R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils

Abstract

Heterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1–2% of all cases of Parkinson’s disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2—either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10Thr73) was measured by quantitative multiplexed immunoblotting for pRab10Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10Thr73 occupancy. We found a significant over fourfold increase in pRab10Thr73 phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10Thr73 phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors.

Distinct profiles of LRRK2 activation and Rab GTPase phosphorylation in clinical samples from different PD cohorts

“…In other words, in vitro kinase activity is a good predictor of LRRK2 mutation status, but not of disease status. In another recent study, combining western immunoblotting and targeted massspectrometry measures of phosphorylated Rab10, it was reported that T73-Rab10 levels in a small sample of carriers of the R1441G were significantly elevated in neutrophils compared to healthy control subjects 12 . Unlike the present study, phosphorylated Rab10 levels from carriers of the G2019S-LRRK2 mutation were not different from healthy controls, regardless of disease status 12 ; however in an earlier study also using an MS-based approach, pT73-Rab10 levels in neutrophils were approximately 2-fold higher in a set of G2019S carriers 15 .…”

“…In a small proof-of-concept study, Rab10 phosphorylation (T73) in neutrophils was not significantly changed in carriers of the G2019S LRRK2 mutation 10 ; and in PBMCs from iPD patients, pT73-Rab10 was not changed compared to healthy controls, despite being correlated with levels of certain cytokines 11 . Interestingly, in neutrophils from carriers of the less common R1441G mutation in LRRK2, a significant increase in pT73-Rab10 is detected in comparison to healthy controls, irrespective of disease status 12 . This is consistent with findings reported in transgenic mice expressing R1441C or G2019S, where a robust increase in Rab10 phosphorylation was observed in R14411C but not G2019S mice 13 .…”

Rab GTPases in Parkinson's disease: a primer

“…However, LRRK2 does not prominently phosphorylate the different Rab substrates. For example, the stoichiometry of LRRK2-mediated Rab10 phosphorylation in various cell lines and in healthy patient-derived neutrophils is around 1% [66,[78][79][80] and increases to approximately 2-5% in the presence of pathogenic LRRK2, respectively [78,79,81]. Whilst it remains possible that such stoichiometry may be higher in disease-relevant cell types, this raises the issue of how such a small change in phospho-Rab protein levels may cause PD in a dominant fashion.…”

The Regulation of Rab GTPases by Phosphorylation

“…However, they found a significant increase in Rab10 phosphorylation in G2019S LRRK2 as well as D620N VPS35 mutation carriers. A recent follow-up study from Fan and colleagues also showed no difference between the controls and the idiopathic PD patients, although a significant increase could be detected in patients harboring the R1441G LRRK2 mutation [ 29 ]. In a recent report by Wang and colleagues, they established a quantitative and high-throughput assay to measure the levels of Rab10 phosphorylation in human PBMCs [ 30 ].…”

“…They detected a substantial increase in the intensity of the phospho-Rab10 staining in the brains of idiopathic PD patients. On the other hand, Fan and colleagues examined the Rab10 phosphorylation in postmortem brains using immunoblotting and mass spectrometry but failed to show any difference between the controls and the PD patients [ 29 ].…”