Study of the effects of macroautophagy deficiency on mitochondrial function, energy metabolism and cell fate

Ph. D. ThesisFunctional cellular metabolism underpins healthy ageing by sustaining cellular signalling and organelle function. In turn, cellular metabolism critically depends on macronutrient availability and organelle health, which are maintained by cellular catabolic pathways, and by autophagy in particular. Importantly, decline in autophagy flux and mitochondrial dysfunction are observed in a variety of age-related chronic disorders and neurodegenerative diseases. I hypothesized that autophagy impairment stands at the root of age-related pathology and set out to test my hypothesis in a genetic model of autophagy deficiency. I have established a model where autophagy 5 (Atg5) knockout cells are forced to produce ATP via oxidative phosphorylation (OXPHOS) in culture and uncovered a rapid loss of viability accompanied by a reduction in nucleotides. In particular, loss of nicotinamide adenine dinucleotide (NAD(H)) levels were most predictive of cellular viability. NADH functions as a cofactor in cellular metabolism, maintenance of mitochondrial membrane potential and ATP production. I identified several compounds that rescue loss of cellular viability due to autophagy dysfunction, of which NAD(H) level normalization by precursor supplementation proved most effective. I tested the translational potential of my findings in cellular models of Niemann Pick Type C1 (NPC1) disease that is characterized by altered lipid metabolism and autophagy impairment, and its clinical presentations include spleen and liver enlargement, and progressive neurodegeneration. In immortalized NPC1 knockout cell lines, NAD(H) precursor treatment rescued cell death in conditions of enforced OXPHOS respiration. Furthermore, I identified NAD(H) depletion and increased susceptibility to exogenous oxidative stress in primary human fibroblasts isolated from NPC1 patients. Corroborating results from other models tested in my study, NAD(H) precursor supplementation successfully boosted NAD(H) levels and improved resistance to oxidative stress in patient cells. In summary, I have demonstrated that autophagy promotes a healthy nucleotide and NAD(H) metabolism and that NAD(H) precursor supplementation could protect cells and tissues affected by autophagy dysfunction.Medical Research Council, EU COST action PROTEOSTASI