In-depth mass-spectrometry reveals phospho-RAB12 as a blood biomarker of G2019S LRRK2-driven Parkinson

Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising disease-modifying therapy for LRRK2-associated Parkinson disease (L2PD) and idiopathic PD (iPD). Yet, pharmaco-dynamic readouts and progression biomarkers for disease modification clinical trials are insufficient. Employing phospho-/proteomic analyses we assessed the impact that LRRK2 activating mutations had in peripheral blood mononuclear cells (PBMCs) from a LRRK2 clinical cohort from Spain (n=174) encompassing G2019S L2PD patients (n=37), non-manifesting LRRK2 mutation carriers of G2019S, here, G2019S L2NMCs (n=27), R1441G L2PD patients (n=14), R1441G L2NMCs (n=11), iPD (n=40), and controls (n=45). We identified 207 differential proteins in G2019S L2PD compared to controls (39 up/ 168 down) and 67 in G2019S L2NMCs (10 up/ 57 down). G2019S down-regulated proteins affected the endolysosomal pathway, proteostasis and mitochondria, e.g., ATIC, RAB9A, or LAMP1. At the phospho-proteome level, we observed increases in endogenous phosphorylation levels of pSer106 RAB12 in G2019S carriers, which were validated by immunoblotting after 1 year of follow-up (n=48). Freshly collected PBMCs from 3 G2019S L2PD, 1 R1441G L2PD, 1 iPD, and 5 controls (n=10) showed strong diminishment of pSer106 RAB12 phosphorylation levels after in-vitro administration of the MLi-2 LRRK2 inhibitor. Using machine learning, we identified an 18-feature G2019S phospho-/protein signature capable of discriminating G2019S L2PD, L2NMCs, and controls with 96% accuracy that correlated with disease severity, i.e., UPDRS-III motor scoring. Our study identified pSer106 RAB12 as an endogenous biomarker in easily accessible PBMCs from G2019S carriers and suggests that phospho-/proteomic findings in human PBMCs such as pSer106 RAB12 can be deployed as a universal pharmaco-dynamic readout for L2PD, L2NMCs, and iPD. Future work may determine whether pSer106 RAB12 could help with patient enrichment and monitoring drug efficacy in LRRK2 clinical trials. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by the Michael J. Fox Foundation for Parkinson Research (MJFF) (MJFF 000858) to RFS, ME, CM, ES, JI, and JRM. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Probands participated in the study after local ethics approval and signed informed consent. The overall study was approved by the Ethical committee of the Hospital Clinic of Barcelona (code HCB/2020/1238). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes We provide full open access to all data generated here through the information included in this article and the open Curtain software.