Cockayne syndrome proteins CSA and CSB maintain mitochondrial homeostasis through NAD+ signaling.

Cockayne syndrome (CS) is a rare premature aging disease, most commonly caused by mutations of the genes encoding the CSA or CSB proteins. CS patients display cachectic dwarfism and severe neurological manifestations and have an average life expectancy of 12 years. The CS proteins are involved in transcription and DNA repair, with the latter including transcription-coupled nucleotide excision repair (TC-NER). However, there is also evidence for mitochondrial dysfunction in CS, which likely contributes to the severe premature aging phenotype of this disease. While damaged mitochondria and impaired mitophagy were characterized in mice with CSB deficiency, such changes in the CS nematode model and CS patients are not fully known. Our cross-species transcriptomic analysis in CS postmortem brain tissue, CS mouse, and nematode models shows that mitochondrial dysfunction is indeed a common feature in CS. Restoration of mitochondrial dysfunction through NAD+ supplementation significantly improved lifespan and healthspan in the CS nematodes, highlighting mitochondrial dysfunction as a major driver of the aging features of CS. In cerebellar samples from CS patients, we found molecular signatures of dysfunctional mitochondrial dynamics and impaired mitophagy/autophagy. In primary cells depleted for CSA or CSB, this dysfunction can be corrected with supplementation of NAD+ precursors. Our study provides support for the interconnection between major causative aging theories, DNA damage accumulation, mitochondrial dysfunction, and compromised mitophagy/autophagy. Together, these three agents contribute to an accelerated aging program that can be averted by cellular NAD+ restoration.

Quantification of DNA Damage and Repair in Mitochondrial, Nuclear, and Bacterial Genomes by Real-Time PCR.

DNA damage caused by genotoxic insults is often used as an indicator of specific diseases, environmental challenges, and metabolic processes. To date, various different methods have been described to detect damaged DNA. Many techniques need high amounts of DNA for the analysis and/or require the exact determination of DNA template concentration. Here, we describe a rapid and quantitative method for the evaluation of the relative levels of damage in mitochondrial, nuclear, and bacterial DNA in comparison to untreated controls. The approach is based on the real-time PCR amplification of DNA fragments of two different lengths in the respective samples. DNA damage detection using this protocol is gene-specific. The technique can also be expanded to monitor DNA repair and to detect genomic hot-spots for DNA lesions.