Asa Abeliovich, Md - US grants

DESCRIPTION (provided by applicant) Parkinson?s disease (PD) is a devastating neurological disorder that afflicts over 1 million North Americans and is characterized by gait difficulty, rigidity, and tremor. Although symptomatic therapies are effective in the early stages of PD, no treatments exist to alter the inexorable progression of the disorder. The recent identification of two genes that underlie familial inherited forms of PD, alpha-Synuclein (alpha-Syn) and Parkin, has afforded new clues regarding the molecular pathogenesis of PD. Interestingly, aggregates of alpha-Syn protein, along with ubiquitin, a protein degradation marker, appear to be major constituents of the intracytoplasmic inclusions that typify PD, termed Lewy bodies. Furthermore, the primary structure of Parkin displays sequence homology to ubiquitin ligases, implicating it directly in the ubiquitin/proteasome pathway. Thus, Parkin and alpha-Syn are both implicated in a common potential mechanism of PD pathology: abnormal protein processing and degradation. The analysis of alpha-Syn deficient animals suggests a role for alpha-Syn in the regulation of dopamine release at central synaptic terminals, but it remains unclear how this relates to the pathological activity of mutations in this gene. I intend to utilize complementary mouse genetic, cellular, and biochemical techniques to investigate the mechanisms of action of Parkin and alpha-Syn. Specifically, I propose to test the hypothesis that these genes play roles in common genetic and biochemical pathways in familial PD. By gaining insight into the normal and pathological functions of these genes, it may be possible to develop disease-altering therapies for PD in the foreseeable future. Questions that will be investigated include: --How do alpha-Syn and Parkin mutations cause dopamine neuron loss? --Do mutations in alpha-Syn and Parkin act within a common genetic pathway? --How do the normal activities of these proteins relate to their pathological functions?

[unreadable] DESCRIPTION (provided by applicant): Defective protein degradation through the ubiquitin proteasome pathway (UPP) has been hypothesized to play a central role in neurodegenerative disorders such as Parkinson's disease (PD). Mutations in Parkin, a putative ubiquitin ligase component, cause a familial, autosomal recessive form of PD characterized by midbrain dopamine neuron loss. It has therefore been hypothesized that inefficient degradation and consequent toxic accumulation of Parkin ubiquitination substrates underlie the loss of dopamine neurons in autosomal recessive Parkinson's disease. We further hypothesize that Parkin may play a direct role in regulating neuronal survival in the CNS. We propose to use molecular and cellular tools to investigate the mechanism of Parkin action in protein ubiquitination and neuronal survival. Our preliminary data indicate that Parkin associates in a multiprotein ubiquitin ligase complex with 2 previously characterized ubiquitin ligase components, the F-box/WD repeat-containing protein hSel- 10, and Cullin-1 (Cul 1). Furthermore, hSel-10 serves to direct this complex to specific substrates including Cyclin E, a putative regulator of neuronal apoptosis. We will test the hypotheses that (1) auxiliary components of the Parkin ubiquitin ligase complex serve to regulate or target this activity, and (2) that, in Parkin-associated familial PD, premature neuronal death is a consequence of defective ubiquitination and the accumulation of neuronal apoptosis-related Parkin complex substrates. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The investigation of familial Parkinson's disease-related syndromes is likely to provide insight into the pathogenesis of sporadic Parkinsons's disease (PD) and may suggest novel therapeutics. Mutations in the amino-terminal repeat domain of a-Synuclein (alpha-Syn) underlie rare autosomal-dominant, familial forms of PD. Familial, autosomal-recessive mutations in Parkin lead to both juvenile and adult-onset forms of PD. Furthermore, Parkin appears to possess a ubiquitin-ligase activity, implicating it in the protein degradation process. Mounting evidence implicates altered protein ubiquitination and degradation by the ubiquitin/proteasome pathway (UPP) in human neurodegenerative disorders. Both pathological and genetic data link altered protein degradation pathways with PD. A pathological hallmark of PD, the Lewy Body (LB), appears to represent intracellular inclusions composed of multiple proteins including ubiquitin, a-Synuclein (alpha-Syn), ubiquitin carboxy-terminal hydrolase (UCH-L1), and Parkin. Both Parkin and UCH-L1 are implicated in protein ubiquitination, whereas alpha-Syn appears to be a substrate of ubiquitination. The goal of this proposal is to gain an understanding of the mechanisms of action of the PD-related genes alpha-Syn, Parkin. Of particular interest are potential relationships among these molecules. I propose to combine complementary biochemical, cellular, and genetic approaches to this end.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a slowly progressive but devastating neurodegenerative disorder that afflicts over 1 million Americans. The etiology of Parkinson's disease has posed a particularly challenging problem to investigate, in large part because of the lengthy disease course. Pathological studies have correlated cellular oxidative stress or protein misfolding with PD. The identification of several genes that underlie familial, inherited forms of PD has allowed for a molecular approach to PD. Recently, mutations in DJ-1 have been found to lead to autosomal recessive, early onset PD. The clinical history of DJ-1-associated familial PD is typical of the disease, including rest tremor, rigidity, gait difficulty, and responsiveness to levodopa treatment. Functional neuroimaging of the brain reveals a presynaptic dopamine deficit consistent with PD. The normal cellular function of DJ-1 is unknown, but the ubiquitous expression of DJ-1 in vertebrates and the high degree of conservation among DJ-1 homologues among diverse species suggest that DJ-1 subserves important cellular functions. DJ-1 homologues have been implicated in several biochemical and cellular roles including protease, amidotransferase, catalase, and chaperone activities, complicating the interpretation of DJ-1 function. The expression of DJ-1 and homologues is induced ii cellular stress responses, suggesting a possible link between DJ-1 mutation and PD pathology. Our preliminary data indicate that DJ-1 is a redox-dependent molecular chaperone that plays an important role in the cellular response to oxidative stress. We propose to investigate the mechanism of DJ-1 activity using complementary biochemical, cellular, and mouse genetic approaches.

