Autophagy promotes cell survival by maintaining NAD(H) levels

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components 1 . As a recycling process, autophagy is also important for the maintenance of cellular metabolites to aid metabolic homeostasis 2 . Loss of autophagy in animal models or malfunction of this process in a number of age-related human pathologies, including neurodegenerative and lysosomal storage diseases, contributes to tissue degeneration 3-9 . However, it remains unclear which of the many cellular functions of autophagy primarily underlies its role in cell survival. Here we have identied an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD+/NADH) levels, which are critical for cellular survival. In respiring cells, loss of autophagy caused hyperactivation of PARP and Sirtuin families of NADases. Uncontrolled depletion of NAD(H) pool by these enzymes resulted in mitochondrial membrane depolarisation and cell death. Supplementation with NAD(H) precursors improved cell viability in autophagy-decient models including human pluripotent stem cell-derived neurons with autophagy deciency or patient-derived neurons with autophagy dysfunction. Our study provides a mechanistic link between autophagy and NAD(H) metabolism, and suggests that boosting NAD(H) levels may have therapeutic benets in human diseases associated with autophagy dysfunction.


Abstract
Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components 1 .As a recycling process, autophagy is also important for the maintenance of cellular metabolites to aid metabolic homeostasis 2 .Loss of autophagy in animal models or malfunction of this process in a number of age-related human pathologies, including neurodegenerative and lysosomal storage diseases, contributes to tissue degeneration [3][4][5][6][7][8][9] .However, it remains unclear which of the many cellular functions of autophagy primarily underlies its role in cell survival.Here we have identi ed an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD+/NADH) levels, which are critical for cellular survival.In respiring cells, loss of autophagy caused hyperactivation of PARP and Sirtuin families of NADases.Uncontrolled depletion of NAD(H) pool by these enzymes resulted in mitochondrial membrane depolarisation and cell death.Supplementation with NAD(H) precursors improved cell viability in autophagy-de cient models including human pluripotent stem cell-derived neurons with autophagy de ciency or patient-derived neurons with autophagy dysfunction.Our study provides a mechanistic link between autophagy and NAD(H) metabolism, and suggests that boosting NAD(H) levels may have therapeutic bene ts in human diseases associated with autophagy dysfunction.

