Fibroblast Biomarkers of Sporadic Parkinson’s Disease and LRRK2 Kinase Inhibition

Abstract

It has been uncertain whether specific disease-relevant biomarker phenotypes can be found using sporadic Parkinson’s disease (PD) patient-derived samples, as it has been proposed that there may be a plethora of underlying causes and pathological mechanisms. Fibroblasts derived from familial PD patients harboring leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1), and Parkin mutations show clear disease-relevant mitochondrial phenotypes, which are exacerbated under conditions of pharmacological stress. We utilized fibroblasts derived from non-familial sporadic PD patients (without LRRK2 mutations) or LRRK2 mutation carriers to directly compare the cellular phenotypes during and after mitochondrial stress. We then determined the effects of pharmacological LRRK2 kinase inhibition using LRRK2-in-1. We found that there were two distinct populations of sporadic PD patient-derived fibroblast lines. One group of sporadic PD lines was highly susceptible to valinomycin-induced mitochondrial depolarization, emulating the mutant LRRK2 phenotype. These lines showed elevated mitochondrial superoxide/ nitric oxide levels, displayed increased mitochondrial and lysosome co-localization, and an increased rate of mitochondrial collapse, which corresponded with changes in mitochondrial fission and fusion proteins. The application of LRRK2-in-1 reversed decreased levels of mitochondrial and lysosome co-localization and partially restored mitochondrial network associated proteins and the mitochondrial membrane potential in the fibroblasts. This study identifies novel mitochondrial biomarkers in sporadic PD patient-derived fibroblast lines, which could be used as preclinical tools in which to test novel and known neuroprotective compounds.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-015-9435-4) contains supplementary material, which is available to authorized users.

LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson’s disease

“…The role of LRRK2 in macroautophagy has also been intensively investigated in previous studies (54), several of which concluded that its effect is proautophagic (10,17,19,(57)(58)(59), and others reporting an inhibitory effect (20,60,61) or no effect (62). Previous attempts to address the impact of LRRK2 on basal mitophagy (52,58), which is unaffected by PINK1 and Parkin (63, 64) showed opposing effects, whereas a single study assessing toxin-induced mitophagy in fibroblasts from patients with sporadic and LRRK2-linked PD, using colocalization of TOMM20 and the lysosomal marker LAMP1 as a supposed "mitophagy index" was inconclusive (65). Only one previous study investigated, in detail, the direct impact of LRRK2 on PINK1/Parkin-dependent mitophagy, and its findings are consistent with our observations (66).…”

Phosphoregulation on mitochondria: Integration of cell and organelle responses

“…The recruitment of Drp‐1 to mitochondrial and subsequent fission in PD patient‐derived fibroblasts and human SH‐SY5Y cells was found to be dependent upon LRRK2 kinase activity . Given the recent identification of Rab GTPases, specifically Rab7, as bonafide LRRK2 substrates, it is likely that mutant LRRK2 activity could drive mitochondrial fragmentation observed in PD patients by assembling the protein machinery and activating membrane fusion …”

“…Fibroblasts from PD patients do exhibit more mitochondrial fragmentation and enhanced mitophagy initially compared to control cells. Thus, the role of LRRK2 in mitophagy will require a closer examination …”

“…Thus, the role of LRRK2 in mitophagy will require a closer examination. 169 Lastly, enhanced LRRK2 kinase activity can affect ROS levels and production through manipulation of mitochondrial substrates.…”

LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

“…Wang et al, 2012). Accordingly, a number of different cell types, including fibroblasts and iPSC-derived neurons from PD patients harboring mutations in LRRK2 exhibit increased oxidative stress and reactive oxygen species and defects in mitochondrial network integrity (Sison et al, 2018;Smith et al, 2016).…”