2004 — 2008 |
Muchowski, Paul J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Modifiers of Huntingtin and Alpha-Synuclein Toxicity @ University of Washington
DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominant inherited disorder characterized by involuntary movements, personality changes and dementia, and is caused by an expansion of a CAG/polyglutamine repeat in the IT-15 gene. A major neuropathological hallmark in HD is the occurrence of intranuclear and cytoplasmic inclusion bodies that contain huntingtin (the protein encoded by IT-15). Cytoplasmic inclusion bodies (Lewy bodies) are also a prominent nature of Parkinson's disease (PD), a neurodegenerative disorder characterized by muscle rigidity, bradykinesia, resting tremor and postural instability. Lewy bodies are composed primarily of the protein alpha-synuclein, and two point mutations in the alpha-synuclein gene cause early-onset, inherited forms of Parkinson's disease. Alpha-synuclein and huntingtin aggregate into ordered fibrillar structures with properties characteristic of amyloid. The 'amyloid hypothesis', developed originally to describe the role of beta-amyloid in Alzheimer's Disease (AD), suggests that the aggregation of proteins into an ordered fibrillar structure is causally related to aberrant protein interactions that culminate in neuronal dysfunction and cell death (Hardy and Selkoe, 2002). The precise roles of protein aggregation, amyloid formation and inclusion bodies in neurodegeneration remain controversial, and it is not yet clear if common molecular mechanisms underlie HD and Parkinson's disease. We have used yeast as a model eukaryotic organism to test the hypothesis that the downstream targets and molecular mechanisms by which huntingtin and ot-synuclein mediate toxicity are unique. Using a genome-wide screening approach in yeast we isolated 52 genes that modify huntingtin toxicity, and 86 genes that modify alpha-synuclein toxicity. 30% of genes that affect huntingtin toxicity are enriched in the functionally related categories of protein folding and cell stress, while 29% of genes that modify alpha-synuclein toxicity are involved in vesicular transport and lipid metabolism. Our preliminary results indicate surprisingly that the genes and cellular pathways that modulate huntingtin and alpha-synuclein toxicity in yeast are completely divergent. Nearly half of the genes we isolated are annotated as having one or more human ortholog, suggesting we may have discovered in yeast conserved cell-biological response pathways to huntingtin and alpha-synuclein that are relevant to HD and Parkinson's disease. Using the resources and information that we have generated, we now wish to advance our understanding of the neurodegeneration that occurs in HD and PD by applying molecular genetic and biochemical techniques to validate (or invalidate) the genetic modifiers we have identified. Our long-term goal is to use the information we gain in these studies to test hypotheses in animal models of HD and Parkinson's disease.
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0.955 |
2006 — 2010 |
Muchowski, Paul J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Elucidation of the Structures and Biological Activities of Huntingtin Oligomers @ J. David Gladstone Institutes
[unreadable] DESCRIPTION (provided by applicant): Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin, which leads to its aggregation into amyloid fibrils. Historically, fibrils were thought to directly mediate neurodegeneration in HD and other neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). However, recent studies in AD and PD suggest an alternative hypothesis whereby potentially diffusible nanoscale assemblies, such as spherical oligomers, protofibrils and pore-like annular structures, are the structural entities that mediate neurodegeneration. Whether the mutant huntingtin protein forms such structures and shares a similar pathogenic mechanism to AD and PD remains poorly understood. In our preliminary data we demonstrate by ex situ atomic force microscopy (AFM) that a mutant huntingtin fragment with an expanded polyQ repeat forms spherical and annular oligomeric structures reminiscent of those formed by proteins linked to AD and PD. We also show that the molecular chaperones Hsp70 and Hsp40, which are protective in animal models of HD and other neurodegenerative disorders, can attenuate the formation of spherical and annular oligomers. Although our preliminary results are provocative, an important caveat to these experiments is that the AFM was not performed in solution. We now wish to advance our understanding of the neurodegeneration that occurs in HD by elucidating the structures and biological activities of different types of polyQ aggregates using in situ AFM under near physiological conditions. We will first characterize the aggregation of mutant huntingtin fragments on various surface chemistries and under different solution conditions. We will next determine the biological properties of different aggregates by characterizing their effects on the function of nuclear pore complexes in nuclear envelopes by combining in situ AFM with patch clamp analyses. Finally, we will also characterize the effects of molecular chaperones and anti-huntingtin antibodies on the structure and biological activities of mutant huntingtin fragments. Our long-term goal is that a fundamental understanding of the structures of aggregates formed by mutant huntingtin, and how they mediate neurodegeneration, will result in the design of potential therapeutics to suppress their toxicity in vivo. [unreadable] [unreadable] [unreadable]
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0.904 |
2008 — 2012 |
Muchowski, Paul J |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Microglial Kynurenine Pathway and Selective Neuronal Vulnerability @ J. David Gladstone Institutes