Main
Autophagy is a cellular tra cking pathway mediated by the formation of double-membraned vesicles called autophagosomes, which ultimately fuse with lysosomes where their cargo is degraded.By sequestering and clearing dysfunctional cellular components, such as protein aggregates and damaged organelles, autophagy maintains cellular homeostasis whilst also providing metabolites and energy during periods of starvation 2 .Studies using a range of laboratory models from yeast to mammals have established that autophagy is essential for cellular and organismal survival.For example, loss of an essential autophagy gene atg5 leads to reduced survival of Saccharomyces cerevisiae in nitrogen starvation conditions and shortened lifespan in Drosophila melanogaster 10,11 .Likewise, inducible knockout of Atg5 results in cell death and neurodegeneration in adult mice 3,12,13 .It remains unclear which of the many physiological functions of autophagy are most important for its role in maintaining cell and organismal survival.Furthermore, the role of autophagy in the quality control of cellular proteins and organelles is likely to impact a plethora of signal transduction and stress response pathways, which in turn affect metabolism, growth, and survival 14,15 .Untangling this complexity in vivo is challenging whilst mechanistic studies of the cellular roles of autophagy in vitro are hindered by the fact that autophagyde cient cells are viable in cell culture 12,13,16 .We hypothesized that this apparent discrepancy between the requirement for functional autophagy in vivo and in vitro could be due to a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis in tissue culture, which could mask an underlying viability defect in autophagy-de cient cells 17 .
Cell death underlying loss of autophagy was associated with NAD(H) depletion A well-established strategy to reverse cellular reliance on energy generation via aerobic glycolysis and promote mitochondrial OXPHOS is to replace glucose, the major carbon source in tissue culture media, with galactose [18][19][20][21][22] .Strikingly, Atg5 -/-but not wild-type mouse embryonic broblasts (MEFs) cultured in galactose media displayed rapid (~24 h) caspase activation and cell death (Fig. 1a, b, Extended Data Fig. 1a).This phenotype was not caused by galactose directly as it was not toxic in the presence of glucose (Extended Data Fig. 1b, c).Instead, it was driven by mitochondrial respiration since its suppression by hypoxia or mitochondrial pyruvate carrier inhibitor UK-5099 was su cient to rescue cell death (Extended Data Fig. 1d-g).Speci city of this phenotype was validated by re-expression of Atg5, and the apoptotic nature of cell death as evidenced by caspase 3 cleavage was con rmed using Z-VAD-fmk (Extended Data Fig. 2a-c).A similar phenotype was observed in cell lines with CRISPR/Cas9 knockout of key autophagy genes such as Atg5, Atg7 or Rb1cc1 (homologue of human FIP200), as well as with the loss of lysosomal cholesterol transporter Npc1 required for e cient autophagy 6 (Fig. 1c, Extended Data Fig. 2d, e).
The rapid nature of cell death suggested an underlying metabolic collapse in autophagy-de cient cells 23 .Loss of autophagy was previously shown to cause depletion of cellular metabolites, although the mechanisms linking these metabolic defects to the cell death phenotype are poorly understood 2 .To investigate the potential metabolic basis of cell death due to autophagy de ciency, we performed an unbiased metabolomics pro ling of wild-type and Atg5 -/-MEFs after 16 h in galactose media, i.e., prior to the onset of cell death.In agreement with a previously proposed general defect in nucleic acid recycling in autophagy-de cient cells 24 , a number of nucleotides were depleted in Atg5 -/-MEFs (Fig. 1d, Extended Data Fig. 3a-c).By plotting the magnitude of change against the measure of signi cance, we identi ed the reduced form of nicotinamide adenine dinucleotide (NADH) as the most signi cantly depleted metabolite in autophagy-de cient cells (Fig. 1d, Extended Data Fig. 3b).NAD + , the oxidized form of the NAD dinucleotide, was also signi cantly decreased, suggesting that autophagy-de cient cells present with a depletion of the total pool of the NAD(H) (Fig. 1d, Extended Data Fig. 3b, c).We further con rmed NAD(H) de cit via a uorescence-based assay in Atg5 -/-MEFs (Fig. 1e).

Evolutionarily conserved role of autophagy in maintaining NAD(H) levels
Autophagy is required for the survival of eukaryotic organisms from yeast to man 25 .We investigated whether the role of autophagy in the maintenance of intracellular NAD(H) pools is evolutionarily conserved.Knockdown of Atg5 in the fruit y, Drosophila melanogaster, reduced autophagic ux as was evident from accumulation of autophagy substrate Ref(2)P 26 , and resulted in signi cant depletion of NAD(H) (Fig. 1f, Extended Data Fig. 3d).We further analyzed nitrogen-deprived Saccharomyces cerevisiae yeast that are dependent on mitochondrial respiration, wherein autophagy de ciency causes loss of respiratory capacity and cell death 11 .We found that nitrogen-deprived atg5Δ yeast exhibited both the loss of autophagy ux as monitored by Atg8-GFP cleavage 26 and a striking depletion of NAD(H) levels (Fig. 1g, Extended Data Fig. 3e).Together with our observations in MEFs, these data imply an evolutionaryconserved role of autophagy in the preservation of NAD(H) levels.

