Nicotinamide Mononucleotide Adenylyltransferase Research Articles

8700 Mono(ADP-Ribosyl)ation of Ribosomal Proteins Promotes Differentiation During Adipogenesis

Abstract Disclosure: X. Tan: None. S. Challa: None. C.V. Camacho: None. T.S. Nandu: None. M.S. Stokes: None. W.L. Kraus: Other; Self; W.L.K. is a founder and stockholder for Ribon Therapeutics, Inc; a founder, member of the SAB, member of the BOD, and a stockholder for and ARase Therapeutics, Inc. He is also coholder of U.S. Patent. ADP-ribosylation, a crucial post-translational modification, involves the covalent attachment of ADP-ribose units to amino acids on proteins through ADP-ribosyl transferase (ART) enzymes, utilizing NAD+ synthesized by nicotinamide mononucleotide adenylyl transferases (NMNATs). We previously showed that NMNAT-2, a cytoplasmic NAD+ synthase, increases during adipogenesis, leading to reduced nuclear NAD+ levels through compartmentalized NMN depletion and enhanced differentiation of preadipocytes. However, the implications of increased cytoplasmic NAD+ synthesis by NMNAT-2 in adipocytes remain unknown. Our current findings demonstrate elevated ribosome MARylation during 3T3-L1 preadipocyte differentiation, driven by NMNAT-2 induction. Inhibiting NAD+ biosynthesis by knockdown of NMNAT-2 or FK866 results in reduced ribosome MARylation, with PARP16 identified as the primary ART mediating MARylation. Knockdown of NMNAT-2 or PARP16 impaired global protein synthesis and preadipocyte differentiation. Treatment with the PARP16 inhibitor DB008 hindered ribosomal MARylation and preadipocyte differentiation. Immunofluorescence and subcellular fractionation analysis demonstrated that MARylated ribosomes are mostly associated with the endoplasmic reticulum. Using TMT mass spectrometry, we identified numerous high-confidence ADP-ribosylation sites on glutamate and aspartate residues in both undifferentiated and differentiated cells. We examined five proteins with specific sites of MARylation that are consistently more modified in the differentiated state. Among these, ADP-ribosylation of RPSA significantly promote preadipocyte differentiation; mutation of the site inhibits this effect. Ongoing investigations involve studying the impact of Parp16 knockout on high fat diet-induced obesity in mice to further determine the biological role of ribosome protein MARylation during adipogenesis. This work is supported by a grant from the NIH/NIDDK (R01 DK069710) and the DOD OCRP (OC200311). Presentation: 6/1/2024

Chronically Low NMNAT2 Expression Causes Sub-lethal SARM1 Activation and Altered Response to Nicotinamide Riboside in Axons.

Nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is an endogenous axon survival factor that maintains axon health by blocking activation of the downstream pro-degenerative protein SARM1 (sterile alpha and TIR motif containing protein 1). While complete absence of NMNAT2 in mice results in extensive axon truncation and perinatal lethality, the removal of SARM1 completely rescues these phenotypes. Reduced levels of NMNAT2 can be compatible with life; however, they compromise axon development and survival. Mice born expressing sub-heterozygous levels of NMNAT2 remain overtly normal into old age but develop axonal defects in vivo and in vitro as well as behavioural phenotypes. Therefore, it is important to examine the effects of constitutively low NMNAT2 expression on SARM1 activation and disease susceptibility. Here we demonstrate that chronically low NMNAT2 levels reduce prenatal viability in mice in a SARM1-dependent manner and lead to sub-lethal SARM1 activation in morphologically intact axons of superior cervical ganglion (SCG) primary cultures. This is characterised by a depletion in NAD(P) and compromised neurite outgrowth. We also show that chronically low NMNAT2 expression reverses the NAD-enhancing effect of nicotinamide riboside (NR) in axons in a SARM1-dependent manner. These data indicate that low NMNAT2 levels can trigger sub-lethal SARM1 activation which is detectable at the molecular level and could predispose to human axonal disorders.

NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage.

