Christopher A. Ross - US grants

The goals of this project will be to clarify the molecular biology and neuroanatomy of brain opioid systems by studying enkephalin convertase (EC), a recently discovered enzyme in the processing pathway for opiate peptides. Opioid systems in the brain participate in the regulation of mood and affect, control of the autonomic nervous system, and the pathophysiology of neuropsychiatric conditions, such as Huntington's disease, Alzheimer's disease, and Parkinson's disease. The first aim will be to map EC and EC mRNA in rat brain and in the periphery using immunohistochemistry and in situ hybridization. Double label studies will investigate the colocalization of EC with opioid and non opioid peptides. Combined retrograde tracer plus immunohistochemical techniques will permit the tracing of EC containing pathways in brain, particularly in areas of the hindbrain involved in cardiovascular regulation, and in areas of the limbic system which may be involved with the action of ECT or antidepressant drugs. The second aim will be to study the regulation of EC in brain, pituitary and adrenal, with manipulations such as insulin or dexamethasome treatment, morphine dependence, or antidepressant treatment. EC enzyme activity, EC immunoreactivity, and EC mRNA levels will be studied coordinately. The final aim will be to define the neuropathology of human opioid systems in neuropsychiatric illnesses, such as Huntington's disease, Parkinson's disease, and Alzheimer's disease. Sections through the amygdala, areas of basal ganglia, and selected brainstem regions in patients brains will be labeled immunocytochemically for EC and compared with controls. These studies should clarify the neuroanatomy and molecular biology of brain opioid systems and their role in the pathophysiology of neuropsychiatric illness.

The goals of this project will be to clarify the molecular biology and neuroanatomy of brain opioid systems by studying enkephalin convertase (EC), a recently discovered enzyme in the processing pathway for opiate peptides. Opioid systems in the brain participate in the regulation of mood and affect, control of the autonomic nervous system, and the pathophysiology of neuropsychiatric conditions, such as Huntington's disease, Alzheimer's disease, and Parkinson's disease. The first aim will be to map EC and EC mRNA in rat brain and in the periphery using immunohistochemistry and in situ hybridization. Double label studies will investigate the colocalization of EC with opioid and non opioid peptides. Combined retrograde tracer plus immunohistochemical techniques will permit the tracing of EC containing pathways in brain, particularly in areas of the hindbrain involved in cardiovascular regulation, and in areas of the limbic system which may be involved with the action of ECT or antidepressant drugs. The second aim will be to study the regulation of EC in brain, pituitary and adrenal, with manipulations such as insulin or dexamethasome treatment, morphine dependence, or antidepressant treatment. EC enzyme activity, EC immunoreactivity, and EC mRNA levels will be studied coordinately. The final aim will be to define the neuropathology of human opioid systems in neuropsychiatric illnesses, such as Huntington's disease, Parkinson's disease, and Alzheimer's disease. Sections through the amygdala, areas of basal ganglia, and selected brainstem regions in patients brains will be labeled immunocytochemically for EC and compared with controls. These studies should clarify the neuroanatomy and molecular biology of brain opioid systems and their role in the pathophysiology of neuropsychiatric illness.

Angiogenesis may play an important role in the physiologic response of the myocardium to ischemia and infarction. In addition, cell growth factors may be implicated in the pathophysiology of myocardial hypertrophy. The mitogenic protein, basic fibroblast growth factor (bFGF), is plentiful in the myocardium, and forms complexes in vivo with cell surface and extracellular matrix heparan sulfate molecules. Heparan sulfates may serve to protect bFGF from proteolytic degradation, maintain it in insoluble form until released to exert its actions, and possibly provide a vehicle for the transmembrane export of newly synthesized bFGF. Detailed structural information regarding specific bFGF-binding proteoglycans is limited, particularly in the heart. Such information may clarify the physiologic relationships between bFGF and heparan sulfate, which will in turn provide insight into the sequence of events leading to myocardial angiogenesis or hypertrophy. The specific aims of the proposed studies are: 1. Obtain partial amino acid sequences from peptide fragments of the core protein from cardiac bFGF-binding proteoglycan. bFGF-binding proteoglycan will be purified from total ventricular plasma membrane using anion-exchange and bFGF-affinity chromatography. The core protein of this proteoglycan will be cleaved with cyanogen bromide, followed by partial sequencing of bFGF-binding peptide fragments. 2. Raise antibodies against portions of the cardiac bFGF-binding proteoglycan. Synthetic peptides and cyanogen bromide cleavage products will be used as the immunogen. Both approaches will be attempted. 3. Identify a cDNA clone coding for the peptide fragment(s) identified in Specific Aim 1. Two methods of screening are proposed. A bovine heart cDNA library will be screened using the antibody(s) raised in Specific Aim 2, as well as cognate oligonucleotide probes synthesized based on partial amino acid sequence data. Information obtained by these studies will lead to the determination of the primary structure of a bFGF-binding proteoglycan present on myocardial cell membranes. cDNA clones identified in Specific Aim 3 will be sequenced and the primary structure of the proteoglycan core protein deduced. Following the identification of a specific cardiac cell type producing the proteoglycan, cell-culture models will be developed which either under- or over-express the bFGF-binding ability of such a proteoglycan.

The Baltimore Huntington's Disease Project (BHDP) is a cohesive research program, which includes research on etiology, pathogenesis, and treatment. In this application, we request funding for years 11-15. Using a variety of new methods, we propose to study the mechanism of gene action in HD. Dr. Orest Hurko (Mitochondrial Function) approaches the question through Dr. Joseph Coyle (Selective Neuronal Vulnerability) uses the rodent HD model and neuronal culture to investigate the basic mechanisms leading to glutamate-induced excitotoxicity and will test drugs in these systems for their ability to prevent neuronal damage. One outcome of this work is a separately funded Experimental Therapeutic Trial of Idebenone. A related theme is the role of extra-striatal pathology in HD. Drs. Chris Ross and John Hedreen (Extra-Striatal Pathology) use neuropathological and neurochemical experiments to elucidate the pathogenetic meaning of cortical and amygdala pathology in HD. Dr. Jason Brandt (Correlates of Frontal- Striatal Degeneration) uses neuropsychological methods to assess the significance of cortical atrophy (using MRI) in cognitive and functional deficits. Dr. David Zee (Ocular Motor Function), uses novel paradigms for testing ocular motor function in HD patients to study the anatomy and function of ocular motor pathways and their changes in HD. The BHDP is coordinated through its Cores, and through the physical proximity of the investigators whose offices and laboratories are in the Meyer Building. The Administrative Core provides both formal and informal forums for discussion of ideas and progress, and provides statistical services for all Projects and Cores. The Clinical Core recruits and maintains approximately 300 well-characterized HD patients. A separately funded research project, Presymptomatic Testing for HD, grew out of the resources of the Clinical Core. The Pathology Core has brain material on over 70 HD patients, most of whom were followed until death by the Clinical Core. Progress will be reviewed by an External Review Committee. Our in-house Ethics Committee reviews questions relating to patient participation in research.