Huntington's Disease (HD) and Alzheimer's disease (AD) are neurodegenerative disorders characterized by the accumulation of misfolded proteins. Sadly, many years of research into the mechanisms of neurodegeneration have failed to produce effective therapies that halt or reverse these diseases. We recently completed a genomic screen in S. cerevisiae with single gene deletion strains that identified kynurenine 3-monooxygenase (KMO), an enzyme in the kynurenine pathway (KP) of tryptophan degradation, as a potent suppressor of mutant huntingtin (htt) toxicity. The brain levels of two neurotoxic metabolites in the KP, quinolinic acid (QUIN) and 3-hydroxykynurenine (3-HK), are increased in the striatum and neocortex in early grade HD; similar increases in QUIN and/or 3-HK are present in three mouse models of HD. We show that brain levels of QUIN are increased in the hippocampus and entorhinal cortex of mouse models of AD, but not in the striatum or other unaffected brain regions. QUIN and 3-HK have long been hypothetically linked to the pathophysiology of neurological diseases including HD and AD. Indeed, intrastriatal injection of QUIN together with 3-HK causes striatal lesions resembling those found in HD that may be mediated by the combination of A/-methyl D-aspartate (NMDA) receptor over-stimulation (excitotoxicity) and free radical formation. Subchronic intraventricular infusion of QUIN in rats produces biochemical changes and memory deficits that may share similarities with those found in AD patients. In our proposal, we present data showing that treatment of a mouse model of HD with Ro 61-8048, a high-affinity, orally bioavailable, small-molecule inhibitor of KMO, improved multiple behavioral outcome measures despite the fact that this compound displayed marginal penetration across the blood brain barrier (BBB). We recently generated a novel series of brain penetrating KMO inhibitors and mice that carry a conditional null allele of Kmo. With these tools in hand, we are for the first time in a position to test rigorously if the microglial KP and excitotoxicity play important roles in mouse models of HD and AD. The following specific aims will begin to test the hypothesis that microglial derived increases in neurotoxic KP metabolites occur in distinct brain microenvironments in a manner that contributes to selective neuronal vulnerability in mouse models of HD and AD: AIM 1. To determine if genetic and pharmacological inhibition of KMO in microglia improves behavioral and pathological outcome measures in mouse models of HD and AD; AIM 2. To identify the regulatory elements and signal transduction pathways that mediate mutant huntingtin (htt)/amyloid (3-protein (A|3)-induced KP activation in microglia; AIM 3. To determine the cellular mechanisms that mediate increases in toxic microglial KP metabolites in discrete brain microenvironments in a mouse model of HD. In summary, these experiments will determine if pharmacological inhibition of KMO may be a bona fide therapeutic approach to treating HD and AD.
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0.904 |
2008 — 2012 |
Muchowski, Paul J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Microglia and the Kynurenine Pathway in Huntington's Disease @ J. David Gladstone Institutes
DESCRIPTION (provided by applicant): Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the protein huntingtin (htt). Sadly, a decade of research into the mechanisms of polyQ- dependent neurodegeneration has failed to produce even a single effective therapy for HD. Although small molecules have been identified that inhibit the aggregation of a mutant htt fragment in vitro, many bind to secondary structures shared by other proteins, and it is not known if any of these molecules will be effective and specific in more complex models of HD. We recently completed a loss-of-function (LOF) genomic screen in S. cerevisiae with single gene deletion strains that identified kynurenine 3-monooxygenase (KMO), an enzyme in the KP of tryptophan degradation, as a potent suppressor of mutant htt toxicity. The brain levels of two neurotoxic metabolites in the KP, quinolinic acid (QUIN) and 3-hydroxykynurenine (3-HK), are increased in the striatum and neocortex in early grade HD;similar increases in QUIN and/or 3-HK are present in three mouse models of HD. QUIN and 3-HK have long been hypothetically linked to the pathophysiology of HD. Indeed, intrastriatal injection of QUIN together with 3-HK causes striatal lesions that may be mediated by the combination of N-methyl D-aspartate (NMDA) receptor over-stimulation (excitotoxicity) and free radical formation. In our proposal, we present data showing that Ro 61-8048, a high-affinity, orally bioavailable small molecule inhibitor of KMO, decreases QUIN, 3-HK and mutant htt toxicity in yeast, and significantly improved neurological index, rotarod performance, locomotor activity and ambulatory distance in a small pilot study using a mouse model of HD. Remarkably, we show KMO is expressed exclusively in microglia. Microglial activation has been documented in postmortem brains of early grade HD patients and in HD mouse models. However, little is known about the role of microglia in HD pathophysiology. We show that primary microglia isolated from HD mice have significantly increased levels of 3-HK. We hypothesize that mutant htt induces a transcriptional defect that activates the KP in microglia, and that inhibiting the KP via pharmacological and genetic approaches will improve behavioral and pathological outcome measures in HD mouse models. We propose to test these hypotheses by studying the role of mutant htt and the KP in cultured microglia and in vivo in mouse models of HD. These experiments will establish whether KMO inhibitors such as Ro 61-4048, which showed promising results in a small pilot study, deserves further consideration for pre-clinical development as a HD therapy. More broadly, our genetic experiments in mice will determine whether pharmacological inhibition of KMO is a bona fide therapeutic approach to treating HD. PUBLIC HEALTH RELEVANCE In this project we will use genetic and pharmacological approaches to determine if blocking a metabolic pathway implicated in Huntington's disease (the kynurenine pathway) confers protection in mouse models of this disorder. The kynurenine pathway is found predominantly in microglia, the macrophages of the brain, which are activated abnormally in pre-symptomatic Huntington's disease patients. If successful, our studies may lead to clinical tests of small molecule inhibitors of the pathway in patients with Huntington's disease.
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0.904 |