The gene encoding nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), a nicotinamide adenine dinucleotide synthetase localized in the cell nucleus, is a causative factor in Leber's congenital amaurosis, which is the earliest onset type of inherited retinal degeneration. We sought to investigate the roles of NMNAT1 in early retinal development. We used human induced pluripotent stem cells (hiPSCs) and established NMNAT1-knockout (KO) hiPSCs using CRISPR/cas9 technology to reveal the roles of NMNAT1 in human retinal development. NMNAT1 was not essential for the survival and proliferation of immature hiPSCs; therefore, we subjected NMNAT1-KO hiPSCs to retinal organoid (RO) differentiation culture. The expression levels of immature hiPSC-specific genes decreased in a similar manner after organoid culture initiation up to 2 weeks in the control and NMNAT1-KO. Neuroectoderm-specific genes were induced in the control and NMNAT1-KO organoids within a few days after starting the organoid culture; PAX6 and TUBB3 were higher in NMNAT1-KO organoids up to 7days than in the control organoids. However, the induction of genes involving retinal early development, such as RAX, which was induced at around day 10 in this culture, was considerably reduced in NMNAT1-KO organoids. Morphological examination also showed failure of retinal primordial structure formation, which became visible at around 2 weeks of the control culture, in the NMNAT1-KO organoids. Decreased intracellular NAD levels and poly(ADP-ribosyl)ation were observed in NMNAT1-KO organoids at 7 to 10days of the culture. Mass spectrometry analysis of inhibited proteins in the poly(ADP-ribosyl)ation pathway identified poly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase 1 (PARP1) as a major protein. These results indicate that NMNAT1 was dispensable for neural lineage differentiation but essential for the commitment of retinal fate differentiation in hiPSCs. The NMNAT1-NAD-PARP1 axis may play a critical role in the appropriate development of human retinal lineage differentiation.

Extracellular NAD+ response to post-hepatectomy liver failure: bridging preclinical and clinical findings

Liver fibrosis progressing to cirrhosis is a major risk factor for liver cancer, impacting surgical treatment and survival. Our study focuses on the role of extracellular nicotinamide adenine dinucleotide (eNAD+) in liver fibrosis, analyzing liver disease patients undergoing surgery. Additionally, we explore NAD+’s therapeutic potential in a mouse model of extended liver resection and in vitro using 3D hepatocyte spheroids. eNAD+ correlated with aspartate transaminase (AST) and bilirubin after liver resection (AST: r = 0.2828, p = 0.0087; Bilirubin: r = 0.2584, p = 0.0176). Concordantly, post-hepatectomy liver failure (PHLF) was associated with higher eNAD+ peaks (n = 10; p = 0.0063). Post-operative eNAD+ levels decreased significantly (p < 0.05), but in advanced stages of liver fibrosis or cirrhosis, this decline not only diminished but actually showed a trend towards an increase. The expression of NAD+ biosynthesis rate-limiting enzymes, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), were upregulated significantly in the liver tissue of patients with higher liver fibrosis stages (p < 0.0001). Finally, the administration of NAD+ in a 3D hepatocyte spheroid model rescued hepatocytes from TNFalpha-induced cell death and improved viability (p < 0.0001). In a mouse model of extended liver resection, NAD+ treatment significantly improved survival (p = 0.0158) and liver regeneration (p = 0.0186). Our findings reveal that eNAD+ was upregulated in PHLF, and rate-limiting enzymes of NAD+ biosynthesis demonstrated higher expressions under liver fibrosis. Further, eNAD+ administration improved survival after extended liver resection in mice and enhanced hepatocyte viability in vitro. These insights may offer a potential target for future therapies.

Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1.