Determine the longitudinal course of neuroanatomical changes in individuals with Huntington's Disease. Determine which regions of the brain are most affected by the disease at various points in its course, including the presymptomatic stage.

The central hypothesis of this proposal is that expansion of trinucleotide repeats in genes expressed in the brain may be one of the etiologies of neuropsychiatric disorders, including manic depressive illness. Expansion of trinucleotide repeats (CTC or CGG) in these genes has recently been discovered to underlie three different disorders (Fragile X Syndrome, Kennedy's disease, and myotonic dystrophy). The pleiotropic manifestations and unusual pattern of inheritance of these latter disorders are reminiscent of some pedigrees of manic depressive illness, schizophrenia, and other disorders, suggesting that at least some subtypes of these illnesses could be caused by a similar mechanism. We have preliminary data showing that there are many additional novel trinucleotide repeat containing genes expensed in the human brain. In Specific Aims #1 and 2 we will clone cDNAs for new human genes with CGG and CTG repeats, and characterize these cDNAs by sequencing them and by analyzing the expression of their mRNAs. In Specific Aim #3 we will determine their chromosomal localizations by doing PCR using human chromosome specific mouse human hybrid cell lines. We will analyze the lengths of the repeats using PCR in the Centre d'Etude du Polymorphisme Humain (CEPH) parents to determine their distribution in a well characterized population background. Based on the preliminary data, we predict that the lengths of these repeats will frequently be polymorphic. Those that are highly polymorphic will be mapped in the CEPH pedigrees and entered into the human genetic linkage map. In Specific Aim #4, we will search for expansions of the repeats in these novel genes in patients with manic depressive illness, as well as schizophrenia, autism, and mental retardation, in collaboration with ongoing fly study investigations at Johns Hopkins. For all genes, we will analyze all the probands in the initial screen using PCR. In addition, for the strongest candidate genes, we will study three affected and three unaffected members (including the parents) of each unilineal pedigree. If there appears to be expansion of the repeats in affected members, we will then analyze the entire pedigree as well as other pedigree.s in detail using PCR and genomic Southern blots. We predict that we will find one or more genes whose trinucleotide repeats are expanded only in affected members and therefore will be genes involved in those forms of manic depressive illness or other neuropsychiatric disorders.

Seven diseases are now known to be caused by expansions of triplet repeats. Four, including the subject of the present proposal, are caused by expansions of CAG repeats, coding for polyglutamine. These include Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA-1), and spinal and bulbar muscular atrophy. All involve degeneration of differing, although overlapping, subsets of neurons. The most recently discovered CAG triplet repeat disease is dentatorubral and pallidoluysian atrophy (DRPLA or Smith's disease). The gene causing this disorder was identified as part of a screening program for triplet repeats in the principal investigator's laboratory (Li et al., 1993). Primer sequences published in our paper were used by two groups of Japanese investigators to demonstrate that triplet repeat expansion at this locus causes DRPLA. We now propose to clone the full length cDNA for this gene, which we are terming "Atrophin-1." We have found that it has an unusual alternating acidic and basic residue protein motif with homology to other proteins, which may shed light on its function. We will clone and sequence full length cDNAs for rat and human versions of the cDNA. We will study the expression of the Atrophin-1 mRNA in human and rat tissues, in development, and in tissues from patients with the disorder, using techniques of RNA blot analysis and in situ hybridization. Based on the predicted protein sequence, we will synthesize unique peptides, couple them to carrier proteins, and raise and affinity purify specific antibodies. We will use these for protein blot and immunohistochemical studies to define the cellular and tissue localization of the Atrophin-1 protein. These studies will be carried out in parallel with ongoing studies in the PI's lab of the HD gene product. Together these studies should shed light on genes involved in these neurodegenerative disorders.

The Baltimore Huntington's Disease (HD) Project is an integrative clinical and basic science program designed to follow the natural history of HD in a cohort of well characterized patients, and perform laboratory studies to better understand the pathophysiology of the disorder. With the recent cloning of the gene whose expanding triplet repeat causes HD, this is now an opportune time to study the cell biology and biochemistry of the gene's protein product, in order to clarify the pathophysiology. In the Longitudinal Core Study, we will follow prospectively a well characterized cohort of 150 HD patients and 40 gene negative controls, based on an epidemiologic sample, in order to determine the influences, including the length of the triplet repeat, on the rate of progression of the disorder. Patients are evaluated and followed through the Clinical Core (B) to autopsy, and their brains are accessioned and studied through the neuropathology Core (C). The Genetics Core (D) assists in diagnosis by determining the length of the triplet repeat in IT-15, the gene which causes HD, and studies other factors which may influence age of onset in HD including possible modifier genes. The Neuropsychology Project (1) will better define cognitive deficits of HD and follow their course longitudinally over time. MRI scans will be performed in the Imaging Project (2) to correlate changes in the brain with functional disability. The Cell Biology Project (3) will elucidate the basic biology of the HD protein produce. We hypothesize that the pathophysiology of the disorder arises from abnormal protein-protein interactions caused by the expanded polyglutamine tract in the HD gene product (Huntington). We will characterize the cell biology of the HD protein and identify and study the proteins with which it interacts. These studies will clarify the clinical features of HD and illuminate the basic biology of the disorder, which should contribute toward the design of rational treatment.

neurotoxicology; protein structure function; protein sequence; Huntington's disease; nucleic acid repetitive sequence; molecular pathology; neurotransmitter transport; open reading frames; intermolecular interaction; gene expression; gamma aminobutyrate; protein glutamine gamma glutamyltransferase; brain metabolism; glutamine; enzyme substrate; tissue /cell culture; nucleic acid sequence; immunocytochemistry; laboratory rat; human tissue; transfection; molecular cloning; histopathology; immunologic assay /test; antisense nucleic acid;

The pathogenesis of Parkinson's Disease (PD) involves death of dopaminergic neurons in the substantia nigra and Lewy bodies in the substantia nigra and other brain regions, including brainstem monominergic neurons, and neurons in the cerebral cortex. Alpha-Synuclein mutations cause autosomal dominant forms of PD clinically indistinguishable from sporadic PD. In addition, alpha-synuclein is a component of the Lewy bodies in both PD and in Diffuse Lewy Body disease. In Specific Aim #1 we will identify protein interactors for alpha-synuclein using the yeast two- hybrid system and confirm the interactions using biochemical techniques. In Specific Aim #2 we will characterize the cellular pattern of expression of these interactors in comparison with alpha-synuclein. In Specific Aim #3 we will make a cell model which reproduces some of the features of PD using transient and stable expression of alpha-synuclein and interacting proteins in cell culture. We will study the pathways of cell death which are activated in this model. Taken together these studies will shed light on the molecular and cellular pathogenesis of PD and provide models for testing of experimental therapeutics.

