Mitochondrial oxidative phosphorylation plasticity/adaptation triggered by disturbances and stresses and targeted by therapies

Mitochondrion is an important organelle for cells survival. In fact, it is responsible for many processes such as cellular metabolism, i.e. oxidative phosphorylation for ATP production, energy homeostasis and regulation of apoptosis and autophagy. Mitochondrion, due to this role, needs to be “plastic” in order to respond and adapt quickly to any perturbation and change of conditions in the different tissues of the human body. The induction of mitochondria biogenesis is required to meet different energetic demands under stress conditions. Thus, mitochondrial plasticity is the mechanism that controls modification in conditions of cellular stress or in response to environmental stimuli like exercise, caloric restriction, cold exposure, oxidative stress, cell division and renewal, and differentiation. Recently, mitochondrial modulation has become also a topic of interest as a therapeutic target. The master regulator gene of mitochondrial biogenesis is PGC1α that, through nuclear transcription factors and subsequent metabolic sensors and other signalling proteins, is capable to modulate mitochondrial abundance, activity and oxidative phosphorylation as a consequence of energy homeostasis unbalance. Mitochondrial plasticity during the last few years was extensively studied in skeletal muscle models, due to its fast adaptation in exercise and rest condition, but also in cancer cachexia, ageing and heart disease. Also in cancer, mitochondrial adaptations have become a fundamental topic, in particular to understand the underling pathogenic mechanism of disease progression, to identify prognostic factors and to design adjuvant therapies targeting mitochondria. In this frame, this PhD Thesis investigates the role and adaptations of mitochondria in different pathophysiological models of skeletal muscle and brain tumors. The expression of some key proteins of the signalling pathways involved in mitochondrial biogenesis regulation, such as PGC1α, LKB1-AMPK an energy sensing axis, Sirt3 a regulator of mitochondrial enzymes functionality, are investigated together with the OXPHOS complexes, HSP60, CS and TOM20 as mitochondrial mass markers. The first model is aimed at testing the expression levels of the protein panel in skeletal muscle biopsies from a cohort of 16 elderly and 7 young people subjected to immobility (bed-rest) causing hypotrophy and subsequent rehabilitation via exercise training. Based on quantitative immunoblot data, there is a down-regulation of PGC1α, Sirt3 and OXPHOS complexes II, III and IV occurring during bed-rest with a subsequent up-regulation after rehabilitation in both groups. AMPK and LKB1 do not change during bed-rest and rehabilitation in elderly and young subjects suggesting that there is not energetic impairment. According to the down-regulation of OXPHOS biogenesis during bed-rest there is the up-regulation of GAPDH evocative of a metabolic shift during hypotrophy from oxidative phosphorylation towards glycolysis, which is reversed by exercise training. OXPHOS complex V is down-regulated in both groups during bed-rest, but after rehabilitation the complex expression does not increase, maybe due to an imbalance between protein biogenesis and degradation. It is tempting to speculate that exercise could regulate complex V activity, as a compensatory response, through deacetylation mediated by Sirt3, which is up-regulated after rehabilitation. CS and TOM20 present the same pattern: in elderly subjects there is a down-regulation during immobility that is counteracted by exercise training, whereas young subjects present a similar pattern but differences do not reach statistical significance. In conclusion, immobility is effective in down-regulation of mitochondria-related protein expression and training protocol counteracts this effect. The pattern is similar in both elderly and young subjects, with some differences for PGC1α, and Sirt3 appearing less responsive to rehabilitation in elderly. Training is a fundamental tool to recover from immobility periods but also to maintain muscle tonicity as a non-pharmacologic therapeutic treatment for chronic heart failure patients (CHF). In the second model is studied the effect of aerobic exercise training (2 months) on mitochondrial respiration in skeletal muscle of CHF transgenic (Tgαq*44) mice, focusing also on the impact of CHF on skeletal muscle of sedentary mice. Oxidative phosphorylation and electron transport system capacity of biopsies from soleus muscle is assayed by high-resolution respirometry. Sedentary CHF mice exhibit in comparison to wild type an impaired complex I – State 3 respiration and ADP-stimulated respiration sustained by Complex I+II, in contrast to rotenone insensitive electron transport system respiration that is unchanged. This suggests an inactivation of complex I rather than an impairment of OXPHOS biogenesis in soleus muscle, also confirmed by unchanged value of PGC1α expression. Exercise training improves exercise performance, but it does not affect mitochondrial respiration. Factors “upstream” of mitochondria are likely mainly responsible for the functional improvement. The third model focuses on the study of ATP synthase Inhibitory Factor 1 as prognostic marker in low-grade astrocytomas (LGA). 19 pairs of surgical specimens of LGA are evaluated for the tumor border zone in which IF1 abundance is significantly lower than in the tumoral zone. Immunohistochemistry analyses of 68 specimens by Tissue-MicroArrays prove a weak association of IF1 with NF-kB p65-subunit and consolidated radiologic indexes of tumor infiltration and resection. Kaplan–Meier estimation of patients overall survival indicates that IF1 may serve as a prognostic marker. Intriguingly, IF1 expression significantly increases in lesions with suspected first signs of anaplastic transformation (LGA*) as showed, in accordance, by immunofluorescence (12 specimens), immunohistochemistry (11) and immunoblot (9) analyses. Finally, immunoblot analyses provide a picture of mitochondrial and glycolytic markers, suggesting no improvement of glycolysis and little changes in mitochondrial mass. On the contrary, OXPHOS complexes show a significant upregulation in LGA*. IF1 expression levels could be proposed as a biomarker of OS in LGA, rare tumors with a good prognosis, which could nonetheless evolve in anaplastic lesions and are still without an adjuvant therapy