Increased NADase activity mediated NAD(H) depletion in autophagy-de cient cells
We further investigated the role of NAD(H) in mediating cytotoxicity underlying autophagy de ciency.Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) involved in NAD biosynthesis via a salvage pathway 27 (Fig. 2a) by FK866 compromised the viability of wild-type MEFs in galactose, but not glucose, medium, indicating that NAD(H) is a limiting factor for the survival of OXPHOS-dependent cells (Extended Data Fig. 4a, b).We therefore investigated the mechanism of NAD(H) depletion in autophagyde cient cells.Activities of two main classes of NAD-consuming enzymes, poly-ADP-ribose polymerases (PARPs) and deacetylases of sirtuin family (SIRTs) 28 , were increased in Atg5 -/-MEFs after 14 h culture in galactose media.This was evident from elevated levels of poly-ADP-ribosylation (PARylation) and reduced protein acetylation, respectively (Fig. 2b).PARylation, but not deacetylation, activity remained elevated after 20 h of culture (Extended Data Fig. 4c), consistent with the reliance of SIRTs but not PARPs on high cellular NAD + levels 15 .SIRTs and PARPs are activated by reactive oxygen species (ROS) and DNA damage that were found to be signi cantly elevated in respiring Atg5 -/-MEFs (Extended Data Fig. 4d-h), likely resulting from mitochondrial dysfunction due to loss of autophagic quality control 14,29 .Indeed, OXPHOS-dependent Atg5 -/-MEFs displayed disrupted mitochondrial morphology, altered levels of electron transport chain supercomplexes, and reduced ATP generation via OXPHOS (and increased reliance on glycolysis in glucose media) (Extended Data Fig. 4i-l).
We did not nd evidence for PARP hyperactivation as a direct cause of cell death which is mediated by the mitochondria-to-nucleus translocation of apoptosis-inducing factor (AIF) (Extended Data Fig. 5a).Therefore, we hypothesized that the loss of cell viability is triggered by NAD(H) exhaustion due to uncontrolled NAD + cleavage.Indeed, pharmacological inhibition of SIRTs with sirtinol or PARPs with olaparib partially rescued both, intracellular NAD(H) levels and cell viability of Atg5 -/-MEFs (Fig. 2a, c, d, Extended Data Fig. 5b-d).The role of NADases was further validated by siRNA-mediated knockdown of Sirt1 or Parp1, indicating involvement of these enzymes in NAD(H) depletion and cell death (Extended Data Fig. 5e-h).

Boosting intracellular NAD(H) rescued viability of autophagy-de cient cells
To test whether boosting intracellular NAD(H) levels is su cient to rescue viability of autophagy-de cient cells, we utilized the native cellular capacity for NAD + synthesis (Fig. 2a).Supplementation of bioavailable NAD + precursors, nicotinamide (NAM) or nicotinamide riboside (NR), led to the recovery of intracellular NAD + and NADH levels, and rescued viability of Atg5 -/-MEFs (Fig. 2e, f, Extended Data Fig. 6a).The effect of NAM (but not NR) was abrogated by FK866 (Fig. 2e, f, Extended Data Fig. 6a), consistent with the requirement for conversion of these precursors to NAD(H) via the salvage pathway (Fig. 2a).Both precursors also inhibited PARP and/or SIRT activity 28 (Extended Data Fig. 6b).However, rescue of NAD(H) and cell viability did not correlate with PARPs/SIRTs activity because FK866, which also suppressed PARylation/deacetylation by reducing NAD(H) levels, did not rescue cell viability (Fig. 2e, f, Extended Data Fig. 6a, b).Furthermore, in the presence of FK866, NR (but not NAM which requires NAMPT for conversion to NAD + ) improved NAD(H) levels and cell viability whilst PARylation/deacetylation activity was instead partially rescued compared to FK866 alone (Fig. 2e, f, Extended Data Fig. 6a, b).We conclude that boosting NAD(H) levels rescued cell death downstream of PARPs/SIRTs.
We performed an unbiased metabolomics pro ling of Atg5 -/-MEF in galactose medium, treated with or without NAM, to assess any correlation between the rescue of cell death and recovery of intracellular metabolites.By plotting the magnitude of rescue against the measure of signi cance, we found NADH to be the only metabolite that primarily correlated with Atg5 -/-MEF viability, i.e., it was rst found to be signi cantly depleted in Atg5 -/-MEF (Fig. 1d, Extended Data Fig. 3b, c) and then signi cantly restored by NAM supplementation (Fig. 2g, Extended Data Fig. 6c, d).NAD + followed a similar trend (Fig. 1d, 2g, Extended Data Fig. 3b, c, 6c, d).Therefore, the rescue of cell death is mediated by increased NAD(H), but not, for example, ATP (Fig. 2g, Extended Data Fig. 6c, d).The role of NAD(H), but not PARP/SIRT directly, was further supported by NAD(H) supplementation via de novo pathway using L-tryptophan (Fig. 2a), which rescued NAD(H) levels and cell death whilst having no inhibitory effect on PARP/SIRT activities (Fig. 2h, i, Extended Data Fig. 6e, f).Together, the salvage and de novo pathways remain active in autophagy-de cient cells and NAD(H) depletion is primarily mediated by its increased consumption.Since Atg5 -/-MEFs manifested with general nucleotide depletion (Fig. 1d, Extended Data Fig. 3b, c), we supplemented cells in galactose medium with ve nucleosides and found that all were able to restore cell viability whilst NAD(H) levels were not rescued (Extended Data Fig. 7a-c).Therefore, NAD(H) decline is independent of the previously proposed purine/pyrimidine depletion mechanism of cell death in autophagy-de cient tumor-derived cells 24 .Inhibition of Nrf2, previously shown to mediate cell death in autophagy de ciency 30 , also rescued viability without affecting NAD(H) levels (Extended Data Fig. 7d-f).Therefore, NAD(H) decline is an additional and previously unidenti ed cause of cell death due to autophagy de ciency.

