Identifying regulatory targets in parkinson’s disease pathology and screening potential therapeutics
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Abstract
Parkinson’s disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc). There are currently only treatments for relieving the symptoms of Parkinson’s disease with no cure. All of the treatments currently available vary in efficacy and usually cause irreversible side-effects including dyskinesia after long term use (PD Med Collaborative Group, 2014). The main cause of the loss of the dopaminergic neurons is the aggregation of proteins called Lewy bodies. The formation of the Lewy bodies is mainly mediated by alpha-synuclein. Most Parkinson’s disease cases are idiopathic. This has made scientists look at other reasons for the formation of Lewy bodies other than genetics such as post-translational modifications (PTM) on proteins. One such post-translational modification that has been looked at as having an important role in PD pathology is the small ubiquitin-like modifier (SUMO). SUMO is a post-translational modification that may be involved in the aggregation and toxicity of alpha-synuclein (Krumova et al., 2011; Zhu et al., 2018) In this study we assess the role of SUMOylation on alpha-synuclein in vivo and in vitro. Our main hypothesis is that the over-expression of SUMO conjugase, Ubc9, protects dopaminergic neurons in the striatum. We used the Ubc9 overexpressing C57Bl/6 mouse model from the John Hallenbeck lab at NINDS (Lee et al., 2012) and injecting them with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) chronically. We then quantified the levels of alpha-synuclein in the striatum through confocal microscopy. We also generated stable N27 cell lines over-expressing wild-type alpha-synuclein-HA and the K96R:K102R mutant alpha-synuclein-HA to assess different protein-protein interactions potentially. After confirming the expression of wild-type and the SUMOless alpha-synuclein mutant, the proteins were immunoprecipitated, run on a gel, and sent out for mass spectrometry. The immunoprecipitation samples were unable to be read, but the inputs were able to be read. The most interesting observation when comparing the two inputs is the difference in ubiquitin. There is more ubiquitin in the wild-type than the mutant input, which supports our hypothesis that SUMO competes with Ubiquitin for binding to lysines in target proteins. Since the current treatment options for PD only involve treating the symptoms and do not focus on the neuroprotection or neurorecovery, we looked at a pharmacological approach to develop novel compounds that could have potential therapeutic effects. In collaboration with AurimMed Pharma Inc., we screened 31 novel small compounds supplied by AurimMed Pharmaceuticals and tested for neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP+) and measured in MTT and LDH assay (courtesy of Dinesh Verma). The screening of the novel compounds resulted in the identification of a few promising compounds that showed neurorecovery effects at all the concentrations used in both MTT and LDH assays. Neurorecovery is the recovery of cells from MPP+ damage. These studies may help elucidate possible biological neuroprotective targets as well as novel compounds that do not simply target the symptoms of PD, but prevent the loss of dopaminergic neurons.