Nicotinamide adenine dinucleotide (NAD) is involved in renal physiology and is synthesized by nicotinamide mononucleotide adenylyltransferase (NMNAT). NMNAT exists as three isoforms, namely, NMNAT1, NMNAT2, and NMNAT3, encoded by Nmnat1, Nmnat2, and Nmnat3, respectively. In diabetic nephropathy (DN), NAD levels decrease, aggravating renal fibrosis. Conversely, sodium-glucose cotransporter-2 inhibitors increase NAD levels, mitigating renal fibrosis. In this regard, renal NAD synthesis has recently gained attention. However, the renal role of Nmnat in DN remains uncertain. Therefore, we investigated the role of Nmnat by establishing genetically engineered mice. Among the three isoforms, NMNAT1 levels were markedly reduced in the proximal tubules (PTs) of db/db mice. We examined the phenotypic changes in PT-specific Nmnat1 conditional knockout (CKO) mice. In CKO mice, Nmnat1 expression in PTs was downregulated when the tubules exhibited albuminuria, peritubular type IV collagen deposition, and mitochondrial ribosome (mitoribosome) excess. In CKO mice, Nmnat1 deficiency-induced mitoribosome excess hindered mitoribosomal translation of mitochondrial inner membrane-associated oxidative phosphorylation complex I (CI), CIII, CIV, and CV proteins and mitoribosomal dysfunction. Furthermore, the expression of hypermethylated in cancer 1, a transcription repressor, was downregulated in CKO mice, causing mitoribosome excess. Nmnat1 overexpression preserved mitoribosomal function, suggesting its protective role in DN.

Highly Bioadaptable Hybrid Conduits with Spatially Bidirectional Structure for Precision Nerve Fiber Regeneration via Gene Therapy.

Peripheral nerve deficits give rise to motor and sensory impairments within the limb. The clinical restoration of extensive segmental nerve defects through autologous nerve transplantation often encounters challenges such as axonal mismatch and suboptimal functional recovery. These issues may stem from the limited regenerative capacity of proximal axons and the subsequent Wallerian degeneration of distal axons. To achieve the integration of sensory and motor functions, a spatially differential plasmid DNA (pDNA) dual-delivery nanohydrogel conduit scaffold is devised. This innovative scaffold facilitates the localized administration of the transforming growth factor β (TGF-β) gene in the proximal region to accelerate nerve regeneration, while simultaneously delivering nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) to the distal region to mitigate Wallerian degeneration. By promoting autonomous and selective alignment of nerve fiber gap sutures via structure design, the approach aims to achieve a harmonious unification of nerve regeneration, neuromotor function, and sensory recovery. It is anticipated that this groundbreaking technology will establish a robust platform for gene delivery in tissue engineering.

Evolutionary adaptation in the laboratory and the process of retrograde engineering augment autotrophic proliferation in Saccharomyces cerevisiae

Genetic engineering provides various avenues for CO2 transformation into biological matter or bio-substances, aiding in mitigating the unsustainable utilization of organic raw materials that contribute detrimentally to global warming. The employment of widely recognized industrial microorganisms, like Saccharomyces cerevisiae, for the creation of innovative carbon (C) biomanufacturing frameworks may detach biological technology from raw materials with potential substitute applications in agricultural output. Lately, the primary C utilization of S. cerevisiae was rewired through a systematic manipulation strategy, enabling the resultant strains to achieve autotrophic growth with a maximum growth rate (μmax) of 0.002 h−1, which was subsequently enhanced to 0.02 h−1 via evolutionary engineering. By employing retrograde gene manipulation to manipulate Copy Number Variations (CNVs) variations happening in the genes encoding hexokinase and nicotinamide mononucleotide adenylyltransferase (NMNAT) following evolution, we assessed their impact on the enhanced self-sustaining characteristics. The modified CNVs induced reduced catalytic capabilities in potential bifurcation reactions and energy-balancing-related chemical processes. Moreover, we demonstrate how subsequent evolution enables peroxisomal intake and augments proliferation in self-sustaining circumstances. The genetically modified S. cerevisiae strains provide a foundation for the advancement of a versatile innovation that harnesses CO2 for the manufacturing of high-value goods, including cellular organic matter, specialized catalysts, and compounds while circumventing the consumption of organic raw materials alongside potential substitute applications in agricultural output. Additionally, the detection and validation of three critical mechanisms might facilitate the assimilation of foreign CBB cycles or analogous routes to foreign microorganisms.