The study is examining brain volumes and psychological measures in a population of patients with Huntington's disease (HD) who have been followed longitudinally. The specific aim of the project is to determine the relationship between subcortical brain volumes and the pattern and rate of cognitive decline. This is a unique project in which we have successfully interpreted a variety of neurocognitive, psychologic, and neuroanatomic data on 114 HD patients and 47 mutation- negative control subjects. As we continue to collect data annually, we hope to further and more specifically define predictors of impairment and patterns of decline over the course of the illness.

This Interdisciplinary Training Program provides postdoctoral research training in areas relevant to the biological bases of mental disorder. The training program is jointly sponsored by the Department of Psychiatry and the Department of Neuroscience at the Johns Hopkins University School of Medicine. The faculty members all have appointments in either of these two departments and have an established record of collaborative interaction. The research interests of the core faculty represent the major sub-disciplines of the neurosciences and biological psychiatry, including molecular and cellular neurobiology, developmental neurobiology, systems neurobiology, behavioral biology, neuroimaging, psychiatric genetics and neuropsychiatry. The primary vehicle for training is supervised research in a laboratory of one of the core faculty members combined with an organized didactic program in relevant aspects of neuroscience and psychiatric research methodology. The training program provides a unique opportunity for MDs and PhDs to obtain training at the postdoctoral level in multidisciplinary research oriented toward clinical psychiatry. The goal of the program is to prepare future academic researchers to undertake a career in the investigation of the biology of mental disorder.

Development of mouse models of neurodegenerative diseases is critical for testing hypotheses about pathogenesis and for experimental therapeutics. A mouse model of Huntington's Disease (HD) has previously been created using genomic DNA. However models using cDNA constructs have the advantage of being much simpler to modify, since cDNA constructs can be altered easily. We have generated transgenic mice expressing an N-terminal fragment of huntington with an expanded repeat. These mice show a phenotype including progressive weight loss, ataxia, and uncoordination, leading to early death. Post mortem examination reveals intranuclear inclusions, the pathologic hallmark of HD. We will characterize the mice with the truncated construct in specific aim 1. We will further define the neuropathologic and neurochemical changes underlying the phenotype. In specific aim number 2 we will generate mice with a full length cDNA construct and determine similarities and differences in the pathology. In specific aim 3 we will use a construct with a myc-his tag in order to define the site of cleavage of huntingtin, and to purify associated proteins. Finally we will engineer inducible constructs which can target the transgene selectively to the regions of the cerebral cortex and striatum believed to be essential for the development of HD pathology. These experiments will clarify the pathogenesis of HD and provide models for future studies of experimental therapeutics.

The goal of this proposal is to investigate the role of synphilin-1 in the pathogenesis of Parkinson's disease (PD). After Dr. Engelender had done her post-doctoral training in Dr. Ross's laboratory, Dr. Engelender had launched an independent carrier in her original country, Brazil. Dr. Engelender has an independent laboratory at the Department of Anatomy and is waiting for a vacant position to become an Associate Professor. While in Dr. Ross's laboratory, Dr. Engelender had the opportunity to become an expert in the yeast two-hybrid system. This collaboration will be important to find and characterize proteins related to the pathogenesis of PD. PD is a common neurodegenerative disease characterized by tremor, rigidity and bradikynesia. Patients with PD have their symptoms due to death of dopaminergic neurons of the substantia nigra of the brain. It was found that some families with autosomal dominant form of PD have mutations in the a-synuclein gene. A-Synuclein is also an intrinsic component of Lewy bodies of patients with sporadic forms of PD, suggesting that a-synuclein would have an important role in the pathogenesis of PD in general. We found that a-synuclein interacts in vivo with a novel protein called synphilin-1. Synphilin-1 leads to the formation of cytoplasmic inclusions when transfected in mammalian cells together with a-synuclein. In addition, we had just found that synphilin-1 is present in Lewy bodies of PD patients, strengthening our hypothesis that synphilin-1 would also be involved in the pathogenesis of PD. However, similar to a-synuclein, the function of synphilin-1 is still unknown. The main objective of this project is to determine the protein partners of synphilin-1. The identification of protein(s) that interact with synphilin-1, other than a-synuclein, will help to understand its normal function and perhaps the role of synphilin-1 and a-synuclein in PD. In order to determine their relevance to PD, we will study synphilin-1 and synphilin-1 interactor(s) by biochemical and cell biology means. We believe that our approach has the ability to produce unique information regarding the function and role of synphilin-1 and a-synuclein in pathophysiological processes related to PD.

This is an integrative program of Huntington's disease research ranging from the molecular to the clinical. One project involves a longitudinal clinical study of patients with HD and related phenotypes from a set of well characterized families deriving from an epidemiologic sample. A second project uses structural and functional imaging for longitudinal measurement of cerebral atrophy and dysfunction with a special interest in early HD and pre-symptomatic gene positive individuals. A third project involves biochemical and cell biologic studies of the pathogenesis of HD, focusing on the potential proteolytic cleavage of huntingtin, the possible involvement of huntingtin associated proteins, and the use of cell models to understand pathways of cell death and develop therapeutic interventions. A fourth project will use pharmacologic and genetic manipulations of our HD transgenic mouse model to determine possible means of ameliorating the phenotype. These projects are supported by four cores. One core provides administrative and statistical support. A second core provides examination, diagnosis and longitudinal follow up of patients. A third core provides neuropathologic examination, diagnosis, and tissue for research. A forth core provides genetic testing and identification of genetic variants which contribute to phenotypes. Overall, the goal of the program is to better understand the molecular and cellular pathogenesis of HD, and develop and test new therapeutic approaches.

DESCRIPTION (summarized from the abstract): This proposal addresses biochemical and cellular mechanisms of pathogenesis in Huntington's Disease. To do this, 3 specific aims are proposed. In the first aim, a characterization of the N-terminus of the huntingtin protein is proposed, and a characterization of proteins that interact with huntingtin. This will define the degree to which the Huntingtin protein is truncated in the cell, and what proteins might interact with the pathological protein. In the second aim, a characterization will be performed of huntingtin and its interacting proteins in cell model experiments using transient transfection techniques. Cleavage sites and interaction domains defined in specific aim 1 will be tested for the effects on protein localization and function. In specific aim 3, the effect of truncation of the huntingtin protein and interaction with other proteins in the cell on toxicity will be addressed. This will be addressed using both cell lines and primary neuronal cultures.

