Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.

No abstract

Neurodegeneration has been correlated with mitochondrial DNA (mtDNA) damage and exposure to environmental toxins, but causation is unclear. We investigated the ability of several known environmental genotoxins and neurotoxins to cause mtDNA damage, mtDNA depletion, and neurodegeneration in Caenorhabditis elegans. We found that paraquat, cadmium chloride and aflatoxin B1 caused more mitochondrial than nuclear DNA damage, and paraquat and aflatoxin B1 also caused dopaminergic neurodegeneration. 6-hydroxydopamine (6-OHDA) caused similar levels of mitochondrial and nuclear DNA damage. To further test whether the neurodegeneration could be attributed to the observed mtDNA damage, C. elegans were exposed to repeated low-dose ultraviolet C radiation (UVC) that resulted in persistent mtDNA damage; this exposure also resulted in dopaminergic neurodegeneration. Damage to GABAergic neurons and pharyngeal muscle cells was not detected. We also found that fasting at the first larval stage was protective in dopaminergic neurons against 6-OHDA-induced neurodegeneration. Finally, we found that dopaminergic neurons in C. elegans are capable of regeneration after laser surgery. Our findings are consistent with a causal role for mitochondrial DNA damage in neurodegeneration, but also support non mtDNA-mediated mechanisms.

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 JK1 107 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 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.

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.

Parkinson's disease (PD) is the most common movement neurodegenerative disorder. Although our understanding of the underlying mechanisms of pathogenesis in PD has greatly expanded, this knowledge thus far has failed to translate into disease‐modifying therapies. Therefore, it is of the utmost urgency to interrogate further the multifactorial etiology of PD. DNA repair defects cause many neurodegenerative diseases. An exciting new PD research avenue is the role that DNA damage and repair may play in neuronal death. The goal of this mini‐review was to discuss the evidence for the types of DNA damage that accumulates in PD, which has provided clues for which DNA repair pathways, such as DNA double‐strand break repair, are dysfunctional. We further highlight compelling data for activation of the DNA damage response in familial and idiopathic PD. The significance of DNA damage and repair is emerging in the PD field and linking these insights to PD pathogenesis may provide new insights into PD pathophysiology and consequently lead to new therapies.

No abstract

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and neuroprotective interventions remain elusive. High throughput biomarkers aimed to stratify patients based on shared etiology is one critical path to the success of disease-modifying therapies in clinical trials. Mitochondrial dysfunction plays a prominent role in the pathogenesis of PD. Previously, we found brain region-specific mitochondrial DNA (mtDNA) damage accumulation in neuronal and in vivo PD models, as well as human PD postmortem brain tissue. In this study, to investigate mtDNA damage as a potential blood biomarker for PD, we describe a novel Mito DNADX assay that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, compatible with assessing large cohorts of patient samples. We found that levels of mtDNA damage were increased in blood derived from early-stage idiopathic PD patients or those harboring the pathogenic LRRK2 G2019S mutation compared to age-matched healthy controls. Given that increased mtDNA damage was also found in non-manifesting LRRK2 mutation carriers, mtDNA damage may begin to accumulate prior to a clinical PD diagnosis. LRRK2 kinase inhibition mitigated mtDNA damage in idiopathic PD models and patient-derived cells. The latter observations further substantiate a mechanistic role for wild-type LRRK2 kinase activity in idiopathic PD and support mtDNA damage reversal as a suitable approach to slow PD-related pathology. In light of recent advances in the field of precision medicine, the analysis of mtDNA damage as a blood-based patient stratification biomarker should be included in future clinical trials.