Inhibitors of NAD+ Production in Cancer Treatment: State of the Art and Perspectives.

The addiction of tumors to elevated nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of cancer metabolism. Obstructing NAD+ biosynthesis in tumors is a new and promising antineoplastic strategy. Inhibitors developed against nicotinamide phosphoribosyltransferase (NAMPT), the main enzyme in NAD+ production from nicotinamide, elicited robust anticancer activity in preclinical models but not in patients, implying that other NAD+-biosynthetic pathways are also active in tumors and provide sufficient NAD+ amounts despite NAMPT obstruction. Recent studies show that NAD+ biosynthesis through the so-called "Preiss-Handler (PH) pathway", which utilizes nicotinate as a precursor, actively operates in many tumors and accounts for tumor resistance to NAMPT inhibitors. The PH pathway consists of three sequential enzymatic steps that are catalyzed by nicotinate phosphoribosyltransferase (NAPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), and NAD+ synthetase (NADSYN1). Here, we focus on these enzymes as emerging targets in cancer drug discovery, summarizing their reported inhibitors and describing their current or potential exploitation as anticancer agents. Finally, we also focus on additional NAD+-producing enzymes acting in alternative NAD+-producing routes that could also be relevant in tumors and thus become viable targets for drug discovery.

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport

BackgroundBioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.MethodsWe generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.ResultsWe provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.ConclusionNMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

The Role of NMNAT2/SARM1 in Neuropathy Development.

Chemotherapy-induced peripheral neuropathy (CIPN) commonly arises as a side effect of diverse cancer chemotherapy treatments. This condition presents symptoms such as numbness, tingling, and altered sensation in patients, often accompanied by neuropathic pain. Pathologically, CIPN is characterized by an intensive "dying-back" axonopathy, starting at the intra-epidermal sensory innervations and advancing retrogradely. The lack of comprehensive understanding regarding its underlying mechanisms explains the absence of effective treatments for CIPN. Recent investigations into axon degeneration mechanisms have pinpointed nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha and TIR motif-containing 1 protein (SARM1) as pivotal mediators of injury-induced axonal degeneration. In this review, we aim to explore various studies shedding light on the interplay between NMNAT2 and SARM1 proteins and their roles in the progression of CIPN.

The concept of gene therapy for glaucoma: the dream that has not come true yet.

Gene therapies, despite of being a relatively new therapeutic approach, have a potential to become an important alternative to current treatment strategies in glaucoma. Since glaucoma is not considered a single gene disease, the identified goals of gene therapy would be rather to provide neuroprotection of retinal ganglion cells, especially, in intraocular-pressure-independent manner. The most commonly reported type of vector for gene delivery in glaucoma studies is adeno-associated virus serotype 2 that has a high tropism to retinal ganglion cells, resulting in long-term expression and low immunogenic profile. The gene therapy studies recruit inducible and genetic animal models of optic neuropathy, like DBA/2J mice model of high-tension glaucoma and the optic nerve crush-model. Reported gene therapy-based neuroprotection of retinal ganglion cells is targeting specific genes translating to growth factors (i.e., brain derived neurotrophic factor, and its receptor TrkB), regulation of apoptosis and neurodegeneration (i.e., Bcl-xl, Xiap, FAS system, nicotinamide mononucleotide adenylyl transferase 2, Digit3 and Sarm1), immunomodulation (i.e., Crry, C3 complement), modulation of neuroinflammation (i.e., erythropoietin), reduction of excitotoxicity (i.e., CamKIIα) and transcription regulation (i.e., Max, Nrf2). On the other hand, some of gene therapy studies focus on lowering intraocular pressure, by impacting genes involved in both, decreasing aqueous humor production (i.e., aquaporin 1), and increasing outflow facility (i.e., COX2, prostaglandin F2α receptor, RhoA/RhoA kinase signaling pathway, MMP1, Myocilin). The goal of this review is to summarize the current state-of-art and the direction of development of gene therapy strategies for glaucomatous neuropathy.