[unreadable] DESCRIPTION (provided by applicant): DentatoRubral and PallidoLuysian Atrophy (DRPLA) is a progressive neurodegenerative disease caused by a polyglutamine expansion in atrophin-1. The clinical features and areas of the brain affected resemble the more common Huntington's disease (HD), and we study the two diseases in parallel, since changes observed in both may represent common pathogenic features for all of the polyglutamine neurodegenerative diseases. In the previous grant period we generated a transgenic mouse model of DRPLA, and gathered some preliminary evidence suggesting the hypotheses that atrophin-1 undergoes proteolytic cleavage and nuclear translocation, resulting in gene transcription changes, as part of the pathogenesis of DRPLA. We gathered cell culture data to suggest that atrophin-1 has nuclear localization and export signals and that these are involved in toxicity. We now propose to conduct mechanistic in vivo experiments to test these ideas. In Specific Aim 1, we will generate transgenic mice expressing mutant full-length atrophin-1 with the putative nuclear localization signal altered, in order to determine whether nuclear localization contributes to pathogenesis in vivo. In Specific Aim 2, we will generate transgenic mice expressing mutant full-length atrophin-1 with the putative nuclear export signal altered, in order to determine whether nuclear localization contributes to pathogenesis in vivo. In Specific Aim 3, we will establish the site of cleavage of atrophin-1 with our collaborators at the Buck Institute, and generate mice transgenic for a construct expressing full-length atrophin-1 with this site altered. We will cross our atrophin-1 transgenic mice with mice hemizygous for a targeted deletion in the CBP gene in order to test the hypothesis that transcriptional misregulation contributes to pathogenesis in vivo. We predict that these experiments taken together will confirm roles in vivo for proteolytic cleavage, and nuclear localization, and support a role for alterations in gene transcription as critical for pathogenesis in DRPLA, and supporting the idea that these are general features of polyglutamine pathogenesis. Proteolytic cleavage would be an especially good target for therapeutic interventions. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Huntington's disease is a neurodegenerative disorder caused by an expanding CAG repeat coding for polyglutamine in the huntingtin gene. In the previous grant period we established and studied a transgenic mouse model of HD using an N-terminal fragment of huntingtin. We have now developed a new inducible transgenic model of HD expressing full-length huntingtin with an N-terminal myc tag to facilitate biochemical and cell biological studies. Preliminary data indicate that the mice have a robust progressive behavioral phenotype and characteristic pathology, as well as the presence of an N-terminal fragment of huntingtin. We believe proteolytic cleavage may be a critical event in HD pathogenesis. We study this HD model in parallel with a mouse model of the closely related polyglutamine disorder DRPLA, in which proteolytic processing appears to be critical for pathogenesis. In specific aim 1 we will breed sufficient numbers of these HD inducible transgenic mice to characterize the time course of the behavioral phenotype and to provide tissue for pathological and biochemical studies. In specific aim 2 we will define the pathology of the mice, including the regional distribution of intranuclear inclusions and other aggregates of the huntingtin protein. In specific aim 3 we will study the time course and regional and cellular distribution of the huntingtin fragment. We will determine its length and use methods of protein purification and mass spectrometry to determine the cleavage site. We will develop a cell model with stable inducible expression of the same mutant huntingtin construct for comparative in vitro studies. In specific aim 4 we will generate new transgenic mice with alterations in the huntingtin protein sequence to eliminate proteolytic cleavage. We predict that these mice will have a substantially decreased HD phenotype. Taken together these studies will establish cleavage as a key event in HD pathogenesis, and as a novel and accessible target for therapeutics. The new mouse model will be useful for studying pathogenic features of HD, and for testing experimental therapeutic interventions.

Huntington's disease; atrophy; basal ganglia; pathologic process; functional ability; cognition disorders; longitudinal human study; dementia; cognition; patient oriented research; clinical research; human subject; psychological tests;

Studies of the pathogenesis of familial PD will likely contribute to understanding the pathogenesis of all forms of PD. Familial PD can be caused by mutations in alpha-synuclein, parkin or DJ-1. We have identified synphilin-1 as an alpha-synuclein interacting protein, and found that it is a component of the ubiquitinated Lewy bodies in PD postmortem brain, and can be a substrate for ubiquitination by parkin. In a collaborative study, we have suggestive evidence that an R621C mutation in synphilin-1 may be a rare cause of PD. We have found that S 129 phosphorylation of alpha-synuclein strongly modulates inclusion formation in cell culture. We are developing inducible cell models of mutant alpha-synuclein toxicity, and have found that expression of A53T mutant alpha-synuclein causes direct cell toxicity. We propose to characterize the mechanisms of cell death caused by mutant alpha-synuclein, and the interactions of these proteins in the pathogenesis of PD. In Specific Aim 1 we will study alpha-synuclein inducible PC 12 cell lines and mechanisms of cell toxicity, including the role of phosphorylation and cleavage of alpha-synuclein (in collaboration with Project 3), and possible modulation of toxicity by DJ-1 (in collaboration with Project 4). In Specific Aim 2 we will characterize the interactions among alpha-synuclein, synphilin-1 and parkin (in collaboration with Project 1 and the Neuropathology Core). We hypothesize that inclusions are preferentially ubiquitinated via K63 linkage rather than K48 linkage, consistent with a potential role in cell signaling as well as protein degradation. In Specific Aim 3 we will study the phosphorylation of alpha-synuclein, and determine whether phosphorylation modulates interactions with synphilin-1, and promotes aggregation or cleavage, in collaboration with Project 1 and 3, and the Neuropathology Core. Finally in Specific Aim 4 we will create synphilin-1 transgenic mice. We will cross them with mice overexpressing alpha-synuclein, and characterize their behavior and pathology, in collaboration with Project 3 and the Transgenic Mouse Core. We will also cross these double transgenic mice with parkin knockout mice. If this looks promising, we can generate mice overexpressing synphilin-1 with an R621C mutation which may be linked to PD, and characterize their phenotype. We will also generate mice overexpressing alpha-synuclein with the S129A mutation, and characterize their phenotype. These studies taken together should shed light on interactions among proteins implicated in PD, and on the mechanisms of PD pathogenesis. They are likely to lead to improved cell and mouse models of the disease, which will facilitate the search for rational therapeutics.

