Screening for Compounds that Lower Intracellular Alpha-Synuclein Levels

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PROJECT SUMMARY/ABSTRACT The goal of this Supplement, as with the parent grant, is to identify agents that reduce the levels and accumulation of alpha-synuclein (αSyn) that underlies the pathogenesis of Parkinson's disease (PD). The premise of this Supplement proposal is supported by preliminary data from the parent grant and by numerous reports including point mutations or duplication/triplication of the SNCA (Synuclein Alpha) gene that results in αSyn increases leading to autosomal-dominant early-onset PD. The finding that the promoter for the SNCA gene is hypomethylated in PD resulting in increased expression and that over-expression of the gene is a factor contributing to onset of PD suggests reducing the levels of αSyn is a promising target for therapeutic intervention. Furthermore, reduction of αSyn levels using agents such as β2-adrenoreceptor (β2AR) agonists has been reported to be neuroprotective in both cell line and rodent models. An epidemiological analysis of a Norwegian population revealed individuals using β2AR agonist, salbutamol for asthma, have a reduced risk of developing PD. In this Supplement, as part of expansion of the aim 1, we will evaluate the effect of validated hits on αSyn oligomerization and formation of intracellular aggregates using the bimolecular fluorescence complementation (BiFC) assay. Such inhibitors could reduce intracellular αSyn levels by downstream effects enhancing αSyn degradation. The Lewy-related pathology (LRP), primarily comprised of αSyn, is not restricted to PD and has been found in a subset of autopsied Alzheimer's disease (AD) brains and brains of Dementia with Lewy Bodies (DLB) patients. Apolipoprotein E4, a risk factor for AD, is also a risk factor for PD and is associated with earlier onset of PD. A recent study also suggests there is a link between higher levels of αSyn in the CSF and early stages of development of cognitive decline in AD. In this Supplement as part of expansion of the aim 3 we will evaluate prioritized hits in iPSC derived neurons for effect on AD biomarkers. Current therapeutics for PD provide only symptomatic relief, there is an urgent need for the development of disease-modifying compounds capable of slowing or halting PD progression. To address this need in PD as well as LRP in AD and DLB, in the parent proposal we are performing high throughput screening (HTS) of the UCLA 200K compound library to identify hits that lower intracellular αSyn levels; these hits will be validated and prioritized by potency, drug-like properties, and brain permeability for further analysis. In this Supplement for Aim 1 Expansion, hits identified from HTS by AlphaLISA that reduce intracellular αSyn in SK-N-MC human neuroepithelioma cells and are validated in primary and secondary assays in the parent grant would be evaluated in αSyn oligomerization using the BiFC assay that we will set up during the Supplement proposal. This will enable further elucidation of the mechanism of action (MoA) of the active hits. In Aim 3 Expansion, select compounds from Aim 2 would be evaluated in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons from PD, AD, and normal donors; we will evaluate for both αSyn levels and AD biomarkers including sAPPα, sAPPβ and Aβ42.