NMNATs expression inhibition mediated NAD+ deficiency plays a critical role in doxorubicin-induced hepatotoxicity in mice

Doxorubicin (DOX) is one of the most widely used antineoplastic drugs with known cardiotoxicity while other organ toxicity, such as hepatotoxicity is not well defined. This study was to explore the role of nicotinamide adenine dinucleotide (NAD+) in DOX-induced hepatotoxicity. DOX (20 mg/kg) induced acute liver injury and oxidative stress in C57BL/6 J mice at 48 h. Notably, the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and NAD(P)H dehydrogenase quinone 1 (NQO1) were downregulated. NAD+ deficiency was confirmed due to DOX exposure. Mechanistically, the downregulation of nicotinamide mononucleotide adenylyl transferase 1 (NMNAT1), NMNAT2 and NMNAT3, while no alteration of nicotinamide phosphoribosyl transferase was proved. As a consequence of NAD+ deficiency, the expression of poly-ADP-ribose polymerase1 (PARP1), CD38 and Sirtuin1 (SIRT1) were reduced. Furthermore, supplementation of NAD+ (200 mg/kg/day) or its precursor nicotinamide mononucleotide (NMN) (500 mg/kg/day) alleviated liver injury, attenuated oxidative stress, and elevated the downregulation of Nrf2 and NQO1. More importantly, compromised expression of NMNAT1–3, PARP1, CD38 and SIRT1 were improved by NAD+ and NMN. In conclusion, NAD+ deficiency due to NMNATs expression inhibition may attribute to the pathogenesis of DOX-induced hepatotoxicity, thus providing new insights for mitigating DOX side effects.

Jian-Pi-Yi-Shen formula alleviates renal fibrosis by restoring NAD+ biosynthesis in vivo and in vitro.

Patients with chronic kidney disease (CKD) lack efficacious treatment. Jian-Pi-Yi-Shen formula (JPYSF) has demonstrated significant clinical efficacy in treating CKD for decades. However, its renoprotective mechanism has not been fully elucidated. This study aimed to determine whether JPYSF could delay renal fibrosis progression in CKD by restoring nicotinamide adenine dinucleotide (NAD+) biosynthesis. Adenine-diet feeding was used to model CKD in C57BL/6 mice. JPYSF was orally administered for 4 weeks. Human proximal tubular epithelial cells (HK-2) cells were stimulated with transforming growth factor-β1 (TGF-β1) with or without JPYSF treatment. Renal function of mice was assessed by serum creatinine and blood urea nitrogen levels. Renal histopathological changes were assessed using Periodic acid-Schiff and Masson's trichrome staining. Cell viability was assessed using a cell counting kit-8 assay. NAD+ concentrations were detected by a NAD+/NADH assay kit. Western blotting, immunohistochemistry, and immunofluorescence were employed to examine fibrosis-related proteins and key NAD+ biosynthesis enzymes expression in the CKD kidney and TGF-β1-induced HK-2 cells. JPYSF treatment could not only improve renal function and pathological injury but also inhibit renal fibrosis in CKD mice. Additionally, JPYSF reversed fibrotic response in TGF-β1-induced HK-2 cells. Moreover, JPYSF rescued the decreased NAD+ content in CKD mice and TGF-β1-induced HK-2 cells through restoring expression of key enzymes in NAD+ biosynthesis, including quinolinate phosphoribosyltransferase, nicotinamide mononucleotide adenylyltransferase 1, and nicotinamide riboside kinase 1. JPYSF alleviated renal fibrosis in CKD mice and reversed fibrotic response in TGF-β1-induced HK-2 cells, which may be related to the restoration of NAD+ biosynthesis.

Huangqi-Danshen decoction protects against cisplatin-induced acute kidney injury in mice.

