Pathogenic LRRK2 regulates ciliation probability upstream of tau tubulin kinase 2 via Rab10 and RILPL1 proteins

Abstract

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We showed 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 the loss of cilia seen upon serum readdition. 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, and 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; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. 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.

Ciliogenesis is Not Directly Regulated by LRRK2 Kinase Activity in Neurons

“…MLi-2 treatment showed an increase as reported [ 14 ], but the increase was very weak at ~1.1-fold ( Fig. 1A ), compared to the 2~3-fold increase previously reported in LRRK2 R1441C/G MEF cells [ 14 , 16 ].…”

“…Further studies disclosed that PPM1H specifically dephosphorylated Rab proteins phosphorylated by LRRK2, and PPM1H knockdown suppressed ciliogenesis [ 15 ]. Sobu et al [ 16 ] reported that treatment of pathogenic LRRK2 R1441C MEF cells with MLi-2, an LRRK2 kinase inhibitor, significantly increased ciliogenesis at least 3-fold. Another study reported that the proportion of ciliated cells in HEK293T cells expressing LRRK2 pathogenic mutants Y1699C, G2019S, and R1441C, but not WT, was decreased and that MLi-2 treatment partially rescued ciliogenesis defects in the same cells expressing mutants only under serum-fed conditions [ 17 ].…”

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

“…Nevertheless, like RILPL1, expression of HA-RILPL2 blocks ciliogenesis, perhaps by sequestering important components (such as MyoVa and/or Rab10) in a manner that interferes with their ability to function. Whereas RILPL1 overexpression blocks cilia formation by interfering with TTBK2 recruitment to the mother centriole (Sobu et al, 2021), RILPL2 overexpression blocks cilia formation by a different mechanism, as centriolar TTBK2 levels were not changed upon RILPL2 overexpression. It seems likely that in wild-type cells, RILPL2 overexpression interferes with MyoVa's ability to support early events in ciliogenesis that trigger CP110 uncapping independent of TTBK2.…”

“…Rab8a Q63L was cloned into a pET14b expression plasmid using Gibson assembly. mKO2-PACT was generated by amplifying the PACT domain of Pericentrin from cDNA of RPE cells as described (Sobu et al, 2021). To generate a plasmid-encoding Rab10 shRNA#1 and #2-resistant wild-type Rab10, inverse PCR was performed with primers (#1 F 59-ACTGCAAATATGGGATACAGCAGG-39, #1 R 59-TTAATCTTCTTTCCTTGTAATTCAACTG-39; #2 F 59-CACGGCATCAGG-TTTTTTGAGACTAG-39, R 59-TTCTCTAGCGATCTGTTCTCCTTTTCC-39) myc-Rab10 plasmid was used as a template, and the PCR product was self-ligated with T4 DNA ligase, and T4 polynucleotide kinase before transformation.…”

“…RPE cells were transfected with Fugene HD (Promega), whereas A549 cells were transfected using Lipofectamine 3000 according to the manufacturer. RPE cells stably expressing mKO2-PACT were generated by lentivirus as described by Sobu et al (2021). Cells were checked routinely for mycoplasma using either MycoALert Mycoplasma Detection Kit (LT07-318; Lonza) or PCR.…”