genetics; laboratory mouse; model

DESCRIPTION (provided by applicant): LRRK2 and Parkinson's disease cell biology Parkinson's disease is a disorder of movement, cognition and emotion, characterized neuropathologically by neuronal degeneration and deposits of protein aggregates termed Lewy bodies. While most cases are sporadic, rare genetic forms of the disease, caused by mutations in alpha-synuclein, parkin, DJ-1 and PINK1, are helping to elucidate pathogenesis. In previous studies, we have defined the role of alpha- synuclein protein interactions (including interactions with synphilin-1 and parkin) in PD-related cell biology. Mutations in leucine-rich repeat kinase 2 (LRRK2) have recently been found to cause autosomal dominant PD. Our overall hypothesis is that identifications of LRRK2 protein interactions will help elucidate pathogeneses of LRRK2 related PD, and possibly sporadic PD. We have identified interactions between LRRK2 and several other proteins, including parkin, synphilin-1, WSB-1 and CARD7. We have found that that mutant LRRK2 causes direct cellular toxicity, and have initial data that for at least some of the LRRK2 mutations, GTP binding and kinase activity are necessary for toxicity. In Specific Aim 1 we will identify LRRK2 interacting proteins using the yeast two-hybrid system and co-immunopreciptation from transfected cells, and define interaction domains of these proteins. In Specific Aim 2 we will study the LRRK2 interactions in expression studies in cells in culture, and in mouse and human tissue, including postmortem human sporadic and mutant LRRK2 PD tissue. We will study the role of these interactors in LRRK2 cellular toxicity, by using siRNA, and by modifying the interaction domains. In Specific Aim 3 we will determine whether LRRK2 kinase activity is critical for cell toxicity, and determine whether LRRK2 can phosphorylate the interactors-and if so, we will determine whether this has a role in toxicity. These studies will help define the role of LRRK2 and its interacting proteins in cellular pathogenesis related to PD, and potentially identify targets for future therapeutic interventions.

Autopsy; CRISP; Cell Line; Cell Lines, Strains; CellLine; Cells; Cellular Inclusions; Cessation of life; Computer Retrieval of Information on Scientific Projects Database; Cysteine; Death; Disease; Disorder; Exons; Funding; Grant; HD protein, human; Half-Cystine; Huntingtin; Huntingtin Protein; Huntingtin protein, human; Huntington disease protein, human; Huntington's disease gene product; IT15 protein, human; Inclusion Bodies; Institution; Investigators; L-Cysteine; Life; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurons; Patients; Research; Research Personnel; Research Resources; Researchers; Resources; Risk; Source; Testing; Therapeutic Intervention; Toxic effect; Toxicities; United States National Institutes of Health; cell imaging; cellular imaging; cultured cell line; disease/disorder; human Huntingtin protein; intervention therapy; necropsy; neuron toxicity; neuronal; neuronal toxicity; neurotoxicity; postmortem

[unreadable] DESCRIPTION (provided by applicant): The search for neuroprotective therapeutic interventions for HD is accelerating. Volumetric MRI studies indicate that neurodegeneration in the basal ganglia begins ten years or more prior to manifest HD. Therapeutic neuroprotective trials in the pre- manifest population aimed at delaying the onset of manifest HD could potentially have a significant impact on this devastating disorder. CoEnzyme Q10 (CoQ) has emerged as one of the leading therapeutic candidates for neuroprotection in manifest HD, with strong near-significant trends for efficacy in several outcome measures in a 30 month study (CARE-HD) of 600 mg per day. While there are substantial data on the tolerability of CoQ at 600 mg/day in symptomatic HD, dose ranging studies suggest the possibility of decreased tolerability in otherwise healthy individuals at higher dosages. We propose to study CoQ at dosages of 600 mg/day, 1200 mg/day and 2400 mg/day to determine, in a population of expansion positive pre-manifest participants, the highest dosage that is tolerable; with the long term objective of developing future preventive therapeutic trials of CoQ at that dosage. In Specific Aim 1, we will establish the safety and tolerability of CoQ at dosages of 600 mg/day, 1200 mg/day and 2400 mg/day in a mutation positive pre-manifest population with the HD CAG repeat expansion, in the context of a randomized double-blind 20 week parallel-group trial. In Specific Aim 2, we will establish that Coenzyme Q10 is biologically active by assessing changes in serum CoQ levels, and 8-Hydroxydeoxyguanosine (8OHdG) and 8-Hydroxyguanosine (8OHrG) levels in the same trial. We will assess the relationships between serum levels of CoQ, biomarkers of oxidative stress, biomarkers of DNA repair mechanisms (OGG1) and dosage levels of CoQ. The proposed trial is significant in that it will be the first study to evaluate a potential therapeutic agent in a population of individuals at 100% risk for developing a neurodegenerative illness, but who are not yet ill. It will allow us to select a dosage of CoQ for future definitive randomized placebo controlled trials in pre-manifest HD to delay or prevent onset of manifest HD, and will give us valuable information about the process and feasibility of such trials in pre-manifest participants. [unreadable]

DESCRIPTION (provided by applicant): Huntington's disease is a progressive neurodegenerative disorder caused by polyglutamine-encoding muta- tional expansion of the huntingtin gene. While symptomatic treatments are available, Huntington's disease is presently not curable;there are no therapeutic agents that address the underlying neuronal cell death asso- ciated with the disorder. Accordingly, there is a critical need to identify new compounds for preclinical devel- opment against Huntington's disease. The long-term goal of this research is to identify therapeutic strategies that help alleviate the devastating impact of Huntington's disease on patients and their families. The central hypothesis of this study is that targeted, assay-guided isolation of natural products, coupled with semi-synthetic optimization of their bioactivity, will yield compounds with curative potential against Huntington's disease. Therefore, the objective of this translational R21 study is responsive to the NINDS program announcement, "Exploratory/Development Projects in Translational Research," as we will identify candidate therapeutics pre- venting cell death by inhibiting and/or disrupting toxicity of the aggregation-prone form of mutant huntingtin. Our preliminary studies, focused on the screening of a chemically and biologically diverse extract library, yielded several lead natural products that are capable of protecting cells against mutant huntingtin toxicity. The rationale for this research is that as a consequence of obtaining 1) additional naturally-occurring congen- ers of the bioactive principles and 2) preparing derivatives of the active leads via semi-synthesis, we will be able to refine our focus to one or two of the most promising candidate compounds that will serve as therapeutic leads for subsequent U01 preclinical studies. Based on the success of our preliminary investigation, the fol- lowing two specific aims will be addressed. In Specific Aim 1, plants and fungi that are biosystematically- related to the organisms which provided the original natural product leads will be subjected to assay-guided isolation to identify new bioactive congeners of the lead compounds. Under Specific Aim 2, the bioactive lead natural products and their analogs will be further probed by subjecting them to a judicious series of derivatiza- tion procedures that expand on their favorable biological activities. All compounds and derivatives emerging from this work will be critically evaluated in a mammalian-cell-based mutant-huntingtin expression system and the best small-molecule candidates will advance for screening in a mouse model of Huntington's disease. This method will generate a focused series of lead compounds exhibiting enhanced potency and improved biophys- ical features (e.g. good predicted blood-brain-barrier penetration) that are well suited for focused preclinical studies. Our approach is innovative because natural products, an exceptional source of bioactive drug-like lead compounds, have not been systematically evaluated for the discovery of neuroprotective agents against Hun- tington's disease. Furthermore, these samples offer an unprecedented opportunity to identify small-molecule leads that are structurally distinct with pharmacologically novel modes-of-action. This research is significant because candidate therapeutic agents evolving from this research will be particularly well suited for detailed preclinical evaluation and development under the auspices of the U01 mechanism. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because 1) there is currently no cure for treating Hunting- ton's disease and 2) candidate compounds emerging from these studies could find applications in the treat- ment of this and other related polyglutamine expansion disorders. Therefore, this research will ultimately have a positive impact on the health and well-being of patients and families afflicted by Huntington's disease and other neurodegenerative illnesses.

