The novel Parkinson's disease locus RIT2 and alpha-synuclein function in intersecting pathways
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Objective: We aim to describe the molecular pathways at the crossroad of etiology and susceptibility in PD, focusing on Rin, coded by the recently associated locus RIT2, and its interaction with alpha-synuclein (aSyn). Background: A disease-modifying therapy based on a pathogenic mechanism currently represents the main clinical need in PD. Accumulation of aSyn is the hallmark pathology of PD but its biology is still unclear. The protein Rin is a small GTPase that couples nerve growth factor (NGF) stimulation to ERK and p38 MAPK pathways. Its role in the PD context is currently unknown. Methods: Ric (the Rin ortholog) was silenced in D. melanogaster expressing human aSyn and subjected to negative geotaxis. Cell lines stably expressing wild-type and A53T aSyn (SK-N-SH) or Rin (SH-SY5Y) were obtained. SH-SY5Y cells expressing wild-type and G2019S LRRK2 were kindly provided by Dr. Evy Lobbestael (University of Leuven). Protein and mRNA levels were analyzed by western blotting and real-time PCR, respectively, while immunocytochemistry was performed to assess single cell protein expression. Kinase activation was measured using the AlphaScreen SURE fire assay (Perkin Elmer). Results: Silencing of Ric in flies ameliorated aSyn-induced climbing deficit. RIT2 mRNA levels are reduced in aSyn and LRRK2 cell lines, but NGF is able to stimulate kinase activation. Further, basal p38 activity is enhanced in Rin cells. Lastly, LRRK2-G2019S (but not wild-type) cells display pSer129-aSyn positive intracellular staining. In a preliminary experiment, transient overexpression of Rin-GFP (but not GFP alone) seems to reduce the percentage of GFP+ cells with pSer129-aSyn staining. Conclusions: We show that Rin functionally affects aSyn biology. In addition, we confirm NGF activates Rin-ERK/p38 MAPK pathways. Future experiments will investigate the role of this pathway on synaptic transmission. Importantly, Rin appears to have a role in the phosphorylation/aggregation of aSyn in cell lines, triggered by another etiological cause of PD, (G2019S-LRRK2). Collectively, a common pathological pathway involving these proteins seems to occur in cells.