Background: Acute kidney injury (AKI) induced by cisplatin remains a major impediment to the clinical application of cisplatin, necessitating urgent exploration for promising solutions. Huangqi-Danshen decoction (HDD), a Chinese herbal preparation, has been shown by our group to have a reno-protective effect in adenine-induced chronic kidney disease mice and diabetic db/db mice. However, the effect of HDD on cisplatin-induced AKI and its underlying mechanisms are unknown. Methods: The AKI model was established by intraperitoneal injection of cisplatin (20mg/kg) in C57BL/6 mice. The mice in the treatment group were administrated with HDD (6.8g/kg/d) for 5 consecutive days before cisplatin challenge. After 72h cisplatin injection, blood and kidney tissue were subsequently collected for biochemical detection, histopathological evaluation, Western blot analysis, immunohistochemical staining, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling assay. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to detect changes in renal metabolites. Results: The results showed that HDD significantly reduced serum creatinine and blood urea nitrogen levels and alleviated renal histopathological injury in cisplatin-induced AKI mice. And HDD treatment demonstrated a significant inhibition in apoptosis, inflammation, and oxidative stress in AKI mice. Moreover, non-target metabolomics revealed that HDD significantly restored 165 altered metabolites in AKI mice. Subsequent enrichment analysis and pathway analysis of these metabolites indicated that nicotinate and nicotinamide metabolism was the primary pathway affected by HDD intervention. Further investigation showed that HDD could upregulate nicotinamide adenine dinucleotide (NAD+) biosynthesis-related enzymes quinolinate phosphoribosyltransferase, nicotinamide mononucleotide adenylyltransferase 1, and nicotinamide phosphoribosyltransferase to replenish NAD+ content in the kidney of AKI mice. Conclusion: In summary, HDD exerted a protective effect against cisplatin-induced AKI and suppressed apoptosis, inflammation, and oxidative stress in the kidney of AKI mice, which may be attributed to the modulation of NAD+ biosynthesis.

The Molecular Mechanisms Involved in Axonal Degeneration and Retrograde Retinal Ganglion Cell Death.

Axonal degeneration is a pathologic change common to multiple retinopathies and optic neuropathies. Various pathologic factors, such as mechanical injury, inflammation, and ischemia, can damage retinal ganglion cell (RGC) somas and axons, eventually triggering axonal degeneration and RGC death. The molecular mechanisms of somal and axonal degeneration are distinct but also overlap, and axonal degeneration can result in retrograde somal degeneration. While the mitogen-activated protein kinase pathway acts as a central node in RGC axon degeneration, several newly discovered molecules, such as sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 and nicotinamide mononucleotide adenylyltransferase 2, also play a critical role in this pathological process following different types of injury. Therefore, we summarize the types of injury that cause RGC axon degeneration and retrograde RGC death and important underlying molecular mechanisms, providing a reference for the identification of targets for protecting axons and RGCs.

THU547 Role Of NAD+ Synthesis Pathway In PARP Inhibitor Resistance

Abstract Disclosure: S. Challa: None. ADP-ribosylation is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes consisting of 17 members that have distinct structural domains, activities, subcellular localizations, and functions. The catalytic activities of PARP enzymes are intimately tied to the synthesis of NAD+, which is consumed during ADPRylation reactions and, thus, must be regenerated. The intracellular salvage pathway utilizing nicotinamide (NAM) is the primary source of NAD+ in the cell. NAM is converted to nicotinamide mononucleotide (NMN) by NAMPT. Nicotinamide mononucleotide adenylyl transferases (NMNATs) catalyze the final step in the NAD+ salvage pathway, combining NMN and ATP to make NAD+. Three different NMNATs, each with a distinct subcellular localization, control the subcellular levels of NAD+ in each compartment: NMNAT-1 in the nucleus, NMNAT-2 is in the outer membrane of golgi, and NMNAT-3 in the mitochondria. We recently demonstrated that such compartment-specific NAD+ synthesis regulates the levels of ADP-ribosylation. Elevated levels of NMNAT-2, the cytosolic NAD+ synthase, reduces nuclear NAD+ levels and PARP1 activity in adipocytes and ovarian cancer cells. In the current study, we aim to delineate the role of compartmentalized synthesis of NAD+ in PARP inhibitor resistance in ovarian cancers. To identify these mechanisms, we generated ovarian cancer cells with acquired resistance to Niraparib, an FDA approved PARP inhibitor (NirR). We found that NirR cells have elevated levels of NAD+ biosynthesis. Moreover, treatment of parental cells with NAD+ precursors reduced the sensitivity to Niraparib while inhibition of NAD+ metabolism in NirR cells increased the sensitivity to Niraparib. Interestingly, the resistant cells have comparable levels of PARP1 activation to the parental cells. These data collectively suggests that NAD+ metabolism confers resistance to Niraparib without altering PARP1 activity. In line with this, NirR cells are sensitive to the effects of depletion of the cytosolic NAD+ biosynthesis, suggesting high NAD+ biosynthesis in NirR cells supports processes regulated by the cytosolic NAD+ which may drive the resistance phenotype. There is an urgent need to identify the mechanisms of resistance to PARP inhibitors to increase their clinical efficacy. This study identifies the NAD+ biosynthesis pathway as a key mechanism of PARP inhibitor resistance. Presentation: Thursday, June 15, 2023

