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Mitochondrial DNA damage as a potential biomarker of LRRK2 kinase activity in LRRK2 Parkinson's disease.

ABSTRACT: Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson's disease (PD) and LRRK2 kinase inhibitors are currently being tested in early phase clinical trials. In order to ensure the highest chance of success, a biomarker-guided entry into clinical trials is key. LRRK2 phosphorylation, and phosphorylation of the LRRK2 substrate Rab10, have been proposed as target engagement biomarkers for LRRK2 kinase inhibition. However, a pharmacodynamic biomarker to demonstrate that a biological response has occurred is lacking. We previously discovered that the LRRK2 G2019S mutation causes mitochondrial DNA (mtDNA) damage and is LRRK2 kinase activity-dependent. Here, we have explored the possibility that measurement of mtDNA damage is a "surrogate" for LRRK2 kinase activity and consequently of kinase inhibitor activity. Mitochondrial DNA damage was robustly increased in PD patient-derived immune cells with LRRK2 G2019S mutations as compared with controls. Following treatment with multiple classes of LRRK2 kinase inhibitors, a full reversal of mtDNA damage to healthy control levels was observed and correlated with measures of LRRK2 dephosphorylation. Taken together, assessment of mtDNA damage levels may be a sensitive measure of altered kinase activity and provide an extended profile of LRRK2 kinase modulation in clinical studies.

SUBMITTER: Gonzalez-Hunt CP

PROVIDER: S-EPMC7557909 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Mitochondrial DNA damage as a potential biomarker of LRRK2 kinase activity in LRRK2 Parkinson's disease.

Gonzalez-Hunt C P CP Thacker E A EA Toste C M CM Boularand S S Deprets S S Dubois L L Sanders L H LH

Scientific reports 20201014 1

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson's disease (PD) and LRRK2 kinase inhibitors are currently being tested in early phase clinical trials. In order to ensure the highest chance of success, a biomarker-guided entry into clinical trials is key. LRRK2 phosphorylation, and phosphorylation of the LRRK2 substrate Rab10, have been proposed as target engagement biomarkers for LRRK2 kinase inhibition. However, a pharmacodynamic biomarker to ...[more]

PMID: 33057100

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Project description:Pathogenic mutations in LRRK2 cause Parkinson's disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a non-selective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, which allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. We further investigated the relationship between LRRK2 kinase activity, mtDNA damage and mitophagy. Levels of mtDNA damage caused by G2019S LRRK2 were fully re-established within 2 h of a LRRK2 inhibitor wash out and recovery experiment, indicating the mtDNA damage phenotype is highly dynamic. G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Abrogation of mtDNA damage with the mutant selective tool inhibitor EB-42168 demonstrates the potential of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

Project description:In the field of neurodegenerative diseases, especially sporadic Parkinson's disease (sPD) with dementia (sPDD), the question of how the disease starts and spreads in the brain remains central. While prion-like proteins have been designated as a culprit, recent studies suggest the involvement of additional factors. We found that oxidative stress, damaged DNA binding, cytosolic DNA sensing, and Toll-Like Receptor (TLR)4/9 activation pathways are strongly associated with the sPDD transcriptome, which has dysregulated type I Interferon (IFN) signaling. In sPD patients, we confirmed deletions of mitochondrial (mt)DNA in the medial frontal gyrus, suggesting a potential role of damaged mtDNA in the disease pathophysiology. To explore its contribution to pathology, we used spontaneous models of sPDD caused by deletion of type I IFN signaling (Ifnb-/-/Ifnar-/- mice). We found that the lack of neuronal IFNβ/IFNAR leads to oxidization, mutation, and deletion in mtDNA, which is subsequently released outside the neurons. Injecting damaged mtDNA into mouse brain induced PDD-like behavioral symptoms, including neuropsychiatric, motor, and cognitive impairments. Furthermore, it caused neurodegeneration in brain regions distant from the injection site, suggesting that damaged mtDNA triggers spread of PDD characteristics in an "infectious-like" manner. We also discovered that the mechanism through which damaged mtDNA causes pathology in healthy neurons is independent of Cyclic GMP-AMP synthase and IFNβ/IFNAR, but rather involves the dual activation of TLR9/4 pathways, resulting in increased oxidative stress and neuronal cell death, respectively. Our proteomic analysis of extracellular vesicles containing damaged mtDNA identified the TLR4 activator, Ribosomal Protein S3 as a key protein involved in recognizing and extruding damaged mtDNA. These findings might shed light on new molecular pathways through which damaged mtDNA initiates and spreads PD-like disease, potentially opening new avenues for therapeutic interventions or disease monitoring.

Dataset Information

Mitochondrial DNA damage as a potential biomarker of LRRK2 kinase activity in LRRK2 Parkinson's disease.

Publications

Mitochondrial DNA damage as a potential biomarker of LRRK2 kinase activity in LRRK2 Parkinson's disease.

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