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SLC25A51 is a mitochondrial protein that affects cellular NAD⁺ distribution, proliferation, and metabolome profiles a, b, qPCR quantification of SLC25A51 mRNA expression in HEK 293T (n = 3) (a) and HeLa cells (n = 3) (b) expressing shRNA targeting SLC25A51. c, d, NAD⁺ content of isolated mitochondria (n = 3) (c) and whole cell lysates (n = 3) (d) from HeLa cells with stable shRNA knockdown of SLC25A51 (KD) and non-targeting control (Ctrl). e, Western blot confirming shRNA targeting murine Slc25a51 reduces SLC25A51 protein expression in cells transfected with cDNA encoding SLC25A51-FLAG. f, Mitochondrial free NAD⁺ levels in mouse embryonic stem cells expressing shRNA against Slc25a51 and non-targeting shRNA (shFF2), as measured with the mitochondrial cpVenus NAD⁺ biosensor (n = 3). g, qPCR quantification of SLC25A51 mRNA expression in HeLa cells transfected with siRNA targeting SLC25A51 (n = 3). h, Western blot confirming protein expression of Flag-tagged mitochondrial carriers. Controls include stable expression of the NAD⁺ biosensor (sensor) and anti-Tubulin for loading. i, Immunofluorescent detection of SLC25A51 and SLC25A52 subcellular localization. Cells were transiently transfected with cDNA encoding Flag-HA-tagged SLC25A51 or SLC25A52 and probed with anti-Flag and the mitochondrial marker, anti-MTC02. Scale bar: 10 μM, 2 μM on inset. Inset represents zoomed view of Flag localization and mitochondria. j–l, Proliferation of HAP1 SLC25A51-KO (n = 8) (j), HEK 293T SLC25A51 shRNA-knockdown (n = 8) (k), HeLa SLC25A51 shRNA-knockdown cells (n = 8) (l) and their respective controls. Proliferation was measured by CyQuant, a fluorescent DNA dye, at 0h and 96h after plating and expressed as fold change. m–o, qPCR quantification of SLC25A52 mRNA expression in HAP1 SLC25A51-KO (m), HEK 293T SLC25A51 shRNA-knockdown (n) and HeLa SLC25A51 shRNA-knockdown cells (n = 3) (o). p, Western blot of whole cell protein lysates from HAP1 wild type (WT) and SLC25A51 knockout (KO) cells confirming SLC25A51 loss. Loading control is total protein measured by Revert 700 Total Protein. q, r, Heat map of top 30 mitochondrial (q) and whole cell metabolites (r) that differ between HAP1 wild type and SLC25A51-KO cells (n = 3). Data represented as mean ± SEM. P values were determined by unpaired, two-tailed Student’s t-test (for two groups) or one-way ANOVA with multiple comparisons analysis using Dunnett’s method (for groups of three or more). *P < 0.05, and ***P < 0.001 vs control or WT (exact P values are provided in the source data). Source data
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Mitochondria require nicotinamide adenine dinucleotide (NAD+) in order to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants but their very existence is controversial in mammals. Here we demonstrate that mammalian mitochondria a...
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... Distinct NAD+ pools exist in the cytosol, mitochondria, and nucleus, each serving specific functions [19]. The identification of separate transporters for NAD+ (SLC25A51) and NMN (Slc25a45) in mitochondria suggests a fascinating level of compartmentalized regulation within cells [43,159]. This implies that cells can precisely control the influx of these molecules based on specific needs, potentially offering a new avenue for understanding and influencing cellular health. ...
Recent years have seen a surge in research focused on NAD+ decline and potential interventions, and despite significant progress, new discoveries continue to highlight the complexity of NAD+ biology. Nicotinamide mononucleotide (NMN), a well-established NAD+ precursor, has garnered considerable interest due to its capacity to elevate NAD+ levels and induce promising health benefits in preclinical models. Clinical trials investigating NMN supplementation have yielded variable outcomes while shedding light on the intricacies of NMN metabolism and revealing the critical roles played by gut microbiota and specific cellular uptake pathways. Individual variability in factors such as lifestyle, health conditions, genetics, and gut microbiome composition likely contributes to the observed discrepancies in clinical trial results. Preliminary evidence suggests that NMN’s effects may be context-dependent, varying based on a person’s physiological state. Understanding these nuances is critical for definitively assessing the impact of manipulating NAD+ levels through NMN supplementation. Here, we review NMN metabolism, focusing on current knowledge, pinpointing key areas where further research is needed, and outlining future directions to advance our understanding of its potential clinical significance.
