Following is information on projects we are currently funding. For summaries of projects funded in previous years, please choose from the links below.
Also available is our 2010/2011 Annual Report.
MicroRNA Profiles in ALS
Victor Ambros, Ph.D.
Program in Molecular Medicine, University of Massachusetts Medical School, United States
Alexey Wolfson, Ph.D., Catherine Sterling, Ph.D., & Rosalind Lee (Senior Research Associate)
Extracellular microRNAs are detected circulating in normal human blood plasma and cerebrospinal fluid (CSF). Circulating microRNAs are easily assayed by QRT/PCR or deep sequencing and provide potentially powerful reporters of the physiological state of their cells of origin. Accordingly, we have used quantitative RT/PCR to determine the levels of all known human microRNAs in samples of CSF from normal subjects and patients with ALS.
Our preliminary findings indicate that more than 100 microRNAs are robustly detected in human CSF, and that there seem to be a set of specific microRNAs whose expression profile in CSF may serve as an indicator of ALS. These preliminary data, as well as others implicating miRNA in homeostasis of neurons, support the view that microRNAs in the central nervous system may be involved in neurodegenerative disorders such as motor neuron disease.
We have therefore started a series of studies to characterize the identities and quantities of microRNAs in various CNS compartments, particularly cerebrospinal fluid, with the goal of developing miRNA profiles of both normal and neurodegenerative CSF. Our goals are to characterize the natural systemic transport of small RNAs in the CNS and do evaluate circulating microRNAs as biomarkers in ALS.
A Multi-center Study for the Discovery and Validation of ALS Biomarkers
James D. Berry, M.D., M.P.H.
Neurology Clinical Trials Unit, Massachusetts General Hospital, United States
Merit E. Cudkowicz, M.D., M.Sc., Robert P. Bowser, Ph.D., Shafeeq Ladha, M.D., Kevin B. Boylan, M.D., Robert H. Brown, Jr., D.Phil., Jonny Salameh, M.D., Robert Ferrante, Ph.D., M.S., Jonathan D. Glass, M.D., David Lacomis, M.D., & Gerry Shaw, Ph.D.
There is an ongoing effort to discover biomarkers of amyotrophic lateral sclerosis (ALS) in blood and cerebrospinal fluid (CSF). Current approaches use cross-sectional analyses, comparing samples from ALS patients to those from healthy volunteers and/or disease controls.
The present study is designed to create a repository of plasma and CSF collected from patients with ALS every four months for at least two years. Rigorous standard operating procedures have been developed to standardize the collection, processing and storage of biofluids, thus reducing preanalytic sample variability. At each visit, extensive clinical information is also collected, including clinical ALS outcome measures such as the ALSFRS-R, vital capacity, Ashworth Spasticity Scale, Hand-Held Dynamometry, and a screening exam for frontotemporal dementia. Once this biorepository and the linked clinical information database have been established, they will be used to explore novel biomarkers of ALS diagnosis and progression in four predefined projects being conducted at the Barrow Neurologic Institute, Emory University, the Mayo Clinic Florida and the University of Massachusetts. These projects will employ unbiased and targeted proteomics, antibody-capture protein identification and RNA isolation techniques to investigate novel biomarkers and attempt to validate previously reported candidate biomarkers. Project coordination and data management is performed through the fifth collaborative center, Massachusetts General Hospital.
At the end of the study, these longitudinally collected samples will become a part of the Northeast ALS Consortium biorepository where they will be shared with other ALS biomarker researchers. Such a resource does not currently exist in the field of ALS and will be a valuable addition.
Investigating a gain of Toxic Function of ALS-linked Mutant FUS/TLS in the Squid Axoplasm Model of Axonal Transport
Daryl A. Bosco, Ph.D.
Department of Neurology, University of Massachusetts Medical Center, United States
Scott Brady, Ph.D., Gerardo Morfini, Ph.D., & Reddy Ranjith K Sama (Graduate Student)
The focus of our studies is to test the hypothesis that mutant-FUS proteins exert a gain of toxic function that is linked to ALS pathogenesis. Mutations in FUS have been linked to both familial and sporadic forms of ALS. Most ALS-linked mutations in FUS cause the protein to mislocalize from the nucleus to the cytoplasm. Using the squid axoplasm assay for fast axonal transport, we found that mutant-FUS inhibits transport in both the retrograde and anterograde directions.
