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

mTORC1 activity is supported by spatial association with focal adhesions

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli

Intrauterine Fetal Demise After Uncomplicated COVID-19: What Can We Learn from the Case?

Background: SARS-CoV-2 infection in pregnant women can lead to placental damage and transplacental infection transfer, and intrauterine fetal demise is an unpredictable event. Case study: A 32-year-old patient in her 38th week of pregnancy reported loss of fetal movements. She overcame mild COVID-19 with positive PCR test 22 days before. A histology of the placenta showed deposition of intervillous fibrinoid, lympho-histiocytic infiltration, scant neutrophils, clumping of villi, and extant infarctions. Immunohistochemistry identified focal SARS-CoV-2 nucleocapsid and spike protein in the syncytiotrophoblast and isolated in situ hybridization of the virus’ RNA. Low ACE2 and TMPRSS2 contrasted with strong basigin/CD147 and PDL-1 positivity in the trophoblast. An autopsy of the fetus showed no morphological abnormalities except for lung interstitial infiltrate, with prevalent CD8-positive T-lymphocytes and B-lymphocytes. Immunohistochemistry and in situ hybridization proved the presence of countless dispersed SARS-CoV-2-infected epithelial and endothelial cells in the lung tissue. The potential virus-receptor protein ACE2, TMPRSS2, and CD147 expression was too low to be detected. Conclusion: Over three weeks’ persistence of trophoblast viral infection lead to extensive intervillous fibrinoid depositions and placental infarctions. High CD147 expression might serve as the dominant receptor for the virus, and PDL-1 could limit maternal immunity in placental tissue virus clearance. The presented case indicates that the SARS-CoV-2 infection-induced changes in the placenta lead to ischemia and consecutive demise of the fetus. The infection of the fetus was without significant impact on its death. This rare complication of pregnancy can appear independently to the severity of COVID-19’s clinical course in the pregnant mother

Serials: Passion or Addiction? Evaluation Based on Quantitative and Case Study.

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Evaluation and limitations of different approaches among COVID-19 fatal cases using whole-exome sequencing data

Abstract Background COVID-19 caused by the SARS-CoV-2 infection may result in various disease symptoms and severity, ranging from asymptomatic, through mildly symptomatic, up to very severe and even fatal cases. Although environmental, clinical, and social factors play important roles in both susceptibility to the SARS-CoV-2 infection and progress of COVID-19 disease, it is becoming evident that both pathogen and host genetic factors are important too. In this study, we report findings from whole-exome sequencing (WES) of 27 individuals who died due to COVID-19, especially focusing on frequencies of DNA variants in genes previously associated with the SARS-CoV-2 infection and the severity of COVID-19. Results We selected the risk DNA variants/alleles or target genes using four different approaches: 1) aggregated GWAS results from the GWAS Catalog; 2) selected publications from PubMed; 3) the aggregated results of the Host Genetics Initiative database; and 4) a commercial DNA variant annotation/interpretation tool providing its own knowledgebase. We divided these variants/genes into those reported to influence the susceptibility to the SARS-CoV-2 infection and those influencing the severity of COVID-19. Based on the above, we compared the frequencies of alleles found in the fatal COVID-19 cases to the frequencies identified in two population control datasets (non-Finnish European population from the gnomAD database and genomic frequencies specific for the Slovak population from our own database). When compared to both control population datasets, our analyses indicated a trend of higher frequencies of severe COVID-19 associated risk alleles among fatal COVID-19 cases. This trend reached statistical significance specifically when using the HGI-derived variant list. We also analysed other approaches to WES data evaluation, demonstrating its utility as well as limitations. Conclusions Although our results proved the likely involvement of host genetic factors pointed out by previous studies looking into severity of COVID-19 disease, careful considerations of the molecular-testing strategies and the evaluated genomic positions may have a strong impact on the utility of genomic testing

Autophagy promotes cell and organismal survival by maintaining NAD(H) pools

Autophagy is an essential catabolic process that promotes clearance of surplus or damaged intracellular components 1 . As a recycling process, autophagy is also important for the maintenance of cellular metabolites during periods of starvation 2 . Loss of autophagy is sufficient to cause cell death in animal models and is likely to contribute to tissue degeneration in a number of human diseases including neurodegenerative and lysosomal storage disorders 3–7 . However, it remains unclear which of the many cellular functions of autophagy primarily underlies its role in cell survival. Here we have identified a critical role of autophagy in the maintenance of nicotinamide adenine dinucleotide (NAD + /NADH) levels. In respiring cells, loss of autophagy caused NAD(H) depletion resulting in mitochondrial membrane depolarisation and cell death. We also found that maintenance of NAD(H) is an evolutionary conserved function of autophagy from yeast to human cells. Importantly, cell death and reduced viability of autophagy-deficient animal models can be partially reversed by supplementation with an NAD(H) precursor. Our study provides a mechanistic link between autophagy and NAD(H) metabolism and suggests that boosting NAD(H) levels may be an effective intervention strategy to prevent cell death and tissue degeneration in human diseases associated with autophagy dysfunction

Autophagy promotes cell survival by maintaining NAD levels

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency

Summary: 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