... Enterocytes (SI + ) were further annotated as 4 distinct populations with smallest cluster, IFN-enterocytes, being enriched in interferon-associated gene signatures (ISG15, RSAD2, IFIT1). The remaining enterocyte clusters were divided based on the expression of solute carrier family members, where enterocyte 1 exhibited highest expression of transporters SLC22A4 and SLC43A2, enterocyte 2 had high expression of SLC40A1, which has a role in iron export, and enterocyte 3 was defined by expression of mitochondrial transporter SLC25A5 and anion transporter SLC25A6 ( Figure 5B) (59,60). A small population of cells enriched in expression of genes associated with tissue structure and integrity (ACTA2, THY1, DCN, COL1A1) were classified as stromal cells (61). ...
Chronic inflammatory enteropathy (CIE) is a common condition in dogs causing recurrent or persistent gastrointestinal clinical signs. Pathogenesis is thought to involve intestinal mucosal inflammatory infiltrates, but histopathological evaluation of intestinal biopsies from dogs with CIE fails to guide treatment, inform prognosis, or correlate with clinical remission. We employed single-cell RNA sequencing to catalog and compare the diversity of cells present in duodenal mucosal endoscopic biopsies from 3 healthy dogs and 4 dogs with CIE. Through characterization of 35,668 cells, we identified 31 transcriptomically distinct cell populations, including T cells, epithelial cells, and myeloid cells. Both healthy and CIE samples contributed to each cell population. T cells were broadly subdivided into GZMAhigh (putatively annotated as tissue resident) and IL7Rhigh (putatively annotated as non-resident) T cell categories, with evidence of a skewed proportion favoring an increase in the relative proportion of IL7Rhigh T cells in CIE dogs. Among the myeloid cells, neutrophils from CIE samples exhibited inflammatory (SOD2 and IL1A) gene expression signatures. Numerous differentially expressed genes were identified in epithelial cells, with gene set enrichment analysis suggesting enterocytes from CIE dogs may be undergoing stress responses and have altered metabolic properties. Overall, this work reveals the previously unappreciated cellular heterogeneity in canine duodenal mucosa and provides new insights into molecular mechanisms which may contribute to intestinal dysfunction in CIE. The cell type gene signatures developed through this study may also be used to better understand the subtleties of canine intestinal physiology in health and disease.
... The protein sequence of F12E12.11 was blasted using BLASTP (NCBI), and the best match in H. sapiens was found to be estradiol 17-beta-dehydrogenase 8 (HSD17B8). HSD17B8 is an NAD-dependent dehydrogenase, and the cytosolic NAD + is converted from nicotinamide mononucleotide [20][21][22] . NMN was also administered to daf-18(−) mutants, and we found that 1 mM NMN significantly suppressed Q cell proliferation in daf-18-deficient L1-arrested worms (Fig. 2d). ...
Loss of the tumor suppressor PTEN homolog daf-18 in Caenorhabditis elegans (C. elegans) triggers diapause cell division during L1 arrest. While prior studies have delved into established pathways, our investigation takes an innovative route. Through forward genetic screening in C. elegans, we pinpoint a new player, F12E12.11, regulated by daf-18, impacting cell proliferation independently of PTEN's typical phosphatase activity. F12E12.11 is an ortholog of human estradiol 17-beta-dehydrogenase 8 (HSD17B8), which converts estradiol to estrone through its NAD-dependent 17-beta-hydroxysteroid dehydrogenase activity. We found that PTEN engages in a physical interplay with HSD17B8, introducing a distinctive suppression mechanism. The reduction in estrone levels and accumulation of estradiol may arrest tumor cells in the G2/M phase of the cell cycle through MAPK/ERK. Our study illuminates an unconventional protein interplay, providing insights into how PTEN modulates tumor suppression by restraining cell division through intricate molecular interactions.
... We observed that endogenous and stably expressed FLAG-tagged SLC25A38 had a half-life of ~4 hrs (Fig. 1c), which is significantly shorter than the median half-life of mitochondrial carriers in mammalian cells of several days (Fig. 1b). Conversely, SLC25A51, a mitochondrial NAD + transporter [16][17][18] , which showed similar protein-to-mRNA abundance as the general proteome exhibited a half-life of >8 hrs (Fig. 1c). This highlights that the turnover of SLC25A38 under basal conditions is specific to this transporter and not due to general destabilization of mitochondrial transporters by CHX. ...
