RESUMO
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.
Assuntos
Dano ao DNA , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/fisiologia , Mitocôndrias/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Biomarcadores , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Linfócitos , Terapia de Alvo Molecular , Mutação , Doença de Parkinson/diagnóstico , Doença de Parkinson/enzimologia , Fosforilação/efeitos dos fármacosRESUMO
5-Fluoro-2'-deoxyuridine (FUdR) is a DNA synthesis inhibitor commonly used to sterilize Caenorhabditis elegans in order to maintain a synchronized aging population of nematodes, without contamination by their progeny, in lifespan experiments. All somatic cells in the adult nematode are post-mitotic and therefore do not require nuclear DNA synthesis. However, mitochondrial DNA (mtDNA) replicates independently of the cell cycle and thus represents a potential target for FUdR toxicity. Inhibition of mtDNA synthesis can lead to mtDNA depletion, which is linked to a number of diseases in humans. Furthermore, alterations in mitochondrial biology can affect lifespan in C. elegans. We characterized the effects of FUdR exposure on mtDNA and nuclear DNA (nucDNA) copy numbers, DNA damage, steady state ATP levels, nematode size, mitochondrial morphology, and lifespan in the germ line deficient JK1107 glp-1(q244) and PE255 glp-4(bn2) strains. Lifespan was increased very slightly by 25 µM FUdR, but was reduced by 400 µM. Both concentrations reduced mtDNA and nucDNA copy numbers, but did not change their ratio. There was no detectable effect of FUdR on mitochondrial morphology. Although both concentrations of FUdR resulted in smaller sized animals, changes to steady-state ATP levels were either not detected or restricted to the higher dose and/or later timepoints, depending on the method employed and strain tested. Finally, we determined the half-life of mtDNA in somatic cells of adult C. elegans to be between 8 and 13 days; this long half-life very likely explains the small or undetectable impact of FUdR on mitochondrial endpoints in our experiments. We discuss the relative pitfalls associated with using FUdR and germline deficient mutant strains as tools for the experimental elimination of progeny.
