Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). Intriguingly, the majority (~60%) of prokaryotic Agos (pAgos) identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that short pAgos from Geobacter sulfurreducens, Caballeronia cordobensis and Paraburkholderia graminis, together with the NAD+-bound Sir2-proteins form a stable heterodimeric Sir2/Ago complex that recognizes invading plasmid or phage DNA through the pAgos subunit and activates Sir2 subunit triggering the endogenous NAD+ depletion and cell death thus preventing the propagation of invading DNA. This is the first demonstration that short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). Intriguingly, the majority (~60%) of prokaryotic Agos (pAgos) identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that short pAgos from Geobacter sulfurreducens, Caballeronia cordobensis and Paraburkholderia graminis, together with the NAD+-bound Sir2-proteins form a stable heterodimeric Sir2/Ago complex that recognizes invading plasmid or phage DNA through the pAgos subunit and activates Sir2 subunit triggering the endogenous NAD+ depletion and cell death thus preventing the propagation of invading DNA. This is the first demonstration that short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion

Defense-associated sirtuins (DSR) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbor an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defense systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

Abstract P319: SARM1 NAD Hydrolase Deficiency Protects Hearts Against Diabetic Cardiomyopathy

NAD depletion is associated with the pathogenesis of diseases such as heart failure. Strategies to replenish cellular NAD levels by activating NAD synthesis pathways have shown promises to treat heart disease. However, how NAD consumption mechanisms lead to NAD depletion are less understood. SARM1 is a novel intracellular NAD hydrolase that degrades NAD and promotes axonal degeneration in neurons. We recently showed that NAD redox imbalance and depletion promote the progression of diabetic cardiomyopathy. Therefore, we hypothesized that SARM1 deficiency might protect hearts against diabetic cardiomyopathy. 16-week diabetic stress initiated by streptozotocin (STZ) injections was applied to wild-type C57BL/6 (WT) and whole-body SARM1-KO mice. Cardiac function was measured after 8-week or 16-week of diabetic stress, and cardiac tissue and plasma samples from these mice were harvested after 16 weeks of diabetes. SARM1 mRNA or protein levels were suppressed in SARM1-KO hearts. STZ induced similar hyperglycemia (~600 mg/dL) in both WT and SARM1-KO male mice after 16 weeks. Chronic diabetic stress led to progressive decline in systolic function (Baseline fractional shortening: 50%; 16-week diabetes: 35%; P<0.05; n=5) in WT male mice, which was ameliorated in SARM1-KO male mice (16-week diabetes: 48%; P<0.05; n=5). Progressive decline in diastolic function induced by chronic diabetes (Baseline WT E’/A’: 1.53; WT 16-week diabetes: 1.12; n=5) was also improved in diabetic SARM1-KO mice (KO E’/A’ ratio at 16-week diabetes: 1.5; P<0.05; n=5). Heart weights were similar in diabetic WT or diabetic SARM1-KO hearts. A similar study is on-going in a female cohort. Despite loss of SARM1 expression, no compensatory changes in expressions of other NAD hydrolases (i.e. Cd38 or Bst1) were observed in diabetic SARM1-KO hearts, while expression levels of genes related to NAD consumption and synthesis pathways were mostly unchanged except Qprt and Haao. Cardiac fibrosis was induced in diabetic WT hearts and were suppressed in diabetic SARM1-KO hearts after 16-week diabetic stress, but these changes were not observed in tissues harvested after 8-week diabetic stress. The results suggest fibrosis is a later event in the progression of diabetic cardiomyopathy. WT and SARM1-KO mice have also been challenged with high fat diet feeding (HFD) for 16 weeks, and increased fasting glucose levels and body weights were similarly observed in HFD-WT and HFD-SARM1-KO mice. Longitudinal cardiac function analyses are on-going. Our data thus far support the protective role of SARM1 deficiency in metabolic stress-induced cardiomyopathy, while pathogenic mechanisms of SARM1 in diabetic hearts remain to be determined.

