Although the mechanisms controlling skeletal muscle homeostasis have been identified, there is a lack of knowledge of the integrated dynamic processes occurring during myogenesis and their regulation. Here, metabolism, autophagy and differentiation were concomitantly analyzed in mouse muscle satellite cell (MSC)-derived myoblasts and their cross-talk addressed by drug and genetic manipulation. We show that increased mitochondrial biogenesis and activation of mammalian target of rapamycin complex 1 inactivation-independent basal autophagy characterize the conversion of myoblasts into myotubes. Notably, inhibition of autophagic flux halts cell fusion in the latest stages of differentiation and, conversely, when the fusion step of myocytes is impaired the biogenesis of autophagosomes is also impaired. By using myoblasts derived from p53 null mice, we show that in the absence of p53 glycolysis prevails and mitochondrial biogenesis is strongly impaired. P53 null myoblasts show defective terminal differentiation and attenuated basal autophagy when switched into differentiating culture conditions. In conclusion, we demonstrate that basal autophagy contributes to a correct execution of myogenesis and that physiological p53 activity is required for muscle homeostasis by regulating metabolism and by affecting autophagy and differentiation.

The differentiation of skeletal myoblasts is characterized by permanent withdrawal from the cell cycle and fusion into multinucleated myotubes. Muscle cell survival is critically dependent on the ability of cells to respond to oxidative stress. Base excision repair (BER) is the main repair mechanism of oxidative DNA damage. In this study, we compared the levels of endogenous oxidative DNA damage and BER capacity of mouse proliferating myoblasts and their differentiated counterpart, the myotubes. Changes in the expression of oxidative stress marker genes during differentiation, together with an increase in 8-hydroxyguanine DNA levels in terminally differentiated cells, suggested that reactive oxygen species are produced during this process. The repair of 2-deoxyribonolactone, which is exclusively processed by long-patch BER, was impaired in cell extracts from myotubes. The repair of a natural abasic site (a preferred substrate for short-patch BER) also was delayed. The defect in BER of terminally differentiated muscle cells was ascribed to the nearly complete lack of DNA ligase I and to the strong down-regulation of XRCC1 with subsequent destabilization of DNA ligase IIIα. The attenuation of BER in myotubes was associated with significant accumulation of DNA damage as detected by increased DNA single-strand breaks and phosphorylated H2AX nuclear foci upon exposure to hydrogen peroxide. We propose that in skeletal muscle exacerbated by free radical injury, the accumulation of DNA repair intermediates, due to attenuated BER, might contribute to myofiber degeneration as seen in sarcopenia and many muscle disorders.

Summary: Cockayne syndrome (CS) is a rare hereditary multisystem disease characterized by neurological and development impairment, and premature aging. Cockayne syndrome cells are hypersensitive to oxidative stress, but the molecular mechanisms involved remain unresolved. Here we provide the first evidence that primary fibroblasts derived from patients with CS‐A and CS‐B present an altered redox balance with increased steady‐state levels of intracellular reactive oxygen species (ROS) and basal and induced DNA oxidative damage, loss of the mitochondrial membrane potential, and a significant decrease in the rate of basal oxidative phosphorylation. The Na/K‐ATPase, a relevant target of oxidative stress, is also affected with reduced transcription in CS fibroblasts and normal protein levels restored upon complementation with wild‐type genes. High‐resolution magnetic resonance spectroscopy revealed a significantly perturbed metabolic profile in CS‐A and CS‐B primary fibroblasts compared with normal cells in agreement with increased oxidative stress and alterations in cell bioenergetics. The affected processes include oxidative metabolism, glycolysis, choline phospholipid metabolism, and osmoregulation. The alterations in intracellular ROS content, oxidative DNA damage, and metabolic profile were partially rescued by the addition of an antioxidant in the culture medium suggesting that the continuous oxidative stress that characterizes CS cells plays a causative role in the underlying pathophysiology. The changes of oxidative and energy metabolism offer a clue for the clinical features of patients with CS and provide novel tools valuable for both diagnosis and therapy.

Risk factors for gastric cancer (GC) include inter-individual variability in the inflammatory response to Helicobacter pylori infection, in the ability of detoxifying DNA reactive species and repairing DNA damage generated by oxidative stress and dietary carcinogens. To evaluate the association between polymorphic DNA repair genes and GC risk, a case–control study including 314 histologically confirmed GC patients and 548 healthy controls was conducted in a GC high-risk area in Tuscany, Italy. Polymorphic variants of base excision repair (APE1-D148E, XRCC1-R194W, XRCC1-R399Q and OGG1-S326C), nucleotide excision repair (XPC-PAT, XPA-23G>A, ERCC1-19007T>C and XPD-L751Q), recombination (XRCC3-T241M) and alkylation damage reversal (MGMT-L84F) were tested for their potential role in the development of GC by using logistic regression models. The same population was also characterised for GSTT1 and GSTM1 variant alleles to search for possible functional interactions between metabolic and DNA repair genotypes by two-way interactions using multivariate logistic models. No significant association between any single DNA repair genotype and GC risk was detected with a borderline association with the XPC-PAT homozygous genotype [odds ratio (OR) = 1.42; 95% confidence interval (CI) 0.94–2.17]. Gene–gene interaction analysis revealed combinations of unfavourable genotypes involving either multiple DNA repair polymorphisms or DNA repair and GST-specific genotypes. The combination of the XPC-PAT and the XPA variant alleles significantly increased GC risk (OR = 2.15; 95% CI 1.17–3.93, P = 0.0092). A significant interaction was also found between the APE1 wild-type genotype and either the single GSTT1 (OR = 4.90; 95% CI 2.38–10.11, P = 0.0079) or double GSTM1-GSTT1 null (OR = 7.84; 95% CI 3.19–19.22, P = 0.0169) genotypes or the XPA-mutant allele (OR = 3.56; 95% CI 1.53–8.25, P = 0.0012). These findings indicate that a complex interaction between host factors such as oxidative stress, antioxidant capacity and efficiency of multiple DNA repair pathways underlies the inter-individual variability in GC risk.

There is a growing body of evidence indicating that the mechanisms that control genome stability are of key importance in the development and function of the nervous system. The major threat for neurons is oxidative DNA damage, which is repaired by the base excision repair (BER) pathway. Functional mutations of enzymes that are involved in the processing of single-strand breaks (SSB) that are generated during BER have been causally associated with syndromes that present important neurological alterations and cognitive decline. In this review, the plasticity of BER during neurogenesis and the importance of an efficient BER for correct brain function will be specifically addressed paying particular attention to the brain region and neuron-selectivity in SSB repair-associated neurological syndromes and age-related neurodegenerative diseases.