DESCRIPTION (provided by applicant): This Interdisciplinary Training Program in Psychiatry and Neuroscience provides postdoctoral research training in areas relevant to the neurobiological bases of mental disorders. The training program is jointly sponsored by the Department of Psychiatry and the Department of Neuroscience at the Johns Hopkins University School of Medicine. The program is poised to take advantage of the tremendous progress in the understanding of the biology of mental disorders over the past few years, and contribute to new discoveries n the upcoming project period. The research interests of the core faculty represent the major sub-disciplines of the basic and clinical neurosciences relevant to major psychiatric disorders, including molecular and cellular neurobiology, mouse genetics, developmental neurobiology, behavioral biology, neuroimaging, psychiatric genetics and clinical neuropsychiatry. The faculty members are all experienced researchers and mentors, andam provides a unique opportunity for MDs and PhDs to obtain training at the postdoctoral level in basic or clinical neuroscience research oriented toward major psychiatric disorders. The goal of the program is to prepare future academic researchers to undertake a career in the investigation of the biology of mental disorders.

DESCRIPTION (provided by applicant): This consortium aims to capitalize on an unprecedented "grand" opportunity to develop a novel and powerful model of Huntington's disease (HD), a fatal neurodegenerative condition with no current treatment. Skin cells from patients with HD can be reprogrammed to pluripotency and then differentiated into specific neuronal and glial cell types, permitting investigation of the effects of the genetic lesion in the susceptible human cell types. We hypothesize that the genetic changes that cause HD lead to specific alterations in neuronal function- perhaps even survival-that will give important clues as to the mechanism and progression of disease. Altered cellular phenotypes will also serve as the foundation for translational research and drug development. Stimulus funding will bring together a highly focused group that (i) has a strong track record of innovative HD research and of working together, (ii) is poised to engage in cutting-edge research with recently generated induced pluripotent stem (iPS) cells derived from HD patients and is committed to broad distribution of findings, protocols and iPS lines, (iii) can capitalize on this stimulus funding through further grant applications and collaborative studies, and (iv) is partnered with CHDI, an HD foundation with dedicated HD stem cell and translational/drug discovery programs. This infusion of funds will accelerate the coordinated analysis of iPS lines and leverage the complementary, synergistic skill sets that will move the field forward more rapidly than would be possible by any group alone. The proposed studies will provide an entirely novel genetically accurate model to test new drugs in the fight against this disease, a unique resource that will benefit the entire HD community. PUBLIC HEALTH RELEVANCE: We hypothesize that the genetic changes that cause Huntington's disease lead to specific alterations in neuronal function-perhaps even survival-that, in turn, give important clues as to the mechanism of disease and its progression and offer a potential basis for small molecule screening assays. Our studies will provide a more authentic way to study the consequences of the HD mutation in human target cells. They represent a unique and timely opportunity to enhance the investigation of disease mechanisms and will generate a validated resource freely available to the HD community to further accelerate HD research toward a successful treatment.

DESCRIPTION (provided by applicant): Parkinson's disease is a common neurodegenerative disease accompanied by significant functional disability, resulting from neurodegeneration in the substantia nigra and other brain regions. LRRK2 mutations constitute the most common identified cause of human PD, accounting for up to 20% of PD in some populations. The most common mutation, G2019S, is present in up to one percent of apparently sporadic PD in European and North American populations, and causes typical late onset PD, with response to L-DOPA, and Lewy body pathology. We have previously reported that expression of mutant LRRK2 causes substantial cell toxicity in cell culture, and we have recently found that toxicity is mediated via kinase activity of mutant LRRK2. We now propose to study mouse models expressing either full-length human LRRK2 with the G2019S mutation or wild-type. We have generated transgenic mice expressing LRRK2 under the control of the prion promoter, which have a moderate phenotype, and we have more recently generated homozygous mice with higher expression levels and normal development. In Specific Aim 1 we will characterize survival and behavior of the homozygous transgenic mice expressing mutant G2019S LRRK2, and perform an initial assessment of neuronal loss and Lewy bodies, or other PD-like neuropathology in appropriate cellular populations. In Specific Aim 2 we will cross the heterozygous mice with mice expressing a truncated fragment of alpha-synuclein using the TH promoter. A new model of PD which reproduces aspects of phenotypes seen in the human disease would be of great benefit. Future studies of the role of kinase activity could clarify pathogenesis. Furthermore LRRK2 kinase may be an excellent therapeutic target, and mouse models would be valuable for preclinical therapeutic trials. PUBLIC HEALTH RELEVANCE: We propose to generate new mouse models of Parkinson's disease. LRRK2 mutations constitute the most common identified cause of human PD, accounting for up to 20% of PD in some populations. We have previously reported that expression of mutant LRRK2 causes substantial cell toxicity in cell culture, and we have recently found that toxicity is mediated via kinase activity of mutant LRRK2. A new mouse model of PD which reproduces aspects of phenotypes seen in the human disease would be of great benefit. Future studies of the role of kinase activity could clarify pathogenesis. Furthermore LRRK2 kinase may be an excellent therapeutic target, and mouse models would be valuable for preclinical therapeutic trials.

