Matthew J. LaVoie - US grants

[unreadable] DESCRIPTION (provided by applicant): I have spent all of my professional training involved in the study of neurodegenerative disease with an early background in Parkinson's disease, and a purposefully sought postdoctoral position in the laboratory of Dr. Dennis Selkoe at the Center for Neurologic Disease (CND). I have begun an independent line of research clearly distinct from that of my mentor, and seek NIH support to further these studies under the continued guidance of Dr. Selkoe. I have chosen Dr. Selkoe as my mentor because: a) he is a recognized leader in the field of neurodegenerative disease-related cell biology with a remarkable history of training successful, independent researchers early in their careers, b) the Selkoe laboratory continues to provide a stimulating environment focused on issues of protein aggregation, protein-protein interactions in neurodegenerative disease and cell signaling and c) the support of the CND, and of Dr. Selkoe in particular, demonstrate a strong commitment to my continued development as an independent investigator. The CND is an ideal environment to conduct basic research in neurodegeneration, as it is the sole focus of its 14 principal investigators. Furthermore, there is liberal access to the vast resources of the Harvard Center for Neurodegenerative Disease and Repair, as well as collaborative and training opportunities throughout Harvard Medical School. [unreadable] [unreadable] My preliminary data demonstrate that the neurotransmitter dopamine (DA) itself adversely affects [unreadable] Parkin function, causing aggregation and oligomerization of the Parkin protein and inactivation of its [unreadable] E3 ligase activity. These data suggest that DA may contribute to a partial Parkin loss of function in [unreadable] idiopathic PD. The focus of the first 2 Aims of this proposal is to characterize the selective [unreadable] vulnerability of Parkin to modification by DA and determine whether this post-translational [unreadable] modification can be found in human brain. The training goals of this K01 application are to expand [unreadable] my expertise to include protein chemistry and mass spectrometric analyses, relevant to the growing [unreadable] interest in post-translational modification (i.e., Parkin) and cleavage (i.e., a-synuclein) of proteins in [unreadable] neurological disease. Additionally, I propose to gain expertise in the study of appptosis and the role [unreadable] mitochondria play in cell death under the tutelage of Dr. Stanley Korsmeyer. This collaboration is [unreadable] directly relevant to the model of dopamine-induced Parkin deficiency outlined herein, and would [unreadable] provide a world-class training experience in the execution of Aim 3: to determine whether Parkin [unreadable] deficiency promotes mitochondria-induced apoptosis. [unreadable] [unreadable] [unreadable]

I have spent all of my professional training involved in the study of neurodegenerative disease with an early background in Parkinson's disease, and a purposefully sought postdoctoral position in the laboratory of Dr. Dennis Selkoe at the Center for Neurologic Disease (CND). I have begun an independent line of research clearly distinct from that of my mentor, and seek NIH support to further these studies under the continued guidance of Dr. Selkoe. I have chosen Dr. Selkoe as my mentor because: a) he is a recognized leader in the field of neurodegenerative disease-related cell biology with a remarkable history of training successful, independent researchers early in their careers, b) the Selkoe laboratory continues to provide a stimulating environment focused on issues of protein aggregation, protein-protein interactions in neurodegenerative disease and cell signaling and c) the support of the CND, and of Dr. Selkoe in particular, demonstrate a strong commitment to my continued development as an independent investigator. The CND is an ideal environment to conduct basic research in neurodegeneration, as it is the sole focus of its 14 principal investigators. Furthermore, there is liberal access to the vast resources of the Harvard Center for Neurodegenerative Disease and Repair, as well as collaborative and training opportunities throughout Harvard Medical School. My preliminary data demonstrate that the neurotransmitter dopamine (DA) itself adversely affects Parkin function, causing aggregation and oligomerization of the Parkin protein and inactivation of its E3 ligase activity. These data suggest that DA may contribute to a partial Parkin loss of function in idiopathic PD. The focus of the first 2 Aims of this proposal is to characterize the selective vulnerability of Parkin to modification by DA and determine whether this post-translational modification can be found in human brain. The training goals of this K01 application are to expand my expertise to include protein chemistry and mass spectrometric analyses, relevant to the growing interest in post-translational modification (i.e., Parkin) and cleavage (i.e., a-synuclein) of proteins in neurological disease. Additionally, I propose to gain expertise in the study of appptosis and the role mitochondria play in cell death under the tutelage of Dr. Stanley Korsmeyer. This collaboration is directly relevant to the model of dopamine-induced Parkin deficiency outlined herein, and would provide a world-class training experience in the execution of Aim 3: to determine whether Parkin deficiency promotes mitochondria-induced apoptosis.

