Sirtuins Modulators Counteract Mitochondrial Dysfunction in Cellular Models of Hypoxia: Relevance to Schizophrenia.

Schizophrenia (SZ) is a neurodevelopmental-associated disorder strongly related to environmental factors, such as hypoxia. Because there is no cure for SZ or any pharmacological approach that could revert hypoxia-induced cellular damages, we evaluated whether modulators of sirtuins could abrogate hypoxia-induced mitochondrial deregulation as a neuroprotective strategy. Firstly, astrocytes from control (Wistar) and Spontaneously Hypertensive Rats (SHR), a model of both SZ and neonatal hypoxia, were submitted to chemical hypoxia. Then, cells were exposed to different concentrations of Nicotinamide (NAM), Resveratrol (Resv), and Sirtinol (Sir) for 48hrs. Our data indicate that sirtuins modulation reduces cell death increasing the acetylation of histone 3. This outcome is related to the rescue of loss of mitochondrial membrane potential, changes in mitochondrial calcium buffering capacity, decreased O2-rad levels and increased expression of metabolic regulators (Nrf-1 and Nfe2l2) and mitochondrial content. Such findings are relevant not only for hypoxia-associated conditions, named pre-eclampsia but also for SZ since prenatal hypoxia is a relevant environmental factor related to this burdensome neuropsychiatric disorder.

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

Decreased Mitochondrial Function, Biogenesis, and Degradation in Peripheral Blood Mononuclear Cells from Amyotrophic Lateral Sclerosis Patients as a Potential Tool for Biomarker Research.

Amyotrophic lateral sclerosis (ALS) is a multifactorial and progressive neurodegenerative disease of unknown etiology. Due to ALS's unpredictable onset and progression rate, the search for biomarkers that allow the detection and tracking of its development and therapeutic efficacy would be of significant medical value. Considering that alterations of energy supply are one of ALS's main hallmarks and that a correlation has been established between gene expression in human brain tissue and peripheral blood mononuclear cells (PBMCs), the present work investigates whether changes in mitochondrial function could be used to monitor ALS. To achieve this goal, PBMCs from ALS patients and control subjects were used; blood sampling is a quite non-invasive method and is cost-effective. Different parameters were evaluated, namely cytosolic calcium levels, mitochondrial membrane potential, oxidative stress, and metabolic compounds levels, as well as mitochondrial dynamics and degradation. Altogether, we observed lower mitochondrial calcium uptake/retention, mitochondria depolarization, and redox homeostasis deregulation, in addition to a decrease in critical metabolic genes, a diminishment in mitochondrial biogenesis, and an augmentation in mitochondrial fission and autophagy-related gene expression. All of these changes can contribute to the decreased ATP and pyruvate levels observed in ALS PBMCs. Our data indicate that PBMCs from ALS patients show a significant mitochondrial dysfunction, resembling several findings from ALS' neural cells/models, which could be exploited as a powerful tool in ALS research. Our findings can also guide future studies on new pharmacological interventions for ALS since assessments of brain samples are challenging and represent a relevant limited strategy. Graphical abstract.

Chemical Screening Approaches Enabling Drug Discovery of Autophagy Modulators for Biomedical Applications in Human Diseases.

Autophagy is an intracellular degradation pathway for malfunctioning aggregation-prone proteins, damaged organelles, unwanted macromolecules and invading pathogens. This process is essential for maintaining cellular and tissue homeostasis that contribute to organismal survival. Autophagy dysfunction has been implicated in the pathogenesis of diverse human diseases, and therefore, therapeutic exploitation of autophagy is of potential biomedical relevance. A number of chemical screening approaches have been established for the drug discovery of autophagy modulators based on the perturbations of autophagy reporters or the clearance of autophagy substrates. These readouts can be detected by fluorescence and high-content microscopy, flow cytometry, microplate reader and immunoblotting, and the assays have evolved to enable high-throughput screening and measurement of autophagic flux. Several pharmacological modulators of autophagy have been identified that act either via the classical mechanistic target of rapamycin (mTOR) pathway or independently of mTOR. Many of these autophagy modulators have been demonstrated to exert beneficial effects in transgenic models of neurodegenerative disorders, cancer, infectious diseases, liver diseases, myopathies as well as in lifespan extension. This review describes the commonly used chemical screening approaches in mammalian cells and the key autophagy modulators identified through these methods, and highlights the therapeutic benefits of these compounds in specific disease contexts.