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might also activate SARM1. Here we show that the nicotinamide analogue 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration and neuronal death. Systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to peripheral nerve induces SARM1-dependent axon degeneration. We also identify a related pyridine derivative, 2-aminopyridine, as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity, and furthermore, suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

Adenylate Kinase 2 deficiency causes NAD+ depletion and impaired purine metabolism during myelopoiesis

Reticular Dysgenesis is a particularly grave from of severe combined immunodeficiency (SCID) that presents with severe congenital neutropenia and a maturation arrest of most cells of the lymphoid lineage. The disease is caused by biallelic loss of function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) as substrate for adenosine triphosphate (ATP) synthesis in the mitochondria. Accordingly, it has long been hypothesized that a decline in OXPHOS metabolism is the driver of the disease. The mechanistic basis for Reticular Dysgenesis, however, remained incompletely understood, largely due to lack of appropriate model systems to phenocopy the human disease. We have used single cell RNA-sequencing of bone marrow cells from 2 reticular dysgenesis patients to gain insight into the disease pathology. Gene set enrichment for differentially expressed genes in different subsets of myeloid and lymphoid progenitor cells pointed to processes involving RNA and ribonucleoprotein assembly and catabolism as well as cell cycle defects. To investigate these findings and precisely mimic the failure of human myelopoiesis in culture, we developed a cell-tracible model of Reticular Dysgenesis based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. In this model, we have identified that AK2-deficienct myeloid progenitor cells exhibit NAD+ depletion and high levels of reductive stress accompanied by an accumulation of AMP and IMP while ADP and ATP are only mildly decreased. Our studies further show that AK2-deficienct cells have decreased de novo purine synthesis and increased purine breakdown, accompanied by decreased RNA and ribosome subunit cellular content. These data highlight the profound impact of mitochondrial dysfunction on the cellular redox state and nucleotide pool and identify the mechanistic basis of Reticular Dysgenesis as a defect in purine metabolism.

The Role of NAD+ in Rejuvenating Human Body

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme and considered an essential cofactor in cellular bioenergetics and adaptive stress responses. It is present in all living cells and governs fundamental biological processes including energy production, DNA repair, gene expression, calcium-dependent secondary messenger signaling and also in immune-regulatory roles. NAD+ depletion has been a subject of intense research due to the reason that it is associated with hallmarks of aging and age-related diseases, such as metabolic disorders, cancer and neurodegenerative diseases. Recent studies have suggested that physiological and pharmacological interventions that elevate cellular NAD+ levels may slow or even reverse the aspects of aging and also delay the progression of age-related diseases. In this min-review, we have described the roles of NAD+ in relationships to aging and major age-related diseases. The emphasis is on the contribution of NAD+ depletion to aging along with strategies to modulate NAD+ metabolism through physiological and pharmacological pathways. Recent human clinical studies on NAD+ boosting are summarized. We have specifically addressed how boosting NAD+ levels could potentially play an important role as a promising therapeutic strategy to counter aging-associated pathologies and accelerated aging. Finally, a brief perspective on the future research direction is presented.

Ozone Exposure Induces Metabolic Disorders and NAD+ Depletion Through PARP1 Activation in Spinal Cord Neurons

Background and Objective: Ozone therapy has shown therapeutic efficacy in different disorders particularly low back pain (LBP). However, ozone therapy has been associated with toxic effects on the respiratory, endocrine, cardiovascular systems as well as nervous system because of its strong oxidizing capacity. Recent studies have reported possible associations between ozone exposure and metabolic disorders, but the findings are controversial and little is known on the mechanisms of action. This study aims to investigate the cytotoxic effects of ozone exposure and possible mechanism of action in the animal model.Methods: Wistar neonate rats with the age of 24 h after birth were sacrificed by cervical dislocation under general anesthesia, then immersed in 75% alcohol and iodophor for 5 min, respectively. The spinal cord was isolated and cut to samples of ~1 mm3 and prepared for further experiments. The spinal cord neurons (SCNs) were exposed to ozone at different concentrations and then cultured in 96-well plates with glass bottom for 7 days. The cell viability, ATP levels and the NAD+ concentration were determined and compared between the different experimental groups and the control group.Results: Analyses of the data by non-targeted liquid chromatography-mass spectrometry (LC-MS) analysis determined the metabolic disorder in SCNs following the ozone exposure. Moreover, our assessments showed that ozone exposure resulted in DNA damage, poly (ADP)-ribose polymerase-1 (PARP1) excessive activation, nicotinamide adenine dinucleotide (NAD+) depletion and decrease of ATP level in SCNs. The PARP1 inhibitor can inhibit the cytotoxic effect of ozone to SCNs without inhibiting the activation of AMP-activated protein kinase (AMPK). Our findings revealed that the cytotoxic effects of ozone to SCNs might be mediated by excessive PARP1 activation and subsequent NAD+ depletion. Moreover, using PARP1 inhibitor can protect SCNs from cytotoxic effects of ozone by preventing NAD+ depletion during ozone exposure.Conclusion: Ozone exposure seems to induce metabolic disorders and NAD+ depletion through excessive PARP1 activation in SCNs.