DESCRIPTION (provided by applicant): Perhaps the greatest obstacle in the development of disease-modifying therapeutics to combat or cure Parkinson's disease (PD) is the lack of predictive animal models. While familial forms of PD have guided the current generation of mouse models, they have thus far failed to recapitulate essential features of the human condition. Most notably lacking from these models is the spontaneous degeneration of the substantia nigra, and the accumulation of a-synuclein in non-synuclein based mice. Given the undeniable need for vertebrate models for target identification and validation in the translational arena, new models must be considered. We have focused our attention toward aspects of idiopathic PD in our rational design of a novel mouse model of this disease. Deficiencies in mitochondrial Complex-1 are routinely reported in idiopathic PD. In addition, chemical inhibitors of Complex-1 reproduce the characteristic selective lesion of the substantia nigra, deposition of the a-synuclein protein, and subsequent parkinsonism in rodents and non-human primates. There is even a small population of humans who ingested a Complex-1 toxin and subsequently presented clinically with an L-DOPA responsive parkinsonism. Numerous genetic studies have likewise implicated familial PD genes in mitochondrial biology, further heightening our interest in the role that mitochondrial dysfunction plays as a primary event in PD. We propose to characterize a novel model of PD by genetically targeting Complex-1 function. Complex-1 is a large integral membrane complex comprised of over 45 subunits whose assembly is aided by several proprietary chaperones. A critical question is which of these ~50 genes would be the most suitable target for a model of PD. We have focused on a Complex-1 gene that, when deficient in humans, results in severe degeneration of the substantia nigra, the same brain region affected in all PD cases. These data strongly suggest this gene to be an ideal target for the generation of an idiopathic PD animal model. The goal of this exploratory research proposal is to examine the downstream molecular consequences of Compex-1 dysfunction in vitro and characterize the basic neuropathological properties of Complex-1 deficiency in a novel genetic mouse model of PD. This work will determine the suitability of our model for further basic and translational efforts to understand and treat the PD disease process. PUBLIC HEALTH RELEVANCE: A major obstacle in the generation of new therapeutics for the treatment or prevention of Parkinson's disease is the lack of suitable animal models of spontaneous, premature nigral degeneration accompanied by other classic features of idiopathic Parkinson's disease. Here we will examine the suitability of a novel genetic model of Parkinson's disease in neuronal cell culture and in mice in an effort to satisfy perhaps the greatest unmet need in the field.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the near selective loss of neurons in the substantia nigra. While the etiology of the disease is unknown, genetic mutations responsible for familial forms of PD will likely provide critical molecular clues to the underlying mechanisms of idiopathic disease. Loss-of-function mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. Parkin is now widely recognized as a pro-survival protein rapidly up-regulated during cell injury. Parkin is believed to possess broad ranging effects on mitochondrial biology that are likely relevant to idiopathic PD. However, the biochemical substrates of these effects are largely unknown. The long-term goal of our work is to uncover the molecular machinery responsible for parkin's influence on mitochondria. This information will not only improve our understanding of an important neuronal stress response protein but will also elucidate new biochemical pathways likely involved in the selective neurodegeneration in PD. The experimental focus of this application is on the mitochondrial defects that result from neuronal deficiencies in parkin and identifying the proteins responsible. There are three specific aims. 1) Analyze the parkin-dependent ubiquitination of two novel parkin substrates involved in mitochondrial fission and apoptosis. We will determine the effects of endogenous parkin on protein half-life and localization, and the contribution of these candidate substrates on mitochondrial defects in parkin deficient neurons. 2) Analyze the mitochondrial consequences of parkin deficiency in primary cultured neurons, and examine enhanced vulnerabilities of parkin null neurons to stressors implicated in idiopathic PD. 3) Examine mitochondria-specific changes in protein trafficking and function that occur in parkin deficient neurons. The completion of these studies will establish mechanistic links between the E3 ligase activity of parkin and its function as a pro-survival stress response protein with mitochondrial influence. This work will elucidate novel biochemical pathways involved in the pathogenesis of PD and potentially uncover innovative targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Parkin is critically involved in a protective neuronal stress response and is causally linked to familial Parkinson's disease. The goal of this study is to understand the precise mechanisms by which parkin maintains integrity of the mitochondria and to define molecular events that can cause Parkinson's disease. These pathways, once discovered, may identify new therapeutic targets for the treatment of this devastating neurological disorder.