Mitochondrial transporters facilitate the exchange of diverse metabolic intermediates across the inner mitochondrial membrane to ensure adequate amounts of substrates and cofactors to support the redox and biosynthetic reactions in the mitochondrial matrix. However, how the abundance of mitochondrial transporters is regulated and the mechanisms controlling mitochondrial metabolite concentrations remain poorly defined. Using protein half-life data and mRNA-protein correlation analysis, we identify SLC25A38, a mitochondrial glycine transporter, as a short-lived protein, whose mitochondrial pool is turned over with a half-life of 4 hours under steady-state conditions. Pharmacological inhibition and genetic depletion of different cellular proteolytic systems revealed that proteasomal and lysosomal systems do not affect the stability of SLC25A38. Instead, we found that the mitochondrial iAAA-protease YME1L1 regulates the abundance and turnover of SLC25A38 under steady-state conditions. Identification of YME1L1 as the molecular player that regulates SLC25A38 levels will open up avenues to explore how cells coordinate mitochondrial substrate uptake with heme synthesis and other glycine-dependent mitochondrial metabolic pathways under physiological state or in response to environmental cues.
... OXPHOS drop can be explained by the role of mitochondrial shuttles in the transfer of reducing equivalents across the inner mitochondrial membrane. This mechanism complements the function of SLC25A51 and SLC25A52 carriers to transport NAD + into the mitochondria to support complex I activity [76][77][78]. In keeping with this notion, a decreased expression of SLC25A1 mRNA [79] or inhibition of its function by CTPI-2 drives mitochondrial dysfunction and OCR drop [80]. ...
Monocyte-derived dendritic cells (MDDCs) are key players in the defense against fungal infection because of their outstanding capacity for non-opsonic phagocytosis and phenotypic plasticity. Accordingly, MDDCs rewire metabolism to meet the energetic demands for microbial killing and biomass synthesis required to restore homeostasis. It has been commonplace considering the metabolic reprogramming a mimicry of the Warburg effect observed in tumor cells. However, this may be an oversimplification since the offshoots of glycolysis and the tricarboxylic acid (TCA) cycle are connected in central carbon metabolism. Zymosan, the external wall of Saccharomyces cerevisiae, contains β-glucan and α-mannan chains that engage the C-type lectin receptors dectin-1/2 and Toll-like receptors. This makes it an optimal fungal surrogate for experimental research. Using real-time bioenergetic assays and [U–¹³C]glucose labeling, central hubs connected to cytokine expression were identified. The pentose phosphate pathway (PPP) exhibited a more relevant capacity to yield ribose-5-phosphate than reducing equivalents of NADPH, as judged from the high levels of isotopologues showing ¹³C-labeling in the ribose moiety and the limited contribution of the oxidative arm of the PPP to the production of ROS by NADPH oxidases (NOX). The finding of ¹³C-label in the purine ring and in glutathione unveiled the contribution of serine-derived glycine to purine ring and glutathione synthesis. Serine synthesis also supported the 10.13039/100004915TCA cycle. Zymosan exhausted NAD⁺ and ATP, consistent with intracellular consumption and/or extracellular export. Poly-ADP-ribosylated proteins detected in the nuclear fractions of MDDCs did not show major changes upon zymosan stimulation, which suggests its dependence on constitutive Fe(II)/2-oxoglutarate-dependent demethylation of 5-methylcytosine by TET translocases and/or demethylation of histone H3 lysine 27 by JMJD demethylases rather than on NOX activities. These results disclose a unique pattern of central carbon metabolism following fungal challenge, characterized by the leverage of glycolysis offshoots and an extensive recycling of NAD⁺ and poly(ADP-ribose).
... However, this is opposite to what would be expected biologically, since AA should result in an increase in glycolysis and reduces the pyruvate to lactate ratio (Fig. S8d), a known marker for the cytosolic NAD + / NADH ratio 24 . A possible explanation is that because antimycin A inhibits respiration and complex I is unable to re-oxidize NADH back to NAD + , NAD + is transported into the mitochondria through the NAD + transporter SLC25A51 25 . This would cause a local decrease in NAD(H) pool size in the nucleus, resulting in the longer NADH lifetime upon AA treatment. ...
... Interestingly, the effect of NAD(H) pool size on NADH FLIM does not only come into play in case of systemic alterations in nucleotide levels. Recently, a mammalian mitochondrial NAD + carrier was identified suggesting active redistribution of nucleotides pools between mitochondria and cytosol 25 . Accordingly, it has been shown that the subcellular distribution of NAD(H) changes with cellular differentiation 44 and metabolic state 45 . ...