FRI019 Mono(ADP-Ribosyl)ation Of Ribosomal Proteins During Adipogenesis

Abstract Disclosure: X. Tan: None. M.S. Stokes: None. T.S. Nandu: None. W.L. Kraus: None. ADP-ribosylation is a post-translational modification of proteins in which ADP-ribose units from NAD+ are covalently linked to various amino acids on substrates by ADP-ribosyltransferase (ART) enzymes. The NAD+ required for these reactions is synthesized by nicotinamide mononucleotide adenylyl transferases (NMNATs), which serve as the key NAD+ synthase enzymes in the salvage pathway. The three cellular NMNATs have different subcellular localizations (NMNAT-1 in the nucleus, NMNAT-2 in the cytoplasm, and NMNAT-3 in the mitochondria). We have shown previously that NMNAT-2 protein is rapidly and robustly induced during adipogenesis through a stabilization mechanism, causing rapid reduction in nuclear nicotinamide mononucleotide (NMN) levels and decreased PARP1 catalytic activity, leading to an increased expression of proadipogenic genes. We also recently demonstrated that NMNAT-2 support the catalytic activity of PARP16, an endoplasmic reticulum-localized enzyme, to maintain protein homeostasis in ovarian cancer cells by mono(ADP-ribosyl)ation (MARylation) of ribosomal proteins. The biological functions of increased cytoplasmic NAD+ synthesis by NMNAT-2 in adipocytes, however, is unknown. Our results demonstrate a reciprocal decrease of nuclear poly(ADP-ribose) (PAR) levels and increase of cytosolic MAR levels during the differentiation of 3T3-L1 preadipocytes, which is driven by the induction of NMNAT-2. The increase in cytosolic MAR levels leads to enhanced ribosome MARylation, and knockdown of NMNAT-2 or PARP16 results in reduced ribosome MARylation. Analysis of ribosomes isolated from differentiated 3T3-L1 cells revealed the enrichment of MARylation in fractions containing monosomes. Using TMT mass spectrometry, we identified numerous high confidence of ADP-ribosylation sites on ribosome proteins in both undifferentiated and differentiated 3T3-L1 cells, with over 30 sites showing significant increases in differentiated adipocytes. We are now examining the relationship between the MARylation of ribosome proteins and translational programming during adipogenesis to expand our understanding of MARylation in physiology and disease. Presentation: Friday, June 16, 2023

Adipocyte NMNAT1 expression is essential for nuclear NAD+ biosynthesis but dispensable for regulating thermogenesis and whole-body energy metabolism