DESCRIPTION (provided by applicant): Huntington disease (HD) is a progressive, fatal, neurodegenerative disease, with movement disorder, psychiatric features, and cognitive decline. The neurodegeneration is regionally heterogeneous with preferential loss of striatal medium spiny neurons, but with significant atrophy in other regions. This leads to the question whether this pattern of regional degeneration is circuit related, reflecting the anatomic connections of the affected neurons, or by contrast is multifocal. To address this question, we will perform statistical shape analysis of basal ganglia and examine white matter structures connecting atrophied regions with affected cortical regions. We hypothesize that there will be heterogeneous atrophy in selected subcortical regions, and that shape analysis may detect some localized changes early, before overall volumes change significantly. We hypothesize that regional globus pallidus atrophy will correlate with specific local basal ganglia connections, but that regional striatal atrophy will not entirely correlate with connections predicted by regional cortical atrophy. We will also perform complementary analysis of white matter structures. Specifically Aim 1 will perform cross- sectional statistical shape analysis (caudate, putamen, thalamus, hippocampus, nucleus accumbens and globus pallidus) in 351 subjects with and without prodromal HD; Aim 2 will perform longitudinal shape analysis on specific subcortical gray matter regions (as listed above) for 351 subjects with scans at 2 time points; and Aim 3 will perform analysis of white matter structures in to determine whether the regions of striatum most affected receive projections from the regions of cortex most affected, and whether the regions of globus pallidus most affected receive projections from the regions of striatum most affected. PUBLIC HEALTH RELEVANCE: Specific sub-regions of atrophy identified in prodromal and early symptomatic Huntington's Disease (HD) will help determine whether neurodegeneration in HD follows a circuit-based pathway connecting brain structures (similar to prion disease, and as hypothesized for Alzheimer's and Parkinson's disease), or is multifocal. These data will be important for planning interventions, which directly target the brain and thus will be directly relevant for HD therapeutics.

DESCRIPTION (provided by applicant): A CAG repeat expansion in exon 1 of the HD gene product, huntingtin, causes Huntington's disease (HD), a fatal neurodegenerative disease for which there is no cure or neuroprotective treatment. Dysregulation of transcription is a major feature of HD pathogenesis, as indicated by a large body of work using RNA array techniques, and work on specific transcription factors and their targets. More recent studies have also suggested a role for huntingtin in RNA processing. Prior work on gene expression alterations in HD brain tissues used 3' biased gene expression arrays. Of increasing importance in many human diseases, particularly neurodegenerative diseases, is the occurrence of aberrant alternative pre-mRNA splicing. However, conventional gene expression techniques are not well suited to quantitative analysis of alternative splicing patterns, and do not sample rare transcript well. Several lines of evidence from our preliminary work suggest global splicing abnormalities in HD. For example, we reported earlier that microRNA miR-124 was significantly reduced in HD brains. Work by Maniatis and colleagues showed that miR-124 promotes neuronal-specific alternative splicing events by down- regulating an important tissue-specific splicing regulator, polypyrimidine tract-binding protein (PTBP1). Consistent with the decrease in miR-124, we have preliminary evidence for significantly increased PTBP1 mRNA levels in HD patient samples. Moreover, preliminary data suggest that several exons in genes regulated by PTBP1 show corresponding changes in exon inclusion/exclusion in HD brain. Inclusion or exclusion of non-constitutive exons can have dramatic effects on transcript stability and protein activity. Thus transcriptome alterations in HD may extend beyond up- and down-regulated genes to include changes in gene and protein isoforms. Assessing these events on a global scale for HD will aid efforts to unravel disease pathophysiology, and may identify new drug targets for therapy. In our work, which encompasses 3 aims, we will move from identification of the altered HD transcriptome, to validation, to in vitro and in vivo studies to test their relevance on HD phenotypes. These studies combine the genomics and bioinformatics expertise of the Xing lab and the HD expertise of the Ross and Davidson labs. The functional relevance of those changes will be elucidated using gain and loss of function studies in the Davidson and Ross labs, where both groups have substantial experience with HD models.

DESCRIPTION (provided by applicant): Cell models of Huntington's disease have been very important for understanding the cell biology of the disorder and for testing potential therapeutics The recent development of human induced pluripotent stem cell (iPSC) models has greatly enhanced our ability to model disease in human cells. As part of the NINDS funded HD iPSC Consortium, we have developed a cell model of HD. However, as valuable as this model is, the differentiation to medium spiny neurons is very long, cumbersome and expensive, making study of the cells and screening for therapeutics very difficult. We therefore now propose to generate immortalized striatal precursors from the HD iPS cells. In Specific Aim 1, we will use HD and control iPS cells differentiated to striatal precursors for immortalization with viral vectors, and optimize protocols for differentiation to neurons with a mature striatal medium spiny neuron phenotype. In Specific Aim 2, we will assess the cells for CAG- repeat-expansion-dependent toxicity and rescue, and format the model as a screenable assay. These studies taken together will permit the development of a novel cell model of HD which will have features similar to the iPS cell model, but be more reproducible and tractable. These cells should be very valuable for studying HD pathogenesis and for screening for novel neuroprotective therapeutics.

DESCRIPTION (provided by applicant): The best validated therapeutic target in HD remains Htt itself. Previously identified PTMs of expanded Htt (e.g. S13/16 and S421) are important modulators of HD pathogenesis. We previously studied proteolytic cleavage of Htt (Ratovitski et al., 2007, 2009, 2011), and more recently have been studying covalent PTMs of Htt, especially phosphorylation. Htt is very likely to have many other sites of PTM besides the currently known ones (described in the Significance section). We plan to characterize Htt PTMs systematically and quantitatively. Furthermore, our experiments include the use of human HD iPS cells for our continuing discovery studies, and a staged program beginning with mass spectrometry for discovery and progressing through in vitro and then in vivo confirmation and functional validation. Phosphorylation which enhances toxicity will be especially promising as a therapeutic target, if relevant kinases can be identified and inhibited. In Aim 1, we will define Htt PTMs usin Htt-N586-82Q mice, HD knock-in mice and human HD iPS cells, and will determine whether the polyQ expansion in Htt leads to changes in PTMs. In Aim 2, we will conduct in vitro functional studies of the effects of Htt PTMs on mutant Htt conformation and cellular toxicity. In Aim 3, we will test the effects of PTMs on mutant Htt toxicity in vivo, using our N-586-82Q transgenic mouse model or stereotactic injection of viral expression vectors encoding Htt with altered PTMs into the striatum of wild-type mice. These studies taken together will identify novel sites of PTM in mutant Htt, and functionally validate their role in pathogenesis in vitro and in vivo. The sites will then be candidate targets for therapeutic development.

