Pathogenic LRRK2 regulates ciliation probability upstream of Tau Tubulin kinase 2

Abstract

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We have shown previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1 and together, these proteins block cilia formation in a variety of cell types including patient derived iPS cells. We have used live cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and loss of cilia seen upon serum re-addition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation. Kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss, but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis. In both cases, failure of CP110 uncapping was due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, recruitment of EHD1, another step important for ciliogenesis, appears unaltered. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation, and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.KEY WORDS: LRRK2, Rab GTPase, primary cilia, Parkinson's disease SIGNIFICANCE STATEMENTMutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases, in particular Rab8 and Rab10. Phosphorylated Rabs bind preferentially to a distinct set of effectors and block in primary ciliation in multiple cell types. We show here that the cilia blockade is upstream of the recruitment of TTBK2 kinase to the mother centriole, a step required for the release of CP110 and subsequent cilia formation. This study provides fundamental information related to how pathogenic LRRK2 interferes with normal cell physiology.

Endogenous Rab29 does not impact basal or stimulated LRRK2 pathway activity

“…Pathogenic LRRK2 mutants decrease primary cilia formation in cell culture in a manner that is rescued upon LRRK2 inhibition [ 26 ]. The ability of LRRK2 to inhibit ciliogenesis requires RILPL1 binding to LRRK2-phosphorylated Rab8A and Rab10 [ 31 , 32 ]. Recent work showed that LRRK2-phosphorylated Rab proteins are dephosphorylated by a highly selective PPM1H protein phosphatase [ 33 ].…”

Endogenous Rab29 does not impact basal or nigericin and monensin stimulated LRRK2 pathway activity

“…Pathogenic LRRK2 mutants decrease primary cilia formation in cell culture in a manner that is rescued upon LRRK2 inhibition [26]. The ability of LRRK2 to inhibit ciliogenesis requires RILPL1 binding to LRRK2phosphorylated Rab8A and Rab10 [31,32].…”

LRRK2-phosphorylated Rab10 sequesters Myosin Va with RILPL2 during ciliogenesis blockade

“…For bacterial expression, MyoVa GTD, Rab10-Q63L (1-181) and Mst3 were kind gifts from Amir Khan (Trinity College, Dublin, Ireland). mKO2-PACT was generated by amplifying the PACT domain of Pericentrin from cDNA of RPE cells as described (Sobu et al, 2020).…”

“…RPE cells were transfected with Fugene HD (Promega) while A549 cells were transfected using Lipofectamine 3000 according to the manufacturer. RPE cells stably expressing mKO2-PACT were generated by lentivirus as described at (Sobu et al, 2020). Cells were checked routinely for Mycoplasma using either MycoALert Mycoplasma Detection Kit (Lonza LT07-318) or PCR.…”