This project investigates the mechanisms underlying pathogenesis of X-linked Parkinson’s disease due to the p.G192R mutation in the gene RAB39B.

The missense mutation p.G192R in the gene RAB39B causes X-linked dominant parkinsonism. Loss of function of Rab39b has been found to cause intellectual disability and autism spectrum disorder. Rab39b encodes a neuronal Ras family GTPase with a role in autophagosome formation and vesicular trafficking. Rab39b is highly conserved in Drosophila, with 75% amino acid sequence similarity with human orthologues, including conservation of p.G192.

Using CRISPR/Cas9 we generated a Rab39G192R fly model by using a single-stranded oligo donor to introduce the mutation into the endogenous Drosophila Rab39. We will evaluate mutant flies for protein aggregation, locomotor deficits, reduced lifespan, and neurodegeneration.

We also generated iPSCs from peripheral blood cells from a kindred with X-linked PD due to the p.G192R variant. We will differentiate iPSCs into neurons, in which endocytic activity and endolysosomal trafficking will be characterized using DQ-Red BSA, immunocytochemistry, and western blot. We will evaluate endolysosomal function by determining whether there are insoluble ubiquitinated protein aggregates or oligomerization of alpha-synuclein.

A dominant negative Rab39S23N transgene was lethal in flies when expressed ubiquitously or in neuronal tissue. We observed increased ubiquitinated protein aggregation when a constitutively active Rab39 transgene was expressed in neuronal tissue. Our G196R Rab39 adult flies appear morphologically normal. Preliminary characterization of neuronal progenitor cells differentiated from iPSCs have not revealed any gross morphological differences.

We are developing a new human iPSC-derived cell culture and Drosophila model of X-linked PD due to the c.586G>C, p.G196R mutation in RAB39B. We will be further characterizing lifespan, locomotor behavior and protein aggregation in the Drosophila Rab39 model. In our human cell culture model, we will be investigating endolysosomal function and protein aggregation to elucidate the role of RAB39B in PD pathogenesis.