NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy.

Mitophagy, the elimination of mitochondria via the autophagy-lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin-dependent mitophagy. We identified redox-sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.

Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2.

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson's disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4-TRPML1-ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca2+ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4-TRPML1-ALG2 pathway was also activated by H2 O2 , indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation-induced lysosomal retrograde transport involved both the TMEM55B-JIP4 and TRPML1-ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy-inducing signal.

Mitochondrial sodium/calcium exchanger (NCLX) regulates basal and starvation-induced autophagy through calcium signaling.

Mitochondria shape intracellular Ca2+ signaling through the concerted activity of Ca2+ uptake via mitochondrial calcium uniporters and efflux by Na+ /Ca2+ exchangers (NCLX). Here, we describe a novel relationship among NCLX, intracellular Ca2+ , and autophagic activity. Conditions that stimulate autophagy in vivo and in vitro, such as caloric restriction and nutrient deprivation, upregulate NCLX expression in hepatic tissue and cells. Conversely, knockdown of NCLX impairs basal and starvation-induced autophagy. Similarly, acute inhibition of NCLX activity by CGP 37157 affects bulk and endoplasmic reticulum autophagy (ER-phagy) without significant impacts on mitophagy. Mechanistically, CGP 37157 inhibited the formation of FIP200 puncta and downstream autophagosome biogenesis. Inhibition of NCLX caused decreased cytosolic Ca2+ levels, and intracellular Ca2+ chelation similarly suppressed autophagy. Furthermore, chelation did not exhibit an additive effect on NCLX inhibition of autophagy, demonstrating that mitochondrial Ca2+ efflux regulates autophagy through the modulation of Ca2+ signaling. Collectively, our results show that the mitochondrial Ca2+ extrusion pathway through NCLX is an important regulatory node linking nutrient restriction and autophagy regulation.

The evolution of selective autophagy as a mechanism of oxidative stress response: The evolutionarily acquired ability of selective autophagy receptors to respond to oxidative stress is beneficial for human longevity.

Ageing is associated with a decline in autophagy and elevated reactive oxygen species (ROS), which can breach the capacity of antioxidant systems. Resulting oxidative stress can cause further cellular damage, including DNA breaks and protein misfolding. This poses a challenge for longevous organisms, including humans. In this review, we hypothesise that in the course of human evolution selective autophagy receptors (SARs) acquired the ability to sense and respond to localised oxidative stress. We posit that in the vicinity of protein aggregates and dysfunctional mitochondria oxidation of key cysteine residues in SARs induces their oligomerisation which initiates autophagy. The degradation of damaged cellular components thus could reduce ROS production and restore redox homeostasis. This evolutionarily acquired function of SARs may represent one of the biological adaptations that contributed to longer lifespan. Inversely, loss of this mechanism can lead to age-related diseases associated with impaired autophagy and oxidative stress.

Systemic Metabolic Alteration Dependent on the Thyroid-Liver Axis in Early PD.

OBJECTIVE: Parkinson's disease (PD) is a common neurodegenerative disease characterized by initial involvement of the olfactory bulb/amygdala or autonomic nerves followed by nigral degeneration. Although autonomic innervation strictly regulates multiorgan systems, including endocrine functions, circulation, and digestion, how dysautonomia in PD affects systemic metabolism has not been identified. In this study, we tried to estimate the pathogenic linkage of PD by nuclear medicine techniques, trans-omic analysis of blood samples, and cultured cell experiments. METHODS: Thyroid mediastinum ratio of 123 I-metaiodobenzylguanidine (MIBG) scintigraphy was measured in 1,158 patients with PD. Furthermore, serum exosome miRNA transcriptome analysis and plasma metabolome analysis followed by trans-omic analysis were performed in patients with de novo PD and age-matched healthy control persons. Additionally, thyroid hormone was administered to skeletal muscle and liver derived cells to evaluate the effect of hypothyroidism for these organs. RESULTS: Sympathetic denervation of thyroid correlating with its cardiac denervation was confirmed in 1,158 patients with PD by MIBG scintigraphy. Among patients with drug-naïve PD, comprehensive metabolome analysis revealed decreased levels of thyroxine and insufficient fatty acid ß-oxidation, which positively correlate with one another. Likewise, both plasma metabolome data and transcriptome data of circulating exosomal miRNAs, revealed specific enrichment of the peroxisome proliferator-activated receptor (PPARα) axis. Finally, association of thyroid hormone with PPARα-dependent ß-oxidation regulation was confirmed by in vitro experiments. INTERPRETATION: Our findings suggest that interorgan communications between the thyroid and liver are disorganized in the early stage of PD, which would be a sensitive diagnostic biomarker for PD. ANN NEUROL 2023;93:303-316.

BRUP-1, an intracellular bilirubin modulator, exerts neuroprotective activity in a cellular Parkinson's disease model.

