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Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin + methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically improved overall survival of OS patients. However, further research is needed to identify new therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme, nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin, doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of NMNAT1−/− cells showed features of both apoptosis and necroptosis, as indicated by the protective effect of the caspase-3 inhibitor z-DEVD-FMK and the necroptosis inhibitor necrostatin-1. Activation of the DNA damage sensor enzyme poly(ADP-ribose) polymerase 1 (PARP1), a major consumer of NAD+ in the nucleus, was fully blocked by NMNAT1 inactivation, leading to increased DNA damage (phospho-H2AX foci). The PARP inhibitor, olaparib, sensitized wild type but not NMNAT1−/− cells to cisplatin-induced anti-clonogenic effects, suggesting that impaired PARP1 activity is important for chemosensitization. Cisplatin-induced cell death of NMNAT1−/− cells was also characterized by a marked drop in cellular ATP levels and impaired mitochondrial respiratory reserve capacity, highlighting the central role of compromised cellular bioenergetics in chemosensitization by NMNAT1 inactivation. Moreover, NMNAT1 cells also displayed markedly higher sensitivity to cisplatin when grown as spheroids in 3D culture. In summary, our work provides the first evidence that NMNAT1 is a promising therapeutic target for osteosarcoma and possibly other tumors as well.

Leber congenital amaurosis type nine is an autosomal recessive retinopathy caused by mutations of the NAD + synthesis enzyme NMNAT1. Despite the ubiquitous expression of NMNAT1, patients do not manifest pathologies other than retinal degeneration. Here we demonstrate that widespread NMNAT1 depletion in adult mice mirrors the human pathology, with selective loss of photoreceptors highlighting the exquisite vulnerability of these cells to NMNAT1 loss. Conditional deletion demonstrates that NMNAT1 is required within the photoreceptor. Mechanistically, loss of NMNAT1 activates the NADase SARM1, the central executioner of axon degeneration, to trigger photoreceptor death and vision loss. Hence, the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, highlighting an unexpected shared mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway and identifies SARM1 as a therapeutic candidate for retinopathies.

Nicotinamide plays a critical role in the prevention and treatment of tumors, and its metabolism is closely associated with tumor progression. The aim of this study was to understand the prognostic and immunological significance of nicotinamide metabolism-related genes in pan-cancer. We downloaded The Cancer Genome Atlas and Genotype Tissue Expression pan-cancer datasets for NMNAT1 from the UCSC database. We analyzed the differential expression, prognosis, genetic alterations, DNA methylation, immune infiltration, and co-expression with RNA modification-related genes and immune checkpoint-related genes. Genes with expression patterns similar to NMNAT1 were identified using the GEPIA library. The GSCA database was used to investigate the correlation between gene expression and drug sensitivity, as assessed by GDSC and CTRP. The CancerSEA database was employed to examine the association of NMNAT1 expression at the single-cell level across different tumors and its relation to 14 functional states. Immunohistochemistry was performed to assess the clinical significance of NMNAT1 expression. NMNAT1 exhibited differential expression across 25 tumor types, including colorectal cancer (CRC), and its expression was significantly associated with the prognosis of 11 tumors. Furthermore, NMNAT1 expression correlated significantly with clinicopathological features. NMNAT1 was strongly associated with immune cells, RNA modification-related genes, and immune checkpoint-related genes in most tumors, affecting immune responses. The expression of NMNAT1 also correlated with sensitivity and resistance to several drugs. Single-cell analysis revealed that NMNAT1 is involved in the progression of retinoblastoma, uveal melanoma, and CRC. Immunohistochemical analysis confirmed that NMNAT1 expression is an independent prognostic factor in patients with CRC. NMNAT1 is a crucial prognostic and immune marker gene for nicotinamide metabolism, particularly in CRC. It has potential as a clinical biomarker and a therapeutic target for cancer treatment.

The molecular mechanisms of blood–brain barrier (BBB) disruption in the early stage after ischemic stroke are poorly understood. In the present study, we investigated the potential role of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) in ischemia-induced BBB damage using an animal middle cerebral artery occlusion (MCAO) model of ischemic stroke. Recombinant human NMNAT1 (rh-NMNAT1) was administered intranasally and Sirtuin 1 (SIRT1) siRNA was administered by intracerebroventricular injection. Our results indicate that rh-NMNAT1 reduced infarct volume, improved functional outcome, and decreased BBB permeability in mice after ischemic stroke. Furthermore, rh-NMNAT1 prevented the loss of tight junction proteins (occludin and claudin-5) and reduced cell apoptosis in ischemic microvessels. NMNAT1-mediated BBB permeability was correlated with the elevation of nicotinamide adenine dinucleotide (NAD + )/NADH ratio and SIRT1 level in brain microvascular endothelial cells. In addition, rh-NMNAT1 treatment significantly decreased the levels of acetylated nuclear factor-κB, acetylated p53, and matrix metalloproteinase-9 in ischemic microvessels. Moreover, the protective effects of rh-NMNAT1 could be reversed by SIRT1 siRNA. In conclusion, these findings indicate that rh-NMNAT1 protects BBB integrity after cerebral ischemia via the NAD + /SIRT1 signaling pathway in brain microvascular endothelial cells. NMNAT1 may be a novel potential therapeutic target for reducing BBB disruption after ischemic stroke.

