Selective Autophagy and Xenophagy in Infection and Disease

Autophagy, a cellular homeostatic process, which ensures cellular survival under various stress conditions, has catapulted to the forefront of innate defense mechanisms during intracellular infections. The ability of autophagy to tag and target intracellular pathogens toward lysosomal degradation is central to this key defense function. However, studies involving the role and regulation of autophagy during intracellular infections largely tend to ignore the housekeeping function of autophagy. A growing number of evidences now suggest that the housekeeping function of autophagy, rather than the direct pathogen degradation function, may play a decisive role to determine the outcome of infection and immunological balance. We discuss herein the studies that establish the homeostatic and anti-inflammatory function of autophagy, as well as role of bacterial effectors in modulating and coopting these functions. Given that the core autophagy machinery remains largely the same across diverse cargos, how selectivity plays out during intracellular infection remains intriguing. We explore here, the contrasting role of autophagy adaptors being both selective as well as pleotropic in functions and discuss whether E3 ligases could bring in the specificity to cargo selectivity

Dysregulation of autophagy as a common mechanism in lysosomal storage diseases

The lysosome plays a pivotal role between catabolic and anabolic processes as the nexus for signalling pathways responsive to a variety of factors, such as growth, nutrient availability, energetic status and cellular stressors. Lysosomes are also the terminal degradative organelles for autophagy through which macromolecules and damaged cellular components and organelles are degraded. Autophagy acts as a cellular homeostatic pathway that is essential for organismal physiology. Decline in autophagy during ageing or in many diseases, including late-onset forms of neurodegeneration is considered a major contributing factor to the pathology. Multiple lines of evidence indicate that impairment in autophagy is also a central mechanism underlying several lysosomal storage disorders (LSDs). LSDs are a class of rare, inherited disorders whose histopathological hallmark is the accumulation of undegraded materials in the lysosomes due to abnormal lysosomal function. Inefficient degradative capability of the lysosomes has negative impact on the flux through the autophagic pathway, and therefore dysregulated autophagy in LSDs is emerging as a relevant disease mechanism. Pathology in the LSDs is generally early-onset, severe and life-limiting but current therapies are limited or absent; recognizing common autophagy defects in the LSDs raises new possibilities for therapy. In this review, we describe the mechanisms by which LSDs occur, focusing on perturbations in the autophagy pathway and present the latest data supporting the development of novel therapeutic approaches related to the modulation of autophagy.</jats:p

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

Establishing human pluripotent stem cell-based platforms with autophagy readouts to investigate the role of autophagy in cellular homeostasis and disease

Autophagy is a vital homeostatic pathway essential for cellular survival and human health. It primarily functions as an intracellular degradation process for the turnover of unwanted cytoplasmic materials such as aggregation-prone proteins, damaged organelles and invading pathogens. Defective autophagy contributes to the pathology of a myriad human diseases including neurodegeneration and infection, and thus therapeutic exploitation of autophagy presents an attractive opportunity. The work described in this thesis involves establishment of human embryonic stem cell (hESC) models of autophagy for studying its homeostatic role and developing screening platforms in physiologically-relevant human cell-types of interest. The autophagy-deficient (ATG5–/–) hESC-based system was used to address how loss of autophagy leads to cytotoxicity; an important question relevant to neurodegeneration. The ATG5–/– hESCs and hESC-derived neurons exhibited basal cytotoxicity accompanied by a specific metabolic defect. Depletion of NADH was found to be mediating cytotoxicity during autophagy deficiency via mitochondrial depolarisation, since boosting cellular NADH levels improved cell viability and mitochondrial membrane potential in ATG5–/– neurons. This intervention was also cytoprotective in a human induced pluripotent stem cell (hiPSC)-based disease model of a neurodegenerative lysosomal storage disorder associated with autophagy dysfunction, and thus could be relevant for related neurodegenerative conditions. The autophagy reporter (mCherry-EGFP-LC3) hESC-based system was developed for identifying potent autophagy inducers enhancing pathogen clearance in macrophages; relevant for combating infection. The hESC-derived macrophages were characterised to confirm their cellular identity. These isogenic platforms can be of basic and biomedical relevance for mechanistic studies of autophagy regulation including human cell-type specific effects, and drug discovery of autophagy modulators for therapeutic applications in human diseases

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