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

Ying Fan, Raja S. Nirujogi, Alicia Garrido, Javier Ruiz-Martínez, Alberto Bergareche-Yarza, Elisabet Mondragón Rezola, Ana Vinagre Aragón, Ioana Croitoru, Ana Gorostidi Pagola, Laura Paternain Markinez, Roy Alcalay, Richard A. Hickman, Jonas Düring, Sara Gomes, Neringa Pratuseviciute, Shalini Padmanabhan, Francesc Valldeoriola, Leticia Pérez Sisqués, Cristina Malagelada, Teresa XimelisLaura Molina Porcel, Maria José Martí, Eduardo Tolosa, Dario R. Alessi, Esther M. Sammler

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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 (pRab10 Thr73) was measured by quantitative multiplexed immunoblotting for pRab10 Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10 Thr73 occupancy. We found a significant over fourfold increase in pRab10 Thr73 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 pRab10 Thr73 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 pRab10 Thr73 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.

Original languageEnglish
Number of pages20
JournalActa Neuropathologica
Early online date14 Jun 2021
DOIs
Publication statusE-pub ahead of print - 14 Jun 2021

Keywords

  • Biomarkers
  • LRRK2
  • LRRK2 kinase inhibitors
  • Parkinson’s disease
  • Protein phosphorylation
  • RabGTPases

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