Christopher E. Henderson - US grants

DESCRIPTION (provided by applicant): Project Summary- Development of effective treatments for patients with amyotrophic lateral sclerosis (ALS) is currently hindered by the lack of validated therapeutic targets. Our long-term goal is to use motor neuron-based cell and animal models to better understand the molecular and cellular mechanisms involved in ALS, and to validate these as targets for clinical intervention. Motor neurons purified from SOD1 mutant mice, a model of familial ALS, show selectively exacerbated sensitivity to activation of a new cell death pathway involving the Fas death receptor and nitric oxide (referred to as the Fas/NO pathway). All elements of the pathway are activated in the spinal cord of presymptomatic SOD1 mice. The project will address four questions: (a) Is the Fas/NO pathway an essential contributor to the disease process in vivo in SOD1 mutant mice? (b) Which cell types are involved in activation of the Fas/NO pathway in vivo? (c) How does mutant SOD1 sensitize motor neurons to activation of the Fas/NO pathway? (d) What is the relevance of the Fas/NO pathway to sporadic ALS in humans? We will cross conditional knockout mice for Fas and FasL to SOD1 mutant mice and measure effects on motor function, survival and pathology. We will analyze the sites of Fas action in vivo using a biomarker approach. Lastly, we will analyze expression of elements of the Fas/NO pathway on sections of spinal cord from human patients with sporadic ALS. Overall, the work will provide new insights into mechanisms of motor neuron degeneration in vitro and in vivo and should allow for validation of new therapeutic targets in both familial and sporadic ALS. Relevance Amyotrophic lateral sclerosis (ALS), better known as Lou Gehrig's disease, leads rapidly to paralysis and death of patients, reflecting progressive loss of motor neurons, the nerve cells in the spinal cord that control muscle movement. This study will use cell and animal models to better understand the molecular events that underlie ALS as a means for developing rational therapeutic strategies.

DESCRIPTION (provided by applicant): Although we do not fully know if disease study of cells in Petri dishes can fully emulate the developmental progression that occurs in human adult neurodegenerative disease like ALS, new described technical ability to generate Induced Pluripotent Cells (iPS) from ALS patients provides an exceptional tool by which we can explore these issues. Many recent insights into the pathophysiology of ALS come from the study of familial forms of this disease. The ability to actually have human cell lines- representing the natural disease in the most relevant cell types- motor neurons and astrocytes- will provide unprecedented tools to 1) study cell- cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. Eventually these ALS cell lines will also be useful to compare common and uncommon pathways between ALS and other neurodegenerative iPS models. But - iPS cell biology is exceptionally new and we do not yet have sufficient information about the reliability of the cells generated, their ability to truly reflect human cell biology, recapitulate the protein, genetic and functional characteristics of native motor neurons and astroglia. Before we can embark on extensive use of these cells for basic/translational research- it would be critical to generate a series of cell lines- all produced under identical conditions, from different fALS mutations, to determine how representative they are for cell type specificity and functional biology. The overall proposal will involve four principal investigators, working in tight collaboration, to generate and evaluate familial ALS (fALS) iPS cell lines. Project 1, led by Dr. Eggan will obtain the skin biopsies from FALS and control patients, generate the fibroblast and ultimately the initial iPS lines. We will employ the aid of iZumi, a biotech company to be a central site for uniform protocol iPS cell generation. iPS cell lines with neural/glial characteristics will be sent to the Project 2 Lab- Motor neuron biology, lead by Chris Henderson and to Project 3 lab, Astrocytes- lead by Jeffrey Rothstein. These two projects/labs will determine which of the fALS iPS cell lines have the appropriate characteristics of motor neurons and astroglia, through a series of sequential analyses. Only those cell lines that meet final criteria (as compared to human ES cell and prior work on human astroglia) will then go on for final genetic analysis in the Project 4 lab, lead by Tom Maniatis. PUBLIC HEALTH RELEVANCE: Understanding the pathophysiology and development of new therapeutics for ALS has been an enormous challenge. The ability to actually have human cell lines- representing the natural disease in the most relevant cell types- motor neurons and astrocytes- will provide unprecedented tools to 1) study cell- cell interactions responsible for disease pathophysiology and 2) provide critical tools for drug discovery and genetic pathway analysis. Eventually these ALS cell lines will also be useful to compare common and uncommon pathways between ALS and other neurodegenerative iPS models. )