Therefore, mutant-FUS proteins impair axonal transport. In contrast, the normal wild-type FUS protein has no effect on transport in this assay. By employing pharmacological inhibitors against cellular kinases, we found that the effect of mutant FUS on transport is modulated by p38 MAPK, a kinase that is normally activated in response to stress. These data raise the possibility that when mutant-FUS mislocalizes to the cytoplasm, it triggers p38 MAPK activation, which in turn impairs axonal transport. Interestingly, our previous studies revealed that mutant- and aberrantly modified forms of SOD1, another ALS-associated protein, also impair axonal through a mechanism that involves p38 MAPK activation. Motor neurons that are degenerated in ALS may be particularly susceptible to defects in axonal transport by virtue of their long axons, which can extend up to one meter in length. We are beginning to recapitulate the effect of mutant-FUS on p38 MAPK activation in mammalian cell culture models.
Identification of FUS/TLS & TDP-43 Transcriptional Targets
Miriam Bucheli, Ph.D.
San Francisco University of Quito, Ecuador
Daniel Day (Graduate Student), Mazhar Adli (Postdoc), Monica Carrasco (Postdoc), Lulu Tsao (Undergraduate Student), Peter Park (Professor) & Tom Maniatis, Ph.D.
Our first objective was to characterize the transcription/RNA processing activities of FUS/TLS and TDP-43 through the identification of their gene targets. There is no knowledge of all the genomic targets that are shared between FUS/TLS and TDP-43. Based on the probability of overlap for similar transcriptional events at some sites, our second objective was to identify those genes that share occupancy by FUS/TLS and TDP-43 as potential sites of misregulation in ALS and to follow up with their characterization.
Results: TDP-43 was first identified as the major protein in cytoplasmic inclusions found in neurons and glia of individuals with sporadic and some familial forms of amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). The genetic association of TDP-43 with ALS is a more recent discovery, with dominant mutations in TARDBP (TDP-43 gene) first identified in several ALS families and later in sporadic cases. The RNA binding protein TDP-43 is a ubiquitously expressed protein that binds and regulates the expression of transcripts important for neuronal development. While most transcripts bound by TDP-43 are nuclear, it is not clear whether binding occurs co-transcriptionally. To answer this question, chromatin immunoprecipitation followed by next-generation sequencing was used to understand the genomic patterns of binding for the normal and mutant M337V TDP-43 in motor neurons (MNs). The MNs were derived from induced pluripotent stem (iPS) cells generated from an ALS patient and a control individual. Our results suggest that the mutant M337V acquires properties distinct from that of the normal TDP-43. The effect of the imperfect binding of the mutant protein on gene expression was assessed in the context of epigenetic modifications that function as signals for the activation (histone 3 lysine-36 tri-methylation) or repression (histone 3 lysine-27 tri-methylation) of genes. Our results reveal how a mutation in the protein may induce subtle alterations in its occupancy of some genes and directly or indirectly lead to changes in the expression of genes involved in neuronal development.
The role of Sarm in Axon Degeneration and ALS
Marc Freeman, Ph.D.
Department of Neurobiology/HHMI, University of Massachusetts Medical School, United States
Jeannette Osterloh (Ph.D. track student) & Timothy Rooney (M.D./Ph.D. track student)
Axon degeneration is a common feature of peripheral neuropathy and neurodegenerative diseases, including ALS. Axonal and synaptic degradation ultimately leads to loss of functional integrity of affected neural circuits and disease progression. Surprisingly little is known about molecular pathways mediating axonal self-destruction in any context. Wallerian degeneration is a very useful model to study axonal degeneration after injury and is mechanistically related to axon destruction in disease, but is fundamentally different from apoptotic cell death. Using Drosophila forward genetics we found that loss of dSarm (Drosophila sterile Armadillo/Toll-Interleukin receptor homology domain protein) blocked the degeneration of severed axons and their synapses for the lifespan of the fly. dSarm is an ancient member of the TIR domain-containing family of proteins which are well-conserved from C. elegans to humans.
Strikingly, we have found that mouse sarm-/- knockout animals also exhibit profound protection of axon after injury: severed axons remain intact after sciatic nerve lesion for over two weeks in vivo, and DRG and cortical neurons are strongly protected after axotomy in vitro. Thus dSarm/SARM is a founding member of a novel and conserved axon death signaling pathway.
