Kinase inhibition of G2019S-LRRK2 enhances autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions

Abstract

The Parkinson's disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alphasynuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance.

RIT2 reduces LRRK2 kinase activity and protects against alpha-synuclein neuropathology

“…It has been extensively reported that both aSyn and LRRK2 affect the autophagic process (Bellomo et al, 2020; Bravo-San Pedro et al, 2013; Ho et al, 2019; Schapansky et al, 2014). Furthermore, we recently found that the reduction of pS129-aSyn-positive inclusions in G2019S-LRRK2 cells, following exposition to a LRRK2 kinase inhibitor, depends on correct lysosomal activity(Obergasteiger et al, 2020). As an unbiased approach we used the RT² Profiler™ PCR Array for autophagy to measure the gene expression levels of 84 genes implicated at different stages of the ALP.…”

“…1 ng of cDNA was used for each reaction to asses RIT2 mRNA gene expression (Hs01046671_m1, FAM), multiplexed with a housekeeping gene (RPP30, HEX, BioRad cat# 10031243). PCR was carried out as suggested by the manufacturer using ddPCR Supermix for probes (BioRad, cat# 11969064001 The autophagy gene expression array (PAHS-084Z, Qiagen) for G2019S-LRRK2 cells without and with RIT2 overexpression was carried out according to the manufacturers protocol and as described in (Obergasteiger et al, 2020). Quantitative PCR was performed in a CFX96 Touch™ Real-Time PCR Detection System (BioRad).…”

“…To investigate lysosome morphology, we utilized the Lysotracker Deep Red dye (Molecular Probes, L12492) following the manufacturer´s instructions, as in (Obergasteiger et al, 2020).…”

“…To study the proteolytic activity of lysosomes, the fluorescent DQ-Red-BSA dye (Molecular Probes, D12051) was used following manufacturer´s instructions, as in (Obergasteiger et al, 2020).…”

“…Here, we investigate the effects of enhanced RIT2 expression on aSyn pathology, ALP regulation, and the underlying molecular mechanism using preclinical in vitro and in vivo PD models. Overexpression of RIT2 in G2019S-LRRK2 neuroblastoma cells restored ALP deficits and reduced accumulation of phosphorylated aSyn (pS129-aSyn), phenocopying the effects of pharmacological LRRK2 kinase inhibition(Obergasteiger et al, 2020). The selective overexpression of Rit2 in DA neurons in the mouse SNc attenuated nigrostriatal neurodegeneration and pS129-aSyn neuropathology induced by virally expressed aSyn.…”