LRRK2 Knockout Confers Resistance in HEK-293 Cells to Rotenone-Induced Oxidative Stress, Mitochondrial Damage, and Apoptosis

Abstract

Leucine-rich repeat kinase 2 (LRRK2) has been linked to dopaminergic neuronal vulnerability to oxidative stress (OS), mitochondrial impairment, and increased cell death in idiopathic and familial Parkinson’s disease (PD). However, how exactly this kinase participates in the OS-mitochondria-apoptosis connection is still unknown. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 LRRK2 knockout (KO) in the human embryonic kidney cell line 293 (HEK-293) to evaluate the cellular response to the mitochondrial inhibitor complex I rotenone (ROT), a well-known OS and cell death inducer. We report successful knockout of the LRRK2 gene in HEK-293 cells using CRISPR editing (ICE, approximately 60%) and flow cytometry (81%) analyses. We found that HEK-293 LRRK2 WT cells exposed to rotenone (ROT, 50 μM) resulted in a significant increase in intracellular reactive oxygen species (ROS, +7400%); oxidized DJ-1-Cys106-SO3 (+52%); phosphorylation of LRRK2 (+70%) and c-JUN (+171%); enhanced expression of tumor protein (TP53, +2000%), p53 upregulated modulator of apoptosis (PUMA, +1950%), and Parkin (PRKN, +22%); activation of caspase 3 (CASP3, +8000%), DNA fragmentation (+35%) and decreased mitochondrial membrane potential (ΔΨm, −58%) and PTEN induced putative kinase 1 (PINK1, −49%) when compared to untreated cells. The translocation of the cytoplasmic fission protein dynamin-related Protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of the outer membrane 20 (TOM20). Outstandingly, HEK-293 LRRK2 KO cells treated with ROT showed unaltered OS and apoptosis markers. We conclude that loss of LRRK2 causes HEK-293 to be resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. Our data support the hypothesis that LRRK2 acts as a proapoptotic kinase by regulating mitochondrial proteins (e.g., PRKN, PINK1, DRP1, and PUMA), transcription factors (e.g., c-JUN and TP53), and CASP3 in cells under stress conditions. Taken together, these observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.

LRRK2 kinase inhibition protects against Parkinson’s disease-associated environmental toxicants

“…We and others have previously shown that environmental toxicants, such as rotenone and TCE, elevate WT LRRK2 kinase activity in the brain to a similar degree as the most well characterized pathogenic LRRK2 mutation, G2019S 22,25,51,52 . Furthermore, growing data suggests that LRRK2 moderates PD-related pathology caused by exposure to toxicants, such as microglial inflammation induced by the heavy metal manganese 35,53,54 .…”

“…While the data here represent several key mechanisms that explain how LRRK2 may partially contribute to toxicant-induced neurodegeneration in PD, there are also limitations that need to be considered. First, the expression levels of endogenous WT LRRK2 necessitated using a HEK cell line that expresses LRRK2 at measurable levels 26,52 . While different from neurons, HEK cells are extensively characterized in the LRRK2 literature 25,26,44,80,81 , have been used as a model system to study the effects of LRRK2 mutations and display similar energetic demands of neurons that rely heavily on mitochondrial function [82][83][84] .…”

Neuroprotective Effects of the Nutraceutical Dehydrozingerone and Its C2-Symmetric Dimer in a Drosophila Model of Parkinson’s Disease

“…This critical scenario is further worsened in the PD genetic models, where LRRK2 mutation caused increased generation of ROS and cell toxicity. A proof of concept has been offered by a recent in vitro work showing that LRRK2 knockout provides resistance to oxidative stress and apoptosis, suggesting LRRK2 as a proapoptotic kinase [ 62 ]. Moreover, previous studies demonstrated that deletion of the WD40 domain prevents autophosphorylation [ 63 , 64 ], and on the other hand, the G2385R polymorphism in the WD40 domain, expressed in our drosophila PD model, increases the sensitivity of cells to hydrogen peroxide, suggesting a pro-apoptotic mechanism [ 65 ].…”

The severity assessment of Parkinson's disease based on plasma inflammatory factors and third ventricle width by transcranial sonography

“…Oxidative stress is another mechanism via which Cys C exerts a protective effect in PD patients 39 . During oxidative stress, the body can cause continuous protease expression in intracranial tissues, causing the degeneration and death of dopaminergic neurons while promoting PD development.…”