Bilirubin, the end product of heme redox metabolism, has cytoprotective properties and is an essential metabolite associated with cardiovascular disease, inflammatory bowel disease, type 2 diabetes, and neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by progressive degeneration of nigral dopaminergic neurons and is associated with elevated oxidative stress due to mitochondrial dysfunction. In this study, using a ratiometric bilirubin probe, we revealed that the mitochondrial inhibitor, rotenone, which is widely used to create a PD model, significantly decreased intracellular bilirubin levels in HepG2 cells. Chemical screening showed that BRUP-1 was a top hit that restored cellular bilirubin levels that were lowered by rotenone. We found that BRUP-1 up-regulated the expression level of heme oxygenase-1 (HO-1), one of the rate-limiting enzyme of bilirubin production via nuclear factor erythroid 2-related factor 2 (Nrf2) activation. In addition, we demonstrated that this Nrf2 activation was due to a direct inhibition of the interaction between Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) by BRUP-1. Both HO-1 up-regulation and bilirubin restoration by BRUP-1 treatment were significantly abrogated by Nrf2 silencing. In neuronal PC12D cells, BRUP-1 also activated the Nrf2-HO-1 axis and increased bilirubin production, resulted in the suppression of neurotoxin-induced cell death, reactive oxygen species production, and protein aggregation, which are hallmarks of PD. Furthermore, BRUP-1 showed neuroprotective activity against rotenone-treated neurons derived from induced pluripotent stem cells. These findings provide a new member of Keap1-Nrf2 direct inhibitors and suggest that chemical modulation of heme metabolism using BRUP-1 may be beneficial for PD treatment.

Targeting the autophagy-NAD axis protects against cell death in Niemann-Pick type C1 disease models.

Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders.

Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule.

Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.

Metabolic function of autophagy is essential for cell survival.

Age-related human pathologies present with a multitude of molecular and metabolic phenotypes, which individually or synergistically contribute to tissue degeneration. However, current lack of understanding of the interdependence of these molecular pathologies limits the potential range of existing therapeutic intervention strategies. In our study, we set out to understand the chain of molecular events, which underlie the loss of cellular viability in macroautophagy/autophagy deficiency associated with aging and age-related disease. We discover a novel axis linking autophagy, a cellular waste disposal pathway, and a metabolite, nicotinamide adenine dinucleotide (NAD). The axis connects multiple organelles, molecules and stress response pathways mediating cellular demise when autophagy becomes dysfunctional. By elucidating the steps on the path from efficient mitochondrial recycling to NAD maintenance and ultimately cell viability, we highlight targets potentially receptive to therapeutic interventions in a range of genetic and age-related diseases associated with autophagy dysfunction.Abbreviations: IMM: inner mitochondrial membrane; NAD: nicotinamide dinucleotide; OXPHOS: oxidative phosphorylation; PARP: poly(ADP-ribose) polymerase; ROS: reactive oxygen species.

The autophagy-NAD axis in longevity and disease.

Autophagy is an intracellular degradation pathway that recycles subcellular components to maintain metabolic homeostasis. NAD is an essential metabolite that participates in energy metabolism and serves as a substrate for a series of NAD+-consuming enzymes (NADases), including PARPs and SIRTs. Declining levels of autophagic activity and NAD represent features of cellular ageing, and consequently enhancing either significantly extends health/lifespan in animals and normalises metabolic activity in cells. Mechanistically, it has been shown that NADases can directly regulate autophagy and mitochondrial quality control. Conversely, autophagy has been shown to preserve NAD levels by modulating cellular stress. In this review we highlight the mechanisms underlying this bidirectional relationship between NAD and autophagy, and the potential therapeutic targets it provides for combatting age-related disease and promoting longevity.

Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons.

Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5-/-) human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology. For complete details on the use and execution of this protocol, please refer to Sun et al. (2023).1.

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.

Plasma taurine is an axonal excitability-translatable biomarker for amyotrophic lateral sclerosis.

Although various body fluid biomarkers for amyotrophic lateral sclerosis (ALS) have been reported, no biomarkers specifically reflecting abnormalities in axonal excitability indices have currently been established. Capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography time-of-flight mass spectrometry were used to perform a comprehensive metabolome analysis of plasma from seven ALS patients and 20 controls, and correlation analysis with disease phenotypes was then performed in 22 other ALS patients. Additionally, electrophysiological studies of motor nerve axonal excitability were performed in all ALS patients. In the ALS and control groups, levels of various metabolites directly associated with skeletal muscle metabolism, such as those involved in fatty acid ß-oxidation and the creatine pathway, were detected. Receiver operating characteristic curve analysis of the top four metabolites (ribose-5-phosphate, N6-acetyllysine, dyphylline, 3-methoxytyrosine) showed high diagnostic accuracy (area under the curve = 0.971) in the ALS group compared with the control group. Furthermore, hierarchical cluster analysis revealed that taurine levels were correlated with the strength-duration time constant, an axonal excitability indicator established to predict survival. No significant effects of diabetes mellitus and treatment (Riluzole and Edaravone) on this relationship were detected in the study. Therefore, plasma taurine is a potential novel axonal excitability-translatable biomarker for ALS.

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.

A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers.

Macroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of "autophagy profiles" revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates.Abbreviations: ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP+: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-ß: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.

Nonthmicin, a Polyether Polyketide Bearing a Halogen-Modified Tetronate with Neuroprotective and Antiinvasive Activity from Actinomadura sp.

Nonthmicin (1), a new polyether polyketide bearing a chlorinated tetronic acid, was isolated from the culture extract of a soil-derived Actinomadura strain. The structure of 1 was elucidated by interpretation of NMR and MS spectroscopic data, and the absolute configuration of 1 was proposed on the basis of the crystal structure of its dechloro congener ecteinamycin (2) also isolated from the same strain. Tetronic acids modified by halogenation have never been reported from natural products. Compounds 1 and 2 were found to have neuroprotective activity and antimetastatic properties at submicromolar concentrations in addition to antibacterial activity.