TRACING SPREAD OF PATHOLOGY WITHIN THE HD BRAIN VIA AUTOMATED NEUROIMAGING Huntington's disease (HD) is a progressive fatal neurodegenerative disorder caused by an expanding CAG repeat in the Huntingtin gene coding for an expanding polyglutamine stretch in the Huntingtin protein. Neurodegeneration in HD is in large part caused by toxic effects of the abnormal Htt protein, and there is increasing evidence that mutant Htt can spread, like prions, and like abnormal proteins in other neurodegenerative diseases, from one neuron to another. Elucidating the sequence and pattern of atrophy in the HD brain is of special current importance, with ?gene silencing? or ?RNA-lowering? trials, using antisense oligonucleotides, or shRNA, or related reagents, in active development. The key to success of these trials will be to know where and when to intervene, since these reagents do not penetrate the blood-brain-barrier, and must be injected into the CNS. Our studies will elucidate the temporal and spatial patterns of the spread of HD neurodegeneration, to elucidate the pathogenesis of HD and to help guide interventional trials. In Specific Aim 1, we will conduct cross sectional and longitudinal analyses of the spatial and temporal pattern of volumetric change and shape change in subregions of HD compared to control brains, using longitudinal T1 and DTI scans from HD cases and controls from the PREDICT-HD study and the TRACK-HD study. Scans will undergo automated processing through MRICloud, segmented into about 400 subregions. We hypothesize that atrophy will begin in the striatum and spread sequentially to adjacent white matter and then to cortical gray matter. Alternatively degeneration may be multifocal. In Specific Aim 2 we will determine clinical correlations of the brain atrophy from Aim 1. In Specific Aim 3 we will use tract-tracing methods to study the spread of pathology in the HD brain. We hypothesize that the spread of atrophy in the HD brain follows patterns of axonal connectivity. Alternatively, it is possible that pathology begins and spreads in a multifocal fashion. Taken together these studies will delineate the longitudinal spread of pathology within the HD brain, and its clinical consequences. This information will elucidate the pathogenesis of HD and will be critical for designing the timing and localization of planned interventional trials.

Patient-derived Huntington's disease iPS cell models represent a substantial advance over previous cell models of HD, but they have a number of disadvantages ? a long, complex and expensive differentiation protocol, heterogeneous differentiated cell phenotypes, variability of experimental results, and difficulty in production of sufficient amounts of differentiated cells for biochemical assays or pharmacological screens. We have therefore developed a strategy to differentiate iPS cells to a striatal precursor stage, and immortalize them, to derive homogeneous clonal lines. These cells can be maintained as routine monolayer cultures and differentiated to a medium-spiny neuron phenotype in only two weeks (versus 3 months for iPSCs). We hypothesize that differentiated immortalized Striatal Precursor Neurons (SPNs) will recapitulate the CAG-repeat-expansion-associated phenotypes of the HD iPSCs, and should have advantages in being more homogenous, and more suitable for development of screenable assays to identify HD therapeutics. In the R21 phase, we will generate an allelic series of SPNs, perform omics analysis of the cell lines, and validate their suitability for screening in a 24-well plate format. In the R33 phase, we will format the assay for 96-well plates and screen with libraries of small molecules including a library of inhibitors of protein kinases for targets identified in our NINDS/CHDI supported project to study posttranslational modifications (PTMs) of huntingtin. We believe these cells will be highly amenable for development into a screenable cell model for drug discovery efforts.

Validation of Novel Pathogenic Post-Translational Modifications of Huntingtin, and of Modifying Enzymes as Therapeutic Targets for Huntington's Disease HD is a progressive neurodegenerative disorder caused by a single gene mutation, the CAG repeat expansion in Huntingtin (Htt). The best-validated therapeutic target in HD remains Htt itself, and RNAi approaches are under development to lower mutant Htt in HD patients. However, these approaches have daunting obstacles of delivery large molecules to the CNS. Small molecule therapeutics remains an important alternative. We have identified near 40 PTMs (phosphorylation and acetylation) on endogenous Htt from human and mouse brain using quantitative proteomics. We find that the PTMs are arranged in clusters, and we have initial evidence for crosstalk among certain sites in these clusters.. Furthermore, we find that alteration of the residues for several of the PTMs abrogates mutant Htt neuronal toxicity, showing that PTMs are modulators of mutant Htt toxicity. These data indicate that identification of the enzymes catalyzing these PTMs will have potential for yielding defined molecular targets for disease-modifying therapy for HD. We now propose the next series of steps in these studies, in order to identify additional PTMs, and to identify enzymes that catalyze modifications, as potential defined molecular targets for rational therapeutics for HD. We will take advantage now of our newly developed HD iPSC-derived immortalized striatal precursor cell lines. In Aim 1, we will identify novel PTMs on Htt using our novel striatal precursor cell lines (SPNs) derived from HD iPS cells. These can be differentiated into neurons with a medium-spiny neuron phenotype. We will generate PTM-specific antibodies to high-priority PTMs, in order to evaluate localization in cellular compartments using immunofluorescence, and cell fractionation followed by Western blot, in SPNs and human brain. In Aim 2 we will identify kinases and other modifying enzymes. In Aim 3 we will confirm the role of PTMs and kinases in HD cellular pathogenesis and their validation as therapeutic targets. We will perform CRISPR/Cas9 PTM alterations for highly-ranked functional PTMs, as well as CRISPR/Cas9 knock-out of most relevant kinases in striatal precursor neuron model in collaboration with Gene Edit BioLab and Xiao and Shihua Li, who are expert at gene editing in relation to HD. Taken together, these studies will further elucidate the basic biology of mutant Htt. The identification of modifying enzymes has the potential to provide defined molecular targets for preclinical therapeutic studies.

Abstract: The LRRK2 locus, coding for the LRRK2 (Leucine Rich Repeat Kinase 2) protein, is a major risk factor for Parkinson?s disease (PD), and mutations in the LRRK2 gene contribute to both genetic and sporadic PD. Intriguingly, the LRRK2 locus also confers increased risk for Crohn?s disease and leprosy, and LRRK2 is expressed in immune cells, neurons and glia. We recently discovered that LRRK2 interacts with NOD-like receptor (NLR) sensor family proteins (NLRPs), NLRP1 and NLRP3. NLRP proteins are key mediators of innate immunity, and are components of inflammasomes. We propose to test the hypothesis that mutant LRRK2 interacts with and activates the NLRP3- and NLRP1-inflammasomes leading to microglial activation and neurodegeneration contributing to PD pathology. In Aim 1, we will characterize the interaction of LRRK2 with NLRP1 and NLRP3 in vitro and in vivo. In Aim 2, we will investigate whether mutant LRRK2 activates the NLRP3- infammasome in microglial activation, leading to neuroinflammation and degeneration in vitro and in vivo. In Aim3, we will examine whether mutant LRRK2 activates the NLRP1-inflammsome in neurons, resulting in neurodegeneration. These studies will elucidate mechanisms underlying LRRK2-linked inflammasome pathway in neurodegeneration and immune dysfunction. They may also have broader implications for other neurological diseases related to neuroinflammation. The understanding of NLRP/LRRK2-linked pathways in PD pathogenesis may reveal potential biomarkers, and may provide novel targets for development of disease-modifying therapeutic strategies. Thus, this project has the potential for significant impact, and benefits those suffering from PD and other neurodegenerative diseases.