Does CSA play a role in mitochondrial quality control?

Cockayne syndrome (CS) is a rare DNA repair defective hereditary disorder characterized by severe developmental and neurological alterations and early death. The molecular bases are still unknown. The proteins responsible of CS, CSA and CSB, are known to be involved in the repair of UV damage from the transcribed strand of active genes as well as in the repair of oxidatively induced DNA damage. Evidence has been provided that human CS cells present an altered redox balance with increased steady-state levels of intracellular ROS and mitochondrial dysfunction (Pascucci et al., 2012; Scheibye-Knudsen et al., 2012). Here, we show that the mitochondrial dysfunction of CS cells is not due to increased accumulation of oxidatively induced DNA damage in the mitochondrial genome. Both basal and induced 8-oxoguanine (8-OH-Gua) mitochondrial DNA levels in CS cells did not exceed those observed in normal fibroblasts whereas increased levels of 8-OH-Gua were confirmed in the nuclear genome of CS cells. A defect in autophagy has been proposed as the underlying mechanism for mitochondrial dysfunction in CS-B cells (Scheibye-Knudsen et al., 2012). To investigate whether autophagy/mitophagy were affected in the absence of CSA, we used a cell line stably expressing the ectopic wild-type CSA (wtCSA) protein obtained from the SV40-transformed CS-A cell line CS3BE. Following exposure to carbonyl cyanide m-chlorophenyl hydrazine (CCCP), that induces mitochondria uncoupling, CS-A cells showed an accelerated mitophagic flux, seen by LC3 accumulation in the presence of lysosomal inhibitors, translocation of Parkin and stabilization of PINK at mitochondria. Overexpression of Parkin protected wt cells from the killing effects of CCCP but much less efficiently CS-A cells. Mitochondria of CS cells presented an altered morphology and distribution. Mitochondria were mostly perinuclear with a less elongated shape then mitochondria of wt cells. Following CCCP exposure in CS-A cells fewer mitochondria were observed with altered morphology. On the basis of these findings we can conclude that in CS-A cells the key players of mitophagy correctly signal altered mitochondria to degradation and therefore alteration of mitophagy does not account for mitochondrial dysfunction. The mechanisms underlying the defective protection from CCCP killing by Parkin are currently under investigation.