Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

Ying Fan, Andrew J. M. Howden, Adil R. Sarhan, Pawel Lis, Genta Ito, Terina N. Martinez, Kathrin Brockmann, Thomas Gasser, Dario R. Alessi (Lead / Corresponding author), Esther Sammler (Lead / Corresponding author)

Research output: Contribution to journalArticle

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Abstract

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson’s disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson’s as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson’s bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.

Original languageEnglish
Pages (from-to)23-44
Number of pages22
JournalBiochemical Journal
Volume475
Issue number1
Early online date10 Nov 2017
DOIs
Publication statusPublished - 2 Jan 2018

Fingerprint

Phosphorylation
Leucine
Parkinson Disease
Neutrophils
Phosphotransferases
Assays
Blood
Pharmacodynamics

Keywords

  • Animals
  • Antibodies, Monoclonal/chemistry
  • Antibodies, Phospho-Specific/chemistry
  • Antibody Specificity
  • Case-Control Studies
  • Clinical Trials as Topic
  • Enzyme Assays
  • Epitopes/chemistry
  • Gene Expression Regulation
  • Genetic Predisposition to Disease
  • Gyrus Cinguli/immunology
  • Humans
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors
  • Leukocytes, Mononuclear/immunology
  • Mutation
  • Neutrophils/immunology
  • Parkinson Disease/enzymology
  • Phosphorylation
  • Protein Kinase Inhibitors/pharmacology
  • Rabbits
  • Signal Transduction
  • rab GTP-Binding Proteins/chemistry

Cite this

Fan, Ying ; Howden, Andrew J. M. ; Sarhan, Adil R. ; Lis, Pawel ; Ito, Genta ; Martinez, Terina N. ; Brockmann, Kathrin ; Gasser, Thomas ; Alessi, Dario R. ; Sammler, Esther. / Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils. In: Biochemical Journal. 2018 ; Vol. 475, No. 1. pp. 23-44.
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author = "Ying Fan and Howden, {Andrew J. M.} and Sarhan, {Adil R.} and Pawel Lis and Genta Ito and Martinez, {Terina N.} and Kathrin Brockmann and Thomas Gasser and Alessi, {Dario R.} and Esther Sammler",
note = "This work was supported by the Michael J. Fox Foundation for Parkinson's research [grant number 12938 (D.R.A.)]; the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.)]; the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer-to D.R.A.). The Academy of Medical Sciences supported ES with a Starter Grant for Clinical Lecturers (award name AMS-SGCL11- Sammler).",
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Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils. / Fan, Ying; Howden, Andrew J. M.; Sarhan, Adil R.; Lis, Pawel; Ito, Genta; Martinez, Terina N.; Brockmann, Kathrin ; Gasser, Thomas ; Alessi, Dario R. (Lead / Corresponding author); Sammler, Esther (Lead / Corresponding author).

In: Biochemical Journal, Vol. 475, No. 1, 02.01.2018, p. 23-44.

Research output: Contribution to journalArticle

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T1 - Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils

AU - Fan, Ying

AU - Howden, Andrew J. M.

AU - Sarhan, Adil R.

AU - Lis, Pawel

AU - Ito, Genta

AU - Martinez, Terina N.

AU - Brockmann, Kathrin

AU - Gasser, Thomas

AU - Alessi, Dario R.

AU - Sammler, Esther

N1 - This work was supported by the Michael J. Fox Foundation for Parkinson's research [grant number 12938 (D.R.A.)]; the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.)]; the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer-to D.R.A.). The Academy of Medical Sciences supported ES with a Starter Grant for Clinical Lecturers (award name AMS-SGCL11- Sammler).

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N2 - There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson’s disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson’s as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson’s bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.

AB - There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson’s disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson’s as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson’s bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.

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KW - Antibodies, Phospho-Specific/chemistry

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KW - Enzyme Assays

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KW - Gyrus Cinguli/immunology

KW - Humans

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KW - Leukocytes, Mononuclear/immunology

KW - Mutation

KW - Neutrophils/immunology

KW - Parkinson Disease/enzymology

KW - Phosphorylation

KW - Protein Kinase Inhibitors/pharmacology

KW - Rabbits

KW - Signal Transduction

KW - rab GTP-Binding Proteins/chemistry

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JF - Biochemical Journal

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