Our work shows that loss of function mutations in specific genes can completely suppress axon degeneration and defines it as a programmed process of auto-destruction. Functional blockade of the human SARM1 signaling pathway now becomes an exciting new therapeutic approach to suppress axonal and synaptic loss in neurodegenerative diseases. We are therefore now exploring whether sarm mutants can suppress mouse models of ALS (SOD-G93A).
Generation and Characterization of Induced Pluripotent Stem Cell Lines Containing Defined Genetic Mutations Implicated in ALS
Fen-Biao Gao, Ph.D.
University of Massachusetts Medical School, United States
Sandra Almeida, Ph.D., & Zhijun Zhang, Ph.D.
How genetic and environmental factors contribute to the pathogenesis of ALS remains poorly understood. Mutations identified in familial ALS cases have been very informative and helped us to dissect the molecular and cellular pathways that are misregulated in ALS. Since the groundbreaking identification of SOD1 mutations in ALS, several new ALS genes have been reported, including TDP-43, FUS and C9orf72. These recent advances raise the possibility that misregulation of RNAs is a major mechanism of ALS pathogenesis.
Despite recent breathtaking advancements in human genetics studies, our understanding of ALS pathogenesis at the molecular and cellular levels is still critically lacking, which significantly hinders efforts to find a cure. Animal models have been useful but many scientific findings in animal models fail to translate into feasible therapies in humans, in part because of species differences in physiology and toxicology. Moreover, assays with human neurons may be one of the best ways to screen drugs for therapeutic interventions.
One way to generate disease-specific human neurons is to create induced pluripotent stem (iPS) cells from human fibroblasts. These iPS cells are genetically identical to the patient and will therefore carry the same disease mutation. Thus, they afford an excellent opportunity to investigate disease mechanisms, screen for drugs, and develop individualized medicines. With the support of ATA and other agencies, we have generated ALS patient–specific iPS cells containing specific genetic mutations. We are continuing to use the iPS cell models to further dissect the molecular pathways misregulated in ALS.
Mechanisms of Mutant FUS-mediated Motor Neuron Disease
Lawrence J. Hayward, M.D., Ph.D.
University of Massachusetts Medical School, United States
Hae Kyung Ko (Ph.D. Candidate) & Hongru Zhou (Research Assistant)
Mutations in FUS, a nucleic acid binding protein involved in gene transcription and RNA processing, cause ~5% of familial ALS cases. The Hayward lab is establishing cellular and animal models to understand how mutant FUS perturbs cellular homeostasis in vivo so that we may identify new targets for ALS therapy. FUS is a multifunctional protein, and possible insults within compartments of the motor neuron (e.g., in the nucleus, soma, dendrites, axons or terminals) or within supporting cell types in the spinal cord also remain undefined. To address important mechanistic questions and to complement transgenic mouse modeling approaches already in progress, we are studying the consequences of mutant FUS in cell cultures and in experimentally accessible zebrafish models of ALS. We are constructing vectors for transgenic expression of normal or ALS mutant FUS and for targeted perturbation of the endogenous zebrafish FUS gene. These models will be analyzed to detect ALS-like abnormalities to discern whether the FUS mutations trigger dominant gain-of-function, loss-of-function or dominant-negative mechanism(s) affecting motor neurons. Moreover, these models may allow us to design one or more assays to screen for small molecule or genetic suppressors of the observed phenotypes.
Proposal to Leonard Tow for National ALS Mouse Model Repository
Michael Hyde, M.Ed., & Cathleen Lutz, Ph.D.
Jackson Laboratory, United States
Research in ALS mouse models has focused primarily on mutations in the SOD1 gene. Now, recent discoveries point to defects in RNA processing proteins as a key to understanding the causes of the disease. Further, mutations in the TARDBP gene have been shown to cause familial ALS in a small proportion of cases. Similar experiments uncovered mutations in a related RNA processing gene called fused in sarcoma (FUS). These discoveries offer new hope for therapeutic intervention. Creation and rapid distribution of mouse models of these disorders is crucial to finding treatments. Unfortunately, many scientists are slow to make new mouse models available, while high costs and complex licensing requirements further block flow of mice to ALS researchers.
