Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterised by motor dysfunction, resulting from loss of dopaminergic neurons in the midbrain. PD has a multifactorial etiology with a-synuclein aggregation, lysosomal dysfunction and altered calcium homeostasis features of disease.

Heterozygous mutations in the gene encoding the lysosomal enzyme GBA are the most common genetic cause of PD. Patient iPSC-derived neurons offer a unique opportunity to understand the biology of dopaminergic neurons in PD in the context of the disease-conducive genetic background. Phenotypic drug screening in patient derived dopaminergic neurons allows novel drugs and targets to be identified which act on both cell-type specific and disease-relevant phenotypes.

We have previously identified several disease relevant phenotypes in iPSC-derived dopaminergic neurons from PD patients with a heterozygous GBA N370S mutation including decreased intracellular Ca2+ release, increased α-synuclein release and decreased GBA activity. To identify drugs which may correct these phenotypes, we have established a medium throughput screening platform in iPSC-derived dopaminergic neurons in 384-well format.

We have optimised a dual primary screening assay to identify compounds which alter intracellular Ca2+ release and GBA activity and screened a 4000 compound library. We have identified >250 compounds which increase ionomycin-induced intracellular Ca2+-release and confirmed >60 of these with dose-responses. These screening hits were prioritised by activity in multiple GBA patient lines and represent 23 putative targets which have been followed up initially using small-molecule approaches. Three of these targets have been confirmed with multiple structurally diverse compounds and CNS-penetrant analogues identified to enable in vitro target validation.

Secondary screening of compounds includes correction of established GBA N370S neuronal phenotypes including α-synuclein release and perturbation of the autophagosomal-lysosomal system, in addition to confirmation using orthogonal models of PD.

We demonstrate a successful medium-throughput phenotypic screening platform using iPSC-derived dopaminergic neuronal cultures from PD patients as a novel platform for identifying novel targets and compounds for drug discovery in Parkinson’s patients.