DESCRIPTION (provided by applicant): Several observations have linked neurodegenerative disorders such as Parkinson's disease (PD) and Alzheimer's disease with altered intracellular protein degradation (1). Recently, autophagy mechanisms leading to lysosomal-mediated protein degradation have come into focus. Autophagy is an essential mechanism of protein degradation that is induced in the context of starvation and other stressors, and is a prominent feature in brain pathology in neurodegenerative diseases (3, 4), likely representing defects in the autophagy-lysosome pathway (5). Furthermore, mutations in lysosomal genes have been found to underlie rare familial inherited forms of Parkinsonism (6) and associated with common sporadic PD(3). Key questions exist: i. Do autophagy defects recapitulate aspects of neurodegenerative disorders? ii. By what mechanism does altered autophagy in mDNs lead to pathological and morphological changes? We hypothesize, based on preliminary data, that: i. Autophagy plays a central role in regulating the survival and morphology of mDNs, and deficiency of autophagy recapitulates key aspects of neurodegenerative pathology, including accumulation of disease-associated proteins. ii. A novel mechanism by which autophagy defects lead to pathology is through direct (but non- canonical) downstream modification of the PI3K/PTEN/AKT/GSK3beta/Tau signaling pathway;this relates specifically to altered accumulation of signaling pathway components. iii. The altered PI3K/PTEN/AKT/GSK3beta/Tau pathway signaling plays a causal role in the phenotypes associated with autophagy deficiency. PUBLIC HEALTH RELEVANCE: Several observations have linked Parkinson

DESCRIPTION (provided by applicant): A number of developmental and adult brain disorders are associated with midbrain dopamine neurons (mDNs), including Parkinson's disease (PD), schizophrenia, autism, dyskinesias, and drug addiction. Thus, the fundamental mechanisms that regulate the development and function of these cells are of great import. In prior studies, we and others have investigated basic regulatory processes, such as regulation of the expression of key enzymes in the dopamine biosynthetic pathway in the context of dopamine neuron development, function, and survival. However, it is clear from these studies that there is a high level of complexity governing all of these processes. For instance, at least 2 transcription factors, Nurr1 and Pitx3, function synergistically to govern expression of late developmental mDN markers in simplified ES cell-based culture models. More recently, in preliminary data and a published manuscript, my lab has found evidence of an added layer of complexity involving post-transcriptional regulation by microRNAs (miRNAs) in the context of mDN development and function. miRNAs are evolutionarily conserved, 18-25 nucleotide non-protein coding transcripts that play an important function in post-transcriptional regulation of gene expression during development. Specifically, we identified microRNA, miR-133b that is enriched in mDNs and functions within a regulatory feedback circuit with Pitx3. Here we propose to more broadly define the level of complexity of gene expression regulation by miRNA in mDNs, and to determine the function of these forms of regulation in vivo. Ultimately, such forms of regulation are likely to play a role in mDN-associated diseases, and furthermore manipulations of these mechanisms offer potential avenues for therapies. We wish to test two hypotheses: 1. miRNAs function in the regulation of mDNs, both within feedback circuits with mDN transcription factors and by the direct regulation of key mDN targets. 2. Such regulatory networks play functionally important roles in mDNs in vivo. PUBLIC HEALTH RELEVANCE A number of developmental and adult brain disorders are associated with midbrain dopaminergic neurons (mDNs), including Parkinson's disease (PD), schizophrenia, autism, dyskinesias, and drug addiction. Thus, the fundamental mechanisms that regulate the development and function of these cells are of great import. Here we propose to unravel the complex molecular regulatory signals that determine the development and function of midbrain dopamine neurons. We focus on the role of microRNAs, which are short RNA molecules that regulate the expression of key dopaminergic neuron genes. We initially use simplified model systems, including embryonic stem cell derived dopamine neurons and primary neuron cultures, which allow for a detailed molecular analysis. Ultimately, we extend these studies to confirming the role of regulatory molecular circuits in the intact behaving rodent.