To alleviate this problem, the Jackson Laboratory requests $630,755 over three years to establish the National ALS Mouse Model Repository at the Jackson Laboratory. The goals of this repository will be to: 1) actively acquire current ALS models; 2) standardize their genetic background; 3) provide genetic and phenotypic quality assurance around the new models; 4) establish embryonic stem (ES) cells for models that prove the most useful; and 5) quickly make these resources available to the scientific community. This project secured the approval of the ALS Association (ALSA) and a number of top ALS investigators from around the world, including Columbia University’s Dr. Tom Maniatis, during an October 2009 meeting convened to assess interest in establishing an ALS mouse repository at the Jackson Laboratory.
ALS Mouse Trial
Robert Molinari, Ph.D., M.B.A.
Retrotope, United States
Oxidative damage initiated by ROS is a major contributor to the functional decline that is characteristic of age-related diseases, including CNS disease. Reactive carbonyls such as HNE, ONE and HHE formed from both omega-3 and omega-6 PUFA oxidation by ROS have been shown to play a role in the etiology of ALS. Modifications of PUFAs in diet are known to play a positive role in reducing the risk of developing ALS and are therefore known to be an effective intervention.
We propose that “reinforcing” essential PUFAs with isotopic replacement via dietary intake will reduce lipid peroxidation and decelerate HNE-mediated toxic cascades that contribute to neuronal degeneration in ALS. We aim to stabilize PUFAs by substituting D (deuterium, the naturally occurring, heavy isotope variant of hydrogen) for H at oxidation-prone bis-allylic sites of these essential fatty acids. Because the D-C bond is more stable due to the isotope effect, this substitution significantly reinforces the C-H bond that is first broken in lipid peroxidation, without changing the chemical identity of PUFAs. A decrease in the formation of intracellular toxic lipid peroxides reduces the amount of reactive carbonyls and diminishes the activation of toxic cellular cascades. We have successfully tested this approach in yeast and PD mouse model. In this proposed study, we will test whether our novel molecular stabilization approach reduces cellular oxidative damage in the ALS SOD1 G93A mouse model.
Mechanisms underlying MAPK Activation and Axonal Transport Deficits in Familial ALS
Gerardo Morfini, Ph.D.
Department of Anatomy and Cell Biology, University of Illinois at Chicago, United States
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving progressive loss of function and dying-back degeneration of motor neurons. Most ALS cases are sporadic (SALS), but familial forms (FALS) result from mutations in the enzyme superoxide dismutase 1 (SOD1) and other unrelated genes. The phenotypic similarities between sALS and fALS suggest the existence of common pathogenic mechanisms, but a molecular basis for these mechanisms remains elusive.
The marked vulnerability of motor neurons observed in ALS contrast sharply with the ubiquitous tissue expression of SOD1, suggesting that alterations in cellular processes particularly critical for the function and survival of these neurons play a central role in ALS pathogenesis. The enormous size and complex cellular architecture of motor neurons cells renders these cells uniquely vulnerable to alterations in intracellular signaling and axonal transport (AT) mechanisms. Accordingly, abnormal activation of protein kinases and reductions in AT represent well-documented ALS hallmarks, but relationships between these pathogenic events remained unknown.
Our recent studies indicate that pathogenic SOD1 polypeptides inhibit kinesin-1 based AT by a mechanism involving activation of the kinase p38 and upstream mitogen-activated protein kinases (MAPKs). Using cellular and animal models of SOD1-related FALS, experiments under ATA funding currently evaluate alterations in AT of specific kinesin-1 isoforms and their transported membrane cargoes. Additionally, pharmacological, biochemical and cell biological approaches are being used to identify specific MAPKs mediating the increase in p38 activity induced by pathogenic SOD1. Results from these experiments will help identify novel therapeutic targets in ALS.
The Pooled Resource Open Access Clinical Trials (PO-ACT) Database
Prize4Life & NEALS, United States
There have been multiple large Phase II and Phase III ALS clinical trials conducted over the past 15 years. While the vast majority of these trials, with the exception of the Riluzole trials, have not resulted in the identification of new therapies for ALS, there is still great value in the patient data collected during the course of these studies. Despite their wealth of information, clinical trial datasets have not historically been made readily available to the ALS research community and comparisons across datasets are extremely difficult.
Pooling data from existing public and private sources of trial data may yield a database of thousands of ALS patient records, which will be far larger than any single trial or existing dataset. Such a set of pooled data would not only yield useful information about many aspects of the conduct of ALS clinical trials, but could also be “data-mined” for unique observations, novel correlations, patterns of disease progress, epidemiological data and a variety of still unconsidered analyses.
