Mitochondrial DNA damage is a pharmacodynamic biomarker of selective LRRK2 kinase inhibition in human peripheral PD patient-derived immune cells Gonzalez-Hunt, C., Thacker, E., Toste, C., Sanders, L.H. Duke University Medical Center, Department of Neurology, Durham, NC 27710
Mutations in the leucine-rich repeat kinase 2 (LRRK2)gene are the most common cause of familial Parkinson's disease (PD). Most pathogenic mutations are located in the LRRK2 kinase or GTPase domain. The LRRK2 G2019S mutation in the kinase domain leads to a three-fold increase in catalytic activity and is thought to underlie its neurotoxic effect. We discovered that iPSC-derived neurons from individuals with PD carrying the LRRK2 G2019S mutation exhibited mitochondrial DNA (mtDNA) damage. This phenotype was abrogated by correcting the mutation via gene editing, indicating that the mtDNA damage was directly caused by the LRRK2 G2019S mutation. Next, we demonstrated that LRRK2 G2019S-induced mtDNA damage is LRRK2 kinase activity-dependent and targeted inhibition restores mtDNA integrity in PD models. In the present study, we determined whether mtDNA damage levels following LRRK2 inhibition could serve as a valid pharmacodynamic biomarker in peripheral immune cells from LRRK2 G2019S patients. LRRK2 G2019S patient-derived lymphoblastoid cell lines (LCL) and healthy controls were treated with three distinct specific LRRK2 inhibitors at a wide range of doses and time points. Immunobloting was first performed to assess and confirm the reduction in LRRK2 phosphorylation at Ser910, Ser935, Ser955 and Ser973. Consistent with previously published results, LRRK2 inhibitors reduced the constitutive phosphorylation of LRRK2 similarly in LRRK2 G2019S and control LCLs at short (1.5 hours) and longer (24 hours) exposures. Given that the magnitude of the decrease is the same in both controls and LRRK2 LCLs after kinase inhibitor treatment, constitutive LRRK2 phosphorylation sites likely represent a promising measure of target engagement, but not of LRRK2 kinase activity per se. In contrast, preliminary data suggest that more than 50% LRRK2 kinase inhibition was required to reduce mtDNA damage levels in LRRK2 G2019S patient-derived cells. Therefore, the mtDNA phenotype may provide a highly sought after pharmacodynamics biomarker in the setting of a clinical trial aimed at evaluating the potential of LRRK2 kinase inhibitors as disease-modifying treatment for PD.
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