DESCRIPTION (provided by applicant): The goal of this application is to investigate the feasibility and requirements of converting existing in vivo, differentiated cells of the adult CNS - such as astroglia - into functional and appropriately integrated neurons, using in situ directed neuronal conversion (IDNC). IDNC is novel and distinct from stem cell approaches. With IDNC, a cocktail of transcription regulators is transduced into mature CNS cells, such as astroglia, using polycistronic lentiviral vectors or otherwise. These factors lead to a rapid and directed conversion into a distinct mature neuronal phenotype. Such directed conversion challenges the basic tenet that differentiated cell phenotypes are inherently stable - particularly in situ in the CNS. Directed conversion potentially overcomes some of the inherent difficulties with other therapeutic cell replacement strategies, such as stem cell approaches. Directly converting existing cells may be least intrusive on the general architecture of existing circuitry. A second and perhaps greater challenge is the functional integration of newly converted neurons into CNS local circuitry. This problem will be address using a variety of physiological and optical imaging approaches.

DESCRIPTION (provided by applicant): We recently described the directed conversion of human skin fibroblasts from unaffected individuals and Alzheimer's disease (AD) patients to a CNS neuron phenotype, termed human induced neuronal cells (hiN) 1. Herein we propose to further develop hiN cells as tools for AD modeling, and subsequently to validate the approach in a more detailed analysis of cellular mechanisms of AD. The focus of the proposed studies is on familial AD (FAD)-associated disease with defined genetic lesions in presenilin-1 (PSEN1) or presenilin-2 (PSEN2). A clear advantage of such an analysis of FAD with defined mutations is that this facilitates genetic 'rescue' studies, as well as genetic dissection of function. Yet ultimately, perhaps the most exciting aspect of the hiN cell technology is that it may permit a cellular analysis of 'sporadic' disease. The overarching hypotheses to be tested in this work are that cellular aspects of FAD pathophysiology are: (1) Cell-autonomous to neurons and maintained through epigenetic reprogramming, and therefore amenable to hiN cell modeling. (2) Include altered intracellular vesicular trafficking at the soma and the synapse. The proposed deliverables for this proposed work are: (1) Novel human neuronal cell models for dissection of AD mechanisms and drug screening. (2) Directed reprogramming tools that may be broadly applied to the study of neurological disease.

DESCRIPTION (provided by applicant): Common genetic variants in the human population play a significant role in the pathogenesis of non-familial ('sporadic') Parkinson's disease (PD). Among such PD risk variants, the alpha-synuclein (aSyn) locus is of particular interest, as SNPs in this locus show the strongest and most robust impact on sporadic PD risk Furthermore, very rare mutations in aSyn as well as triplication of the aSyn gene locus lead to familial inherited forms of PD. aSyn is thus an attractive therapeutic target for PD, with most strategies aimed at reducing its level or aggregation. Our preliminary data point to a novel regulatory mechanism that we hypothesize to impact aSyn physiological and pathological functions: aSyn messenger RNA (mRNA) transcript differential 3' untranslated region (3'UTR) usage. Longer transcript isoforms (aSynL) correlate with increased protein accumulation, intraneuronal protein redistribution, and pathological functions, both in human brain and in model systems. This ultimately may provide a novel therapeutic approach by targeting specifically pathological rather than physiological functions of aSyn. aSyn 3'UTR usage is modified by dopamine exposure as well as by aSyn locus common genetic single nucleotide polymorphism (SNP) variants that increase PD risk. The 2 mechanisms appear largely separate. Whereas a small segment of the 3'UTR (sufficient to confer dopamine sensitivity) is conserved in rodent aSyn, most of the 3'UTR sequences are unique to human. Our specific hypothesis is that longer mRNA transcript isoforms of human aSyn, with extended 3'UTRs, aSynL, play important pathological roles, by impacting the accumulation of aSyn protein. The goals of this proposal are to (i) define regulatory mechanisms of the aSyn 3'UTR and (ii) relate the molecular properties of different aSyn mRNA 3'UTR isoforms to pathological aSyn functions in vivo. The impact of this proposal is potentially high, as pinpointing a specific pathogenic transcript would present a novel therapeutic target. Such regulation could be especially amenable to high-content drug screens. The deliverables of the project are (i) to provide a structure/function analysis of aSyn 3'UTR sequences with respect to aSyn regulation, and (ii) to potentially identify novel drug targets for PD and other synucleinopathies, by identifying molecular mechanisms that mediate the process.