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Year 4 Pilot Projects

Identification of DJ-1 substrates and DJ-1-targeting compounds for Parkinson disease therapeutic discovery

Lih-Shen Chin, Ph.D., Principal Investigator; Haian Fu, Ph.D., Co-investigator

Parkinson disease (PD) is the most common neurodegenerative movement disorder that has been known for two centuries, yet the pathogenic mechanisms of PD remain unclear and there is currently no effective treatment to halt progression of this devastating disease. Human genetic studies have identified mutations in DJ-1 as a cause for autosomal recessive, early-onset PD. In addition, emerging evidence indicates that DJ-1 is a susceptibility gene for late-onset sporadic PD, and we have shown that oxidative damage to DJ-1 is associated with sporadic PD. Despite the progress in characterization of DJ-1, the precise biochemical function of DJ-1 and how loss of this function triggers neurodegeneration remain unclear. To gain insight into DJ-1 function, we and others have solved the crystal structure of human DJ-1, which shows that DJ-1 exhibits conspicuous structural similarity to the PfpI family of bacterial intracellular proteases. Our recent studies revealed that DJ-1 is synthesized as a latent protease zymogen and can be converted into a catalytically active cysteine protease by carboxyl-terminal cleavage. We found that DJ-1 protease activity is critically involved in cellular defense against oxidative stress. We propose to capitalize on our findings and use the activated form of DJ-1 protease and its “substrate-trap” mutant to screen for endogenous substrate proteins of DJ-1 protease. Furthermore, we will test the hypothesis that PD-linked DJ-1 mutations cause neurodegeneration by impairing the catalytic function and/or substrate-binding property of DJ Finally, we will leverage our knowledge gained from studying DJ-1 to develop high-throughput screening assays and perform a pilot screen to identify DJ-1-targeting compounds for PD therapeutic discovery. Completion of the proposed experiments will yield valuable insights into the mechanism of DJ-1 action in health and disease and provide key preliminary data to support the effort to develop novel therapies for PD and related neurodegenerative disorders.

Motor effects of a novel mGluR4 PAM in parkinsonian monkeys

Gunasingh Masilamoni Jeyaraj, Ph.D., Principal Investigator; Stella M. Papa, M.D., Co-investigator

Long-term L-DOPA therapy of Parkinson’s disease (PD) is usually associated with motor complications including disabling response fluctuations and L-DOPA-induced dyskinesias (LID). Based on significant changes of glutamate transmisi.on in dysfunctional basal ganglia circuits in PD, glutamatergic treatments may provide a strategy to reduce L-DOPA requirements and ameliorate LID. Particularly, agents acting on metabotropic glutamate receptors (mGluRs) with modulatory effects may be suitable for clinical use. Additionally, mGluRs that are mostly presynaptic and control neurotransmitter release may be expressed more selectively in basal ganglia regions. The group III mGluR4 is predominantly expressed in the external pallidum reducing striatopallidal transmission. Significantly, this pahway is overactive in PD suggesting that mGluR4 activating drugs may have antiparkinsonian effects. mGluR4 is also expressed in corticostriatal terminals playing a role in striatal dysregulation and altered L-DOPA responses. Therefore, this receptor may be an excellent target to develop non-dopaminergic treatments with efficacy on both parkinsonian symptoms and LID. In rodents, mGluR4 agonists administered directly into the external pallidum showed antiparkinsonian effects. However, earlier mGluR4 agents had poor brain penetration that limited further tests in animal models. Recently, new positive allosteric modulators (PAMs) with improved pharmacological properties have been developed, providing tools to investigate the therapeutic potential of this target. The selective PAM VU0418506 (Vanderbilt Center for Neuroscience Drug Discovery) demonstrated excellent brain exposure following systemic administration in rodents. Also, VU0418506 showed antiparkinsonian efficacy in rats with nigrostriatal 6-OHDA lesion. With Dr. Conn’s collaboration providing this novel compound, we now have the opportunity to extend preclinical studies. We propose to examine the antiparkinsonian and antidyskinetic effects of VU0418506 in the primate MPTP model of PD. These studies will determine VU0418506 efficacy following monotherapy and L-DOPA co-administration. Outcomes of this project will provide grounds to continue studies for target validation and advance this therapeutics to Phase I clinical trials.

Therapeutic intervention in cell death pathways prematurely activated in dopaminergic neurons.

Edward S. Mocarski, Ph.D., Principal Investigator; Malu G. Tansey, Ph.D., Co-investigator

Premature dopaminergic (DA) neuron death remains a key hallmark of Parkinson’s Disease (PD). Therapeutic intervention may provide benefits if appropriately selective. Over the course of the last decade, caspase-dependent apoptosis and caspase-independent cell death pathways have been implicated as the underlying cause of inflammatory processes in neuronal disease. Over the last few years one caspase-independent pathway has become well-recognized as an alternate to apoptosis. Programmed necrosis controlled by receptor interacting protein kinase 1 (RIP1 or RIPK1) and RIP3 may be unleashed to cause cell death and inflammation during embryogenesis and in disease states. Here, we will establish the contribution of RIP1/RIP3-mediated programmed necrosis in DA neuron death, employing the combination of genetic tools and pharmacological interventions that have emerged from our ongoing research into basic cell death signaling pathways. The relevance of caspase 8 (Casp8)-dependent extrinsic apoptosis and Casp8-dependent regulation of necrotic death will be determined in the process of this study. Mice with kinase domain mutations in RIP1 and RIP3 will be combined with small molecule inhibitors of these kinases to fully dissect the potential for therapeutic intervention. The co-PIs have complementary expertise to make this a highly incisive pilot likely to yield an understanding of the disease process and open practical therapeutic directions to treat PD and other chronic inflammatory diseases affecting the central nervous system (CNS). Out of this pilot, we expect to be in a position to pursue foundation and NIH grant support for this important area with highly translational and practical significance.