With an aging population, the incidence of age‐related hearing loss (ARHL) continues to increase. Aging cells exhibit reduced nicotinamide adenine dinucleotide (NAD+) levels and impaired autophagy; however, the mechanisms underlying these processes remain largely unclear. In our study, we assessed the role of nicotinamide nucleotide adenylate transferase 1 (NMNAT1) in cochlear hair cell aging using D‐galactose (D‐gal)‐induced aging HEI‐OC1 cells and cochlear explants. We observed a significant reduction in NMNAT1 expression in HEI‐OC1 cells and cochlear hair cells treated with D‐gal. Notably, NMNAT1 overexpression activated autophagy and decelerated hair cell aging. Metabolomic analysis revealed a dysregulated tricarboxylic acid cycle in Nmnat1‐knockout cells, indicating that NMNAT1 regulates autophagy and metabolic pathways that affect hair cell aging. These findings offer novel insights into the association between autophagy and metabolism during aging and highlight NMNAT1 as a potential therapeutic target for the prevention and treatment of ARHL. The study presents mechanistic insights into cochlear hair cells aging using cellular and explant models. The assessment into NMNAT1 regulated NAD+ metabolism, autophagy, and aging in ARHL. NMNAT1 overexpression restores autophagic activity and decelerates aging in cochlear hair cells.

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.

Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD + homeostasis for health and function, the specific roles of subcellular NAD + pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD + synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, transcriptomic and immunostaining approaches reveal dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

The homeostasis of NAD+ anabolism is indispensable for maintaining the NAD+ pool. In mammals, the mainly synthetic pathway of NAD+ is the salvage synthesis, a reaction catalyzed by nicotinamide mononucleotide adenylyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNATs) successively, converting nicotinamide (NAM) to nicotinamide mononucleotide (NMN) and NMN to NAD+, respectively. However, the relationship between NAD+ anabolism disturbance and diabetic nephropathy (DN) remains elusive. Here our study found that the disruption of NAD+ anabolism homeostasis caused an elevation in both oxidative stress and fibronectin expression, along with a decrease in Sirt1 and an increase in both NF-κB P65 expression and acetylation, culminating in extracellular matrix deposition and globular fibrosis in DN. More importantly, through constitutively overexpressing NMNAT1 or NAMPT in human mesangial cells, we revealed NAD+ levels altered inversely with NMN levels in the context of DN and, further, their changes affect Sirt1/NF-κB P65, thus playing a crucial role in the pathogenesis of DN. Accordingly, FK866, a NAMPT inhibitor, and quercetin, a Sirt1 agonist, have favorable effects on the maintenance of NAD+ homeostasis and renal function in db/db mice. Collectively, our findings suggest that NMN accumulation may provide a causal link between NAD+ anabolism disturbance and diabetic nephropathy (DN) as well as a promising therapeutic target for DN treatment.

This study aims to genetically characterize a two-year-old patient suffering from multiple systemic abnormalities, including skeletal, nervous and developmental involvements and Leber congenital amaurosis (LCA). Genetic screening by next-generation sequencing identified two heterozygous pathogenic variants in nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) as the molecular cause of the disease: c.439+5G>T and c.299+526_*968dup.This splice variant has never been reported to date, whereas pathogenic duplication has recently been associated with cases displaying an autosomal recessive disorder that includes a severe form of spondylo-epiphyseal dysplasia, sensorineural hearing loss, intellectual disability and LCA (SHILCA), as well as some brain anomalies. Our patient presented clinical manifestations which correlated strongly with this reported syndrome. To further study the possible transcriptional alterations resulting from these mutations, mRNA expression assays were performed in the patient and her father. The obtained results detected aberrant alternative transcripts and unbalanced levels of expression, consistent with severe systemic involvement. Moreover, these analyses also detected a novel NMNAT1 isoform, which is variably expressed in healthy human tissues. Altogether, these findings represent new evidence of the correlation of NMNAT1 and SHILCA syndrome, and provide additional insights into the healthy and pathogenic expression of this gene.

Key Clinical Message The discovery of compound heterozygous NMNAT1 mutations (c.245T>C; p.Val82Ala and c.575A>G; p.Asp192Gly) provides a genetic explanation for Leber congenital amaurosis 9 in an Iranian patient. The proband's symptoms—including severe visual impairment, nystagmus, night blindness, and retinal degeneration—align with Leber congenital amaurosis 9 clinical features. This case underscores the value of exome‐sequencing in diagnosing rare genetic disorders and highlights its role in guiding personalized genetic counseling and potential treatments. Leber congenital amaurosis is a severe early‐onset inherited retinal dystrophy. This study delves into the genetic basis of Leber congenital amaurosis, pinpointing compound heterozygous mutations in the NMNAT1 gene as significant causative factors. While one mutation validates previous findings (c.245T>C; p.Val82Ala), the second (c.575A>G; p.Asp192Gly) proves novel, expanding the genetic landscape of Leber congenital amaurosis 9. Both mutations, inherited independently from nonconsanguineous parents, contribute to the intricate genetic basis of light on Leber congenital amaurosis 9 in this case. The identified mutations shed light on Leber congenital amaurosis genetics in the Iranian population, showcasing the efficacy of exome‐sequencing for molecular diagnoses in hereditary retinal degeneration. These findings provide valuable insights for tailored genetic counseling and potential therapeutic interventions.