DESCRIPTION (provided by applicant): Missense mutations within the multi-domain kinase, LRRK2, are the most common cause of familial Parkinson's disease (PD), accounting for up to 40% of cases in some populations. LRRK2 mutations also appear in an unprecedented number of sporadic PD patients, implicating this protein in all forms of PD. While the genetic links between LRRK2 dysfunction and PD pathogenesis are clear, less is known about the physiological activity of LRRK2 or how it is regulated. The weak penetrance of LRRK2 disease mutations, however, does suggest that other genes may potently influence LRRK2 function and play a role in its regulation. Recent data indicate that like other kinases, LRRK2 can form a functional dimer within the cell. We've shown that the LRRK2 dimer is several-fold more active than its monomeric counterpart. In addition, the majority (~75%) of total LRRK2 is found within the cytoplasm at rest, while the membrane fraction is selectively enriched in the LRRK2 dimer. This is highly reminiscent of other proteins involved in intracellular signaling, where various kinases and GTPases shuttle to and from the membrane, and can be activated through dimerization. Therefore, we hypothesize that these newly discovered biochemical properties of LRRK2 are consistent with canonical mechanisms used throughout nature to regulate kinase activity and intracellular signaling. However, the precise importance of these processes to LRRK2 are not known. Our long-term goal is to uncover the biochemical nature of PD-linked LRRK2 mutants. However, this has remained elusive, partly due to our incomplete understanding of LRRK2 biology. At this time, a broader understanding of the physiological function and regulation of wild-type LRRK2 is critically needed. The goal of this application is to optimize a newly developed, quantitative assay of LRRK2 dimerization, and use this tool to uncover critical aspects of LRRK2 regulation. Specifically, we will 1) develop a protein-fragment complementation assay of LRRK2 dimerization, and 2) identify cellular and genetic determinants of LRRK2 dimerization in the intact cell. This work will not only inform us as to the pathways involved in intracellular LRRK2 signaling, but also provide novel tools to examine the underlying mechanisms of several pathogenic LRRK2 mutations in future studies. PUBLIC HEALTH RELEVANCE: Mutations in the LRRK2 kinase are the most common genetic cause of Parkinson's disease. The goal of this application is to optimize a novel, high-throughput assay of LRRK2 biology to uncover how cells regulate LRRK2 function. Through this new resource, and an improved understanding of the pathways controlling LRRK2 activity, we hope to gain valuable insights into the physiological and pathological functions of LRRK2.

DESCRIPTION (provided by applicant): Autosomal dominant mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Notably, LRRK2-associated PD is clinically indistinguishable from idiopathic PD, and generally is accompanied by the presence of the classic intracellular inclusions of a-synuclein called Lewy bodies, the pathological hallmark of PD. However, identifying biochemical links between a-synuclein aggregation and LRRK2 function has proven difficult. Prior work may have been thwarted by the assumption that the common pathways involving these two disease-linked proteins must occur within the same cell. Recent evidence demonstrates the expression and potent regulation of LRRK2 in non-neuronal cells including microglia, particularly during neuroinflammation. ?ynuclein, on the other hand, is exclusively expressed by neurons in the brain but is secreted by a yet unidentified mechanism. Recent data from multiple labs suggest that extracellular a-synuclein can serve as a ligand to activate microglial cells, which express LRRK2. In this application we will screen primary cultured murine microglia for the effects of multiple conformations and assemblies of a- synuclein, identifying those that activate microglial LRRK2, and confirm these effects in cultured human microglia. Next, using two distinct knock-in mouse models expressing endogenous levels of mutant LRRK2 (R1441C and G2019S), we will determine how pathogenic mutations in LRRK2 alter the a-synuclein- dependent microglial responses, both in vitro and in vivo. This will involve an analysis of how pathogenic LRRK2 mutations influence microglial responses to pathogenic a-synuclein exposure probing important biochemical properties of the LRRK2 protein and the effects on microglial behavior and function. This exploratory project will test a novel hypothesis regarding the functional interactions between LRRK2 and a- synuclein, and may add valuable insight into potential pathways integrating what are arguably the two most important PD-linked gene products.