nucleus, cytoplasmic sequestration of TDP-43 and FUS/TLS could lead to a self-perpetuating cascade of protein overexpression that may be a causative event for disease.
To date, we have shown that TDP-43 mRNA contains introns in its 3’-UTR and is subject to NMD. We have also demonstrated that a significant fraction of FUS/TLS transcripts are degraded in a translation-dependent manner. We are currently investigating the effects of TDP-43 or FUS/TLS transgene overexpression on endogenous protein levels, using both wild type proteins and ALS-causative mutations. We hope that these experiments will prove illuminative as to the effects of ALS-causing mutations on TDP-43 and FUS/TLS protein levels.
Project: FUS/TLS in FALS by Studies in a Model Organism and Yeast modeling of FUS ALS
Investigator: Gregory Petsko, Brandeis University, United States
The aim of this project was to develop a model in a simple, genetically-tractable organism for the role of the human gene FUS/TLS in familial amyotrophic lateral sclerosis (FALS). Mutations in FUS/TLS have been shown to cause inheritable ALS, but the connection with the cell death of spinal motor neurons is not understood.
In humans, the disease is associated with mislocalization of FUS/TLS from the nucleus to the cytoplasm, combined with the formation of discrete clumps of aggregated protein. We therefore attempted to create a model that recapitulated these phenotypes plus cell death. We chose for our model organism budding yeast because it has most of the cellular machinery found in complex organisms and has been used successfully to model Parkinson’s and Huntington’s diseases in the past.
We overexpressed both wild-type and mutant human FUS/TLS in yeast and found that high levels of expression of either caused the protein to mislocalize from the nucleus to the cytoplasm and to form punctate ag gregates; overexpression also caused cell death. We systematically deleted various parts of the FUS/TLS gene and found that the reason both wild-type and mutant protein caused these phenotypes was that the nuclear localization signal contained in the C-terminal part of the protein is not effective in yeast, so high levels of expression of even the wild-type form cannot be retained in the nucleus. This observation suggested that the disease-causing variants in humans act by disrupting this signal, and we were able to show that this is indeed the mechanism.
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