The LRRK2 signaling network converges on a centriolar phospho-Rab10/RILPL1 complex to cause deficits in centrosome cohesion and cell polarization

Abstract

The Parkinsońs disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases including Rab8 and Rab10, which enhances their binding to RILPL1 and RILPL2. The nascent interaction between phospho-Rab10 and RILPL1 blocks ciliogenesis in vitro and in the intact brain, and interferes with the cohesion of duplicated centrosomes in dividing cells. We show here that regulators of the LRRK2 signaling pathway including vps35 and PPM1H converge upon causing centrosomal deficits. The cohesion alterations do not require the presence of other LRRK2 kinase substrates including Rab12, Rab35 and Rab43 or the presence of RILPL2. Rather, they depend on the RILPL1-mediated centrosomal accumulation of phosphorylated Rab10. RILPL1 localizes to the subdistal appendage of the mother centriole, followed by recruitment of the LRRK2-phosphorylated Rab proteins to cause the centrosomal defects. The centrosomal alterations impair cell polarization as monitored by scratch wound assays which is reverted by LRRK2 kinase inhibition. These data reveal a common molecular pathway by which enhanced LRRK2 kinase activity impacts upon centrosome-related events to alter the normal biology of a cell.

Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases

“…In our study, RAB10 appears to increase the recruitment and/or activation of kinesin on LC3+ and LAMP1+ puncta. RAB10, like many other RABs, is regulated by phosphorylation with phospho-RAB10 (especially T73) being generally more active and membrane-associated (Yan et al, 2018; Lara Ordóñez et al, 2022; Kluss et al, 2022; Wauters et al, 2020; Waschbüsch et al, 2020; Homma et al, 2021). RAB10, especially phospho-RAB10, regulates the motility of multiple kinesin-1 and kinesin-3 cargoes within cells (Etoh and Fukuda, 2019; Deng et al, 2014; Taylor et al, 2015; Zajac and Horne-Badovinac, 2022).…”

Targeting Rab‐RILPL interactions as a strategy to downregulate pathogenic LRRK2 in Parkinson's disease

“…Homozygous LRRK2-R1441C knock-in murine embryonic fibroblasts (R1441C-LRRK2 MEFs) were generated from mice at E12.5 and spontaneously immortalized by prolonged passaging, and they were a generous gift from Dario Alessi [13]. R1441C-LRRK2 MEFs were chosen as they express high endogenous levels of LRRK2 and display centrosome cohesion and ciliogenesis defects as compared to littermate wildtype MEFs, which are reverted by the LRRK2 kinase inhibitor MLi2 [21,27,28]. Cells were grown in full medium consisting of DMEM containing high glucose (Gibco, 11960-044), 10% fetal bovine serum (Gibco, 10438-026), 1 mM sodium pyruvate (Gibco, 11360-070), nonessential amino acids (Gibco, 11140-050), 2 mM L-glutamine (Gibco, 25030-081), and 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, 15140-122).…”

“…The centrosomal cohesion deficits mediated by pathogenic LRRK2 correlate with an accumulation of pRabs at the centrosome in a manner dependent on RILPL [17,19,21,22]. To determine whether the RIP peptides disrupt the pRab-RILPL interaction in intact cells, we treated R1441C-knock-in MEF cells with the different peptides and quantified the percentage of cells displaying pRab staining (Figure 6A,B).…”

“…Higher vertebrates have two homologous forms of RILPL: RILPL1 and RILPL2 [18]. The interaction between phosphorylated Rab8a and Rab10 and RILPL proteins at the mother centriole/ciliary base was found to cause both ciliary and centrosomal cohesion defects in various cell types in vitro [13,17,[19][20][21][22]. Physiologically, these pRab-RILPL complexes also cause deficits in centrosome cohesion including in primary or immortalized lymphocytes from LRRK2-PD patients as compared to healthy controls [20,23,24].…”

A potential patient stratification biomarker for Parkinso’s disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

“…Phosphorylation of Rab10 impairs its normal function in membrane trafficking (38,39) but allows it to interact with a new set of effector proteins including RILPL1 (16). RILPL1 is localized at the mother centriole and recruits phosphorylated Rab10 to this location (40,41). The mother centriole forms the base upon which cilia are formed, and the centriolar phospho-Rab10/RILPL1 complex blocks cilia formation in a variety of cell types in vitro (16,40,42,43).…”