Dataset Information

Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons.

ABSTRACT:

Background

Inherited mutations in the LRRK2 protein are common causes of Parkinson's disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10.

Objective

To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies.

Methods

LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern ('sleep'), and courtship memory determined in aged flies.

Results

Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons.

Conclusion

We conclude that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson's disease.

SUBMITTER: Fellgett A

PROVIDER: S-EPMC8609683 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.