Nicotinamide adenine dinucleotide (NAD+) functions as an essential cofactor regulating a variety of biological processes. The purpose of the present study was to determine the role of nuclear NAD+ biosynthesis, mediated by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), in thermogenesis and whole-body energy metabolism. We first evaluated the relationship between NMNAT1 expression and thermogenic activity in brown adipose tissue (BAT), a key organ for non-shivering thermogenesis. We found that reduced BAT NMNAT1expression was associated with inactivation of thermogenic gene program induced by obesity and thermoneutrality. Next, we generated and characterized adiponectin-Cre-driven adipocyte-specific Nmnat1 knockout (ANMT1KO) mice. Loss of NMNAT1 markedly reduced nuclear NAD+ concentration by approximately 70% in BAT. Nonetheless, adipocyte-specific Nmnat1 deletion had no impact on thermogenic (rectal temperature, BAT temperature and whole-body oxygen consumption) responses to β-adrenergic ligand norepinephrine administration and acute cold exposure, adrenergic-mediated lipolytic activity, and metabolic responses to obesogenic high-fat diet feeding. In addition, loss of NMNAT1 did not affect nuclear lysine acetylation or thermogenic gene program in BAT. These results demonstrate that adipocyte NMNAT1 expression is required for maintaining nuclear NAD+ concentration, but not for regulating BAT thermogenesis or whole-body energy homeostasis.

Deletion of Nmnat1 in Skeletal Muscle Leads to the Reduction of NAD+ Levels but Has No Impact on Skeletal Muscle Morphology and Fiber Types.

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme that mediates many redox reactions in energy metabolism. NAD+ is also a substrate for ADP-ribosylation and deacetylation by poly (ADP-ribose) polymerase and sirtuin, respectively. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) is a NAD+ biosynthesizing enzyme found in the nucleus. Recent research has shown that the maintaining NAD+ levels is critical for sustaining muscle functions both in physiological and pathological conditions. However, the role of Nmnat1 in skeletal muscle remains unexplored. In this study, we generated skeletal muscle-specific Nmnat1 knockout (M-Nmnat1 KO) mice and investigated its role in skeletal muscle. We found that NAD+ levels were significantly lower in the skeletal muscle of M-Nmnat1 KO mice than in control mice. M-Nmnat1 KO mice, in contrast, had similar body weight and normal muscle histology. Furthermore, the distribution of muscle fiber size and gene expressions of muscle fiber type gene expression were comparable in M-Nmnat1 KO and control mice. Finally, we investigated the role of Nmnat1 in muscle regeneration using cardiotoxin-induced muscle injury model, but muscle regeneration appeared almost normal in M-Nmnat1 KO mice. These findings imply that Nmnat1 has a redundancy in the pathophysiology of skeletal muscle.

NMNAT1 and hereditary spastic paraplegia (HSP): expanding the phenotypic spectrum of NMNAT1 variants

In the NAD biosynthetic network, the nicotinamide mononucleotide adenylyltransferase (NMNAT) enzyme fuels NAD as a co-substrate for a group of enzymes. Mutations in the nuclear-specific isoform, NMNAT1, have been extensively reported as the cause of Leber congenital amaurosis-type 9 (LCA9). However, there are no reports of NMNAT1 mutations causing neurological disorders by disrupting the maintenance of physiological NAD homeostasis in other types of neurons. In this study, for the first time, the potential association between a NMNAT1 variant and hereditary spastic paraplegia (HSP) is described. Whole-exome sequencing was performed for two affected siblings diagnosed with HSP. Runs of homozygosity (ROH) were detected. The shared variants of the siblings located in the homozygosity blocks were selected. The candidate variant was amplified and Sanger sequenced in the proband and other family members. Homozygous variant c.769G>A:p.(Glu257Lys) in NMNAT1, the most common variant of NMNAT1 in LCA9 patients, located in the ROH of chromosome 1, was detected as a probable disease-causing variant. After detection of the variant in NMNAT1, as a LCA9-causative gene, ophthalmological and neurological re-evaluations were performed. No ophthalmological abnormality was detected and the clinical manifestations of these patients were completely consistent with pure HSP. No NMNAT1 variant had ever been previously reported in HSP patients. However, NMNAT1 variants have been reported in a syndromic form of LCA which is associated with ataxia. In conclusion, our patients expand the clinical spectrum of NMNAT1 variants and represent the first evidence of the probable correlation between NMNAT1 variants and HSP.