Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson’s Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Ying Fan, Francesca Tonelli, Shalini Padmanabhan, Marco Baptista, Lindsey Riley, Danielle Smith, Connie Marras, Andy Howden, Dario Alessi, Esther Sammler (Lead / Corresponding author)

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Abstract

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson’ disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections. In particular, as the LRRK2-controlled Rab10 phosphorylation assay has already been used to identify individuals with hyperactivation of the LRRK2 kinase pathway, they might eventually benefit from future treatment with LRRK2 kinase inhibitors.
Original languageEnglish
Article numbere58956
JournalJoVE: Journal of Visualized Experiments
Issue number157
DOIs
Publication statusPublished - 22 Mar 2020

Keywords

  • Biochemistry
  • Biomarkers
  • Issue 157
  • LRRK2 kinase
  • Parkinson's disease
  • Peripheral blood neutrophils
  • Protein phosphorylation
  • Rab proteins
  • Vesicle trafficking

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