With the support of the ATA, PRO-ACT Database project will design and build an ALS clinical trial database with merged datasets and an underlying searchable data platform. The PRO-ACT Database will be freely available, exclusively for research purposes, to members of the research and development (R&D) community (industry, government and academe), which will provide the ALS research community with an invaluable resource in the effort to develop treatments and a cure.
Characterizing the Surface Hydrophobicity of ALS Mutants of SOD1 by Novel Fluorescent Probes
Ashutosh Tiwari, Ph.D.
Department of Chemistry, Michigan Technological University, United States
Shilei Zhu, Ph.D. (Postdoc), Nethaniah Dorh (Graduate Student) & Claire Drom (Undergraduate Researcher)
In neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), prion diseases and Huntington’s disease (HD), where protein misfolding is implicated, intracellular or extracellular aggregates are observed at the end stage of the disease. The proteins involved in these diseases are expected to adopt non-native conformations due to mutation or stress and the exposed hydrophobic surfaces can result in aberrant interactions with themselves or other cellular constituents. Although hydrophobic fluorophore such as 1-anilinonaphthalene-8-sulfonic acid (ANS) has been widely used as a probe for hydrophobic exposure of proteins, it provides only a global measure of hydrophobicity. Furthermore, effective probes for identifying specific amino acids that are aberrantly exposed on protein surfaces due to mutation or misfolding are lacking. We propose to synthesize novel water soluble, neutral, hydrophobic fluorescent probes with high quantum yield having reactive functional groups that can covalently bind to the side chain of amino acids in the vicinity of the exposed hydrophobic region. Using these novel compounds, we will characterize the aberrantly exposed hydrophobic surface of mutant Cu/Zn Superoxide Dismutase (SOD1) associated with ALS.
Research Conference of International Consortium on Superoxide Dismutase and ALS
Lawrence Hayward, M.D., Ph.D., & Daryl Bosco, Ph.D.
University of Massachusetts Medical School, United States
The 9th Research Conference of the International Consortium on Superoxide Dismutase and ALS (ICOSA) was held at the University of Massachusetts Medical School on May 20-22, 2010. The meeting was organized by Dr. Lawrence Hayward and Dr. Daryl Bosco and included 70 participants from UMass Medical School, Brandeis University, the University of California at Las Angeles, the University of Texas, the University of Liverpool (UK), Umeå University (Sweden), Stockholm University, the University of Kentucky, Brookhaven National Laboratory, Massachusetts General Hospital, Harvard University and Brown University. Sponsored by the ALS Therapy Alliance, the agenda included scientific sessions on superoxide dismutase biochemistry in relation to ALS, mechanisms of altered RNA processing in neurodegeneration, animal models of ALS, and novel therapeutic approaches to ALS. Presentations included ground-breaking studies by graduate students and postdoctoral fellows, and a special session allowed time for the scientists and students to interact directly with individuals living with ALS.
Small-molecule HTS for SOD1-mediated ALS
Osman Bilsel, Ph.D.
University of Massachusetts Medical School, United States
Jill Ann Zitzewitz, Ph.D.
Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease with an average disease duration of less than five years. There is currently only one FDA approved therapeutic, providing a modest increase in average life expectancy. This proposal is aimed toward meeting the need for a small-molecule therapeutic for ALS by performing a high-throughput screen (HTS) that targets the dimeric protein superoxide dismutase (SOD1). Mutations in the gene for this protein have been implicated in approximately 25% of familial ALS (fALS) cases. Although the mechanism of toxicity in fALS is not completely understood, the toxicity is increasingly attributed to the monomeric forms of SOD1. Biophysical studies on disease causing forms of human SOD1 demonstrate that destabilization of the native dimeric form relative to the monomers (folded or unfolded) is a common property. One strategy toward a therapeutic for ALS is to use small molecules to shift the monomer-dimer equilibrium to favor the native dimer and minimize the population of potentially toxic monomeric species. This approach has shown promise in a computer-based screen by the Lansbury/Ray lab.
Our experimental HTS assay is unique in its ability to selectively detect the shift in the monomer-dimer equilibrium upon binding of a small molecule, a sensitivity afforded by a custom fluorescence lifetime based instrument that offers sensitivity beyond what is commercially available. We anticipate these studies to progress to a larger scale screen that will identify and optimize lead compounds that may ultimately identify drugs that slow the progression of ALS.