Objective: The greatest unmet need in Parkinson’s disease (PD) are disease-modifying treatments. The discovery of monogenic forms of PD has provided invaluable insights into pathomechanisms and in turn opportunities for mechanistic patient stratification.

Background: All pathogenic LRRK2 mutations and the PD-causing VPS35-D620N variant exert their effect by augmenting LRRK2 kinase activity, resulting in hyperphosphorylation of its endogenous substrates, including Rab10^1-3. Heterozygous variants in GBA, encoding the lysosomal enzyme glucocerebrosidase, are among the commonest risk factors for PD likely causing lysosomal dysfunction that is also implicated in LRRK2- and idiopathic PD (iPD)⁴. Bis(monoacylglycerol)phosphate isoforms (BMPs) involved in lipid and membrane degradation serve as potential biomarkers of lysosomal dysfunction and urine BMPs are elevated in lysosomal storage disorders and LRRK2-G2019S mutation carriers⁵.

Method: Here, we isolated neutrophils, monocytes and PBMCs from fresh peripheral blood and urine from 110 individuals with manifesting and non-manifesting PD mutations (LRRK2-G2019S / R1441G, VPS35-D620N, several different GBA variants), iPD and controls. We analysed LRRK2-dependent Rab10^Thr73 phosphorylation across all cellular matrices by quantitative immunoblotting and urine BMPs by liquid chromatography–tandem mass spectrometry (MS).

Results: Our results show that LRRK2-R1441G and VPS35-D620N significantly augment LRRK2-dependent Rab10^Thr73 phosphorylation in mutation carriers with and without PD, while no significant difference was seen in LRRK2-G2019S and GBA mutation carriers as well as iPD compared to controls (Figure 1). We confirm that urine BMP levels are elevated in LRRK2-G2019S, and also in LRRK2-R1441G and VPS35-D620N mutation carriers (Figure 2).

Conclusion: This is the first combined biomarker analysis for LRRK2 kinase pathway activity and lysosomal dysfunction in participants with monogenic and iPD, asymptomatic mutation carriers, as well as controls. We provide further evidence that PD-causing LRRK2 and VPS35 mutations map into the same mechanistic pathway. In future experiments, we will deploy a novel highly sensitive multiplexed targeted parallel reaction monitoring MS-assay for simultaneous quantification of a subset of LRRK2-phosphorylated Rab GTPases, as well as total and phospho-LRRK2⁶, for a more comprehensive LRRK2 dependent signature.

References: 1. Alessi DR, Sammler E. LRRK2 kinase in Parkinson’s disease. Science 2018;360(6384):36-37. 2. Mir R, Tonelli F, Lis P, et al. The Parkinson’s disease VPS35[D620N] mutation enhances LRRK2 mediated Rab protein phosphorylation in mouse and human. Biochem J 2018;475(11):1861-1883. 3. Fan Y, Nirujogi RS, Garrido A, et al. R1441G but not G201S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils. medRxiv 2021:2021.2001.2028.21249614. 4. Do J, McKinney C, Sharma P, Sidransky E. Glucocerebrosidase and its relevance to Parkinson disease. Mol Neurodegener 2019;14(1):36. 5. Alcalay RN, Hsieh F, Tengstrand E, et al. Higher Urine bis(Monoacylglycerol)Phosphate Levels in LRRK2 G2019S Mutation Carriers: Implications for Therapeutic Development. Mov Disord 2019. 6. Nirujogi RS, Tonelli F, Taylor M, et al. Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites. Biochem J 2021;478(2):299-326.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/correlating-lrrk2-activation-status-in-peripheral-blood-to-urine-bmps-in-monogenic-and-idiopathic-parkinsons-disease/