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a uniformly fatal neurodegenerative disease for which there is still no effective treatment. One explanation for this is that very few therapeutic targets - molecular events in the disease pathway whose inhibition confers benefit - have been identified, hindering rational translational approaches. One means of discovering novel targets is high-throughput in vitro screening of chemical libraries to identify compounds that can ameliorate disease-related phenotypes. Enzymes inhibited by such compounds constitute candidate therapeutic targets. However, to validate them for further development, it is necessary to demonstrate that their inhibition delays disease onset or progression in vivo. Among the cellular events that occur early in the ALS disease process is the physical die-back of motor axons from motor endplates, leading directly to muscle paralysis that spreads progressively throughout the body. Early muscle denervation is observed in mutant SOD1 rodent models as well as in patients with either sporadic or familial forms of the disease. Preventing axonal degeneration, or stimulating regrowth, would be predicted to delay disease onset or progression. However, since the underlying mechanisms remain unclear it has not been possible to test this therapeutic hypothesis directly. We recently screened a library of ~50,000 compounds to identify agents that enhance motor axon regeneration in an inhibitory context in vitro. The strongest hits were the statins, which enhanced axonal growth by up to 5- fold at concentrations 100-fold lower than with benchmarking compounds. Statin effects on axonal growth depend entirely on inhibition of their known target enzyme HMG-CoA reductase (HMGCR; 3-hydroxy-3-methyl- glutaryl-CoA reductase), which is the rate-limiting step for cholesterol synthesis and protein prenylation pathways. Our data identify HMGCR as a novel candidate therapeutic target in ALS. In this two-year project, we propose to validate HMGCR as a target in vivo using two potent ligands, cerivastatin and simvastatin, as probes. Our experiments will use compartmentalized motor neuron cultures to determine whether HMGCR inhibition needs to occur in the cell body or in the axon terminal. We will then establish protocol for statin administration in vivo that lead to significant HMGCR inhibition in motor neurons in the spinal cord. Finally, we will determine whether statin administration can delay muscle denervation in the mutant SOD1 mouse model of ALS. Overall, our experiments should allow us to determine whether HMGCR and the pathways downstream of it are valid therapeutic targets in ALS. They should also provide proof of principle for the idea that enhancing motor axon growth can delay functional denervation of muscle. Lastly, our data should stimulate further research into the specific processes modulated by statins in motor neurons, and thereby help identify more selective targets - such as protein prenylation - for future drug development.

DESCRIPTION (provided by applicant): Despite multiple clinical trials, there is still no effective therapy for the adult-onset neurodegenerative disease ALS (amyotrophic lateral sclerosis). One major reason for this is that, aside from the genes that are causal in familial ALS, no therapeutic targets have been validated. Examples of targets would be enzymes that play a critical role in disease progression and whose inhibition retards disease onset or slows progression. Strikingly, even in late-stage patients with amyotrophic lateral sclerosis (ALS), eye movement and continence are preserved, reflecting the near-complete resistance of motor neurons in oculomotor and Onuf's nuclei to the disease process. If it were possible to confer even a fraction of this resistance upon the normally vulnerable spinal motor neurons, there would be significant therapeutic benefit. Understanding the mechanisms of resistance therefore provides a method for defining new targets. In preliminary studies, we identified novel genes expressed in ALS-susceptible but not in ALS-resistant motor neurons, or vice versa, using laser-capture microdissection and microarray analysis. One of these is MMP-9 (matrix metalloproteinase-9), an extracellular enzyme which is absent from resistant oculomotor and Onuf's nuclei. We showed that its expression in different motor neuron subsets is tightly correlated with their vulnerability. Strikingly, we find that inactivation of the mmp9 gene in ALS model mice - whose normal lifespan is ~6 months - leads to a >3-month delay in muscle denervation and a 24% increase in survival. Significant benefit was observed even in mice that were heterozygotes for mmp9. MMP-9 is therefore a strong candidate as a potential therapeutic target in ALS. The overall goal of the proposed project is to understand the cellular and molecular mechanisms through which MMP-9 triggers motor neuron degeneration and to provide initial evaluation of potential therapeutic strategies to block this. The proposal is structured around three main questions. First, we will determine the molecular mechanism(s) through which MMP-9 triggers motor neuron degeneration, focusing on candidate pathways involving the Fas receptor and glutamate excitotoxicity. Second, we will investigate the cellular site of action of MMP-9, using different routes of administration of viral vectors expressing mmp9 shRNA. Third, we will ask whether inhibition of the enzymatic activity of MMP-9 is sufficient to confer benefit, or whether is known non-enzymatic modes of action are also implicated. Overall, the results should provide novel insights into the mechanisms of motor neuron degeneration in ALS and important preclinical indications as to the potential of MMP-9 as a therapeutic target for future development.