ATM mediated DNA double‐strand breaks accumulate in LRRK2 G2019S Parkinson's disease

Abstract

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting over one million people in the US. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited and idiopathic PD. We were the first to show that mitochondrial DNA (mtDNA) damage is caused by the most common mutation in LRRK2 (G2019S) and inhibition of LRRK2 kinase activity restores mtDNA integrity in PD models. However, whether aberrant LRRK2 kinase activity due to PD-linked mutations has broad impact on nuclear genome integrity is unknown. Using LRRK2G2019S/G2019S knock-in (KI) human embryonic kidney 293 (HEK293) cells obtained by CRISPR/Cas9 gene editing, our preliminary results indicate nuclear DNA damage is increased, including DNA double-strand breaks (DSBs) as assayed by a neutral comet assay. Consistent with DSB accumulation, we observed significantly increased γ-H2AX and 53BP1 foci. ATM is activated by DSBs and phosphorylates several key proteins that initiate the DNA damage response, cell cycle arrest, DNA repair or apoptosis. We found that basal levels of ATM pS1981 are increased in this in vitro LRRK2 G2019S model, which is an autophosphorylation site that correlates with DNA damage-mediated activation. Additionally, downstream substrates CHK2 (pT68), and P53 (pS15) are similarly increased in LRRK2G2019S/G2019S KI cells compared to isogenic wild-type control cells, substantiating that the ATM-mediated DNA damage response pathway has been up-regulated with the LRRK2 G2019S mutation. Blocking either LRRK2 or ATM kinase activity pharmacologically, significantly reversed LRRK2 G2019S-induced γ-H2AX foci. Overall these results suggest DSBs accumulate in LRRK2 PD, which in turn activate a sustained ATM mediated DNA damage response, which may lead to cell cycle arrest, aberrant DNA repair, and/or cell death. Further understanding of the functional relationship between LRRK2 and ATM offers new molecular insights into PD pathogenesis.