NAD(H) depletion mediated cell death via mitochondrial depolarization
NADH was predominantly detected in a mitochondria-enriched cell fraction where it was depleted in Atg5 -/-MEFs cultured in galactose medium (Fig. 3a).Oxidation of NADH generates mitochondrial membrane potential (ΔΨm) across the inner mitochondrial membrane and we hypothesized that depletion of mitochondrial NADH is the cause of mitochondrial depolarization and apoptosis 14,31 .Indeed, boosting NAD(H) levels with NAM rescued membrane depolarization in Atg5 -/-MEFs (Fig. 3b, c).Furthermore, preventing dissipation of ΔΨm by suppressing ATP synthase activity (using a low dose of oligomycin) 32 , partially prevented depletion of NADH and rescued cell death (Fig. 3b-e, Extended Data Fig. 8a).Consistent with no effect of oligomycin on NAD + levels (Fig. 3d), SIRT and PARP activities remained unaffected (Extended Data Fig. 8b), indicating that oligomycin acted downstream.To further test the role of mitochondrial NADH in the cell death phenotype, we overexpressed a non-proton pumping alternative NADH oxidase, NDI1 33 .In galactose medium, overexpression of NDI1 led to an increased oxidation state of the NAD(H) pool which was su cient to enhance the apoptotic phenotype of Atg5 -/-MEFs (Fig. 3f, g, Extended Data Fig. 8c, d).We conclude that NADH is the limiting factor in the survival of autophagyde cient cells.

Loss of NAD(H) underlies cytotoxicity in human neurons with autophagy de ciency
We next investigated the mechanistic link between NAD(H) and cell survival in human neurons.While hESCs rely on glycolysis for energy production and pluripotency 35 , differentiated cells like neurons with high energy demands are dependent on mitochondrial OXPHOS for ATP generation 36 .This allows analysis in neurons to be performed at basal state by eliminating the need for medium switch to galactose.We found that NAD(H) levels were signi cantly depleted in ATG5 -/-hESC-derived neurons 3 weeks after differentiation (Fig. 4d), further supporting the evolutionarily conserved role of autophagy in maintaining NAD(H) pool (Fig. 1e-g).Since oxidation of NADH generates ΔΨm 14 , depletion of NADH was associated with mitochondrial depolarization (Extended Data Fig. 13a).Autophagy-de cient neurons also exhibited elevation in ROS (Fig. 4e), mitochondrial fragmentation and increased mitochondrial load (Extended Data Fig. 13b-f), presumably due to accumulation of damaged mitochondria arising from abrogation of autophagic clearance.In agreement with a role of NAD-consuming enzymes in depleting NAD(H) levels, PARylation and deacetylation activities of PARPs and SIRTs, respectively, were increased in ATG5 -/-hESC-derived neurons (Fig. 4f).Notably, increased cell death was observed at a basal level in ATG5 -/-neurons between 2 and 4 weeks after differentiation (Fig. 4g-i, Extended Data Fig. 13g, h).
Collectively, these data in autophagy-de cient human neurons were consistent with our nding in Atg5 -/- MEFs.
Pharmacological elevation of NAD(H) is cytoprotective in human neurons with autophagy de ciency or dysfunction Next, we evaluated the effects of pharmacologically modulating NAD(H) levels (Fig. 2a) on cell viability of ATG5 -/-neurons.NAM increased NAD(H) levels, and consequently restored ΔΨm in ATG5 -/-hESCderived neurons (Fig. 5a, b).These changes were associated with improvement in axonal length and rescue of cell death in ATG5 -/-neurons after NAM treatment, which did not affect ATP or ADP levels (Fig. 5c-f, Extended Data Fig. 14a-c).NR also rescued the viability of ATG5 -/-neurons (Extended Data Fig. 14d,   e).Inhibition of NAD(H) production by FK866 further augmented cell death in ATG5 -/-neurons (Extended Data Fig. 15a-d) and abolished the cytoprotective effect of NAM (Fig. 5f), thus implying a role of NAD(H) in governing neuronal survival.
Finally, we utilized disease-affected human neurons differentiated from patient-derived human induced pluripotent stem cells (hiPSCs) of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease.As previously reported 37 , NPC1 neurons presented with a severe autophagic defect (Extended Data Fig. 16a-c) and cell death between 3 and 4 weeks after differentiation (Fig. 5g).Similar to our ndings in autophagy-de cient models, autophagy dysfunction in NPC1 neurons correlated with NAD(H) depletion (Fig. 5h).Treatment with NAM was able to rescue both, NAD(H) levels and cell death phenotype in NPC1 neurons (Fig. 5g, h), thus showing therapeutic bene ts.

Discussion
We found that autophagy de ciency in respiring cells results in cell death.Whilst an accumulation of dysfunctional mitochondria contributes to increased cellular stress, the cell death is caused indirectly by the hyperactivity of stress responsive NAD + -dependent enzymes such as SIRTs and PARPs, which ultimately lead to the loss of NAD(H) homeostasis.Exhaustion of NADH within mitochondria appears to be the weakest link in this chain of events as it ultimately triggers mitochondrial membrane depolarization and activation of apoptosis (Fig. 5i).This model where the demise of autophagy de cient cells is mediated by stress response pathways is conceptually analogous to the previously proposed mechanism involving hyperactivation of Nrf2 30 .How this and other mechanisms, such as depletion of the general nucleotide pool in autophagy-de cient cells 24 , integrate with the critical role of NAD(H) depletion during autophagy de ciency remains to be investigated (Fig. 5i).
Ageing and age-related diseases have long been associated with a decline in both autophagy and NAD + levels 38 .Nutritional or pharmacological activation of autophagy is currently a subject of intense research for the development of small molecule modulators 7 .However, due to the varied nature of autophagy dysfunction in genetic and age-related sporadic diseases, including impairment in lysosomal degradative capability, the development of a universal modulator remains unlikely [7][8][9] .In contrast, boosting the levels of NAD + by precursor supplementation in animal models was found to have a positive impact on agerelated phenotypes, which is at least in part mediated by upregulation of autophagy 38 .Crucially, our data show that autophagy is, in turn, required for NAD(H) maintenance and that increasing NAD(H) levels protects cells by preventing the loss of ΔΨm even in the absence of functional autophagy.As such, our investigations de ne a novel mechanism linking autophagy, NAD(H) metabolism and ageing.Finally, our studies establish several points of intervention that could be targeted therapeutically in order to alleviate cellular pathology in a range of diseases associated with autophagy dysfunction.