NADH autofluorescence imaging is a promising approach for visualizing energy metabolism at the single-cell level. However, it is sensitive to the redox ratio and the total NAD(H) amount, which can change independently from each other, for example with aging. Here, we evaluate the potential of fluorescence lifetime imaging microscopy (FLIM) of NADH to differentiate between these modalities.
We perform targeted modifications of the NAD(H) pool size and ratio in cells and mice and assess the impact on NADH FLIM. We show that NADH FLIM is sensitive to NAD(H) pool size, mimicking the effect of redox alterations. However, individual components of the fluorescence lifetime are differently impacted by redox versus pool size changes, allowing us to distinguish both modalities using only FLIM. Our results emphasize NADH FLIM’s potential for evaluating cellular metabolism and relative NAD(H) levels with high spatial resolution, providing a crucial tool for our understanding of aging and metabolism.
... Distinct NAD+ pools exist in the cytosol, mitochondria, and nucleus, each serving specific functions [19]. The identification of separate transporters for NAD+ (SLC25A51) and NMN (Slc25a45) in mitochondria suggests a fascinating level of compartmentalized regulation within cells [43,175]. This implies that cells can precisely control the influx of these molecules based on specific needs, potentially offering a new avenue for understanding and influencing cellular health. ...
Nicotinamide mononucleotide (NMN), a key NAD+ precursor, exhibits distinct metabolic characteristics and potential health benefits. Recent investigations underscore the pivotal role of gut bacteria and specialized uptake mechanisms in NMN metabolism. While human trials demonstrate NMN's safety and promising health outcomes, challenges persist. Variability in results is likely influenced by lifestyle, health conditions, and individual genetic and gut microbiota differences. Understanding how these factors affect NMN metabolism is crucial for personalized approaches and maximizing NMN's potential. Advances in delivery systems show promise for enhancing NMN bioavailability, but further understanding of optimal NAD+ levels and reliable measures of NMN's effects is necessary. This comprehensive approach is key to optimizing NMN's impact on human health and longevity.
... Conversely, knocking out NMNAT1 elevated cellular NMN and activated SARM1, which was reversed by re-expression of NMNATs ( Supplementary Fig. S1e), verifying that the metabolic regulation of SARM1 by the cellular levels of NMN and NAD 21,24,34 is indeed operational in cells. The less effectiveness of NMNAT3 may be due to the low level of mitochondrial transporter of NAD, SLC25A51 in HEK-293T 35 , hampering the transfer of the mitochondrial NAD to the cytosol. ...
Sterile alpha and Toll/interleukin-1 receptor motif containing 1 (SARM1), a nicotinamide adenine dinucleotide (NAD)-utilizing enzyme, mediates axon degeneration (AxD) in various neurodegenerative diseases. It is activated by nicotinamide mononucleotide (NMN) to produce a calcium messenger, cyclic ADP-ribose (cADPR). This activity is blocked by elevated NAD level. Here, we verified this metabolic regulation in somatic HEK-293T cells by overexpressing NMN-adenyltransferase to elevate cellular NAD, which resulted not only in inhibition of their own SARM1 from producing cADPR but, surprisingly, also in the 5–10 neighboring wildtype cells in mixed cultures via connexin (Cx)-43. Direct visualization of gap junction intercellular communication (GJIC) was achieved by incubating cells with a permeant probe, PC11, which is converted by SARM1 into PAD11, a fluorescent NAD analog capable of traversing GJs. Extending the findings to dorsal root ganglion neurons, we further showed that CZ-48, a permeant NMN analog, or axotomy, activated SARM1 and the produced PAD11 was transferred to contacting axons via GJIC. The gap junction involved was identified as Cx36 instead. This neuronal GJIC was demonstrated to be functional, enabling healthy neurons to protect adjacent axotomized axons from degeneration. Inhibition of GJIC in mice by AAV-PHP.eB-mediated knockdown of Cx36 in brain induced neuroinflammation, which in turn activated SARM1 and resulted in axon degeneration as well as behavioral deficits. Our results demonstrate a novel intercellular regulation mechanism of SARM1 and reveal a protective role of healthy tissue against AxD induced by injury or neuroinflammation.
Classifications: BIOLOGICAL SCIENCES -- Neuroscience; Biochemistry; Cell Biology.
... Consistently, neither a reduction in mitochondrial NAD + concentrations nor impairments of glycolysis or citric acid cycle have been detected in skeletal muscle tissues of Nmnat3-KO mice [102]. In keeping with the mitochondrial uptake of cytosolic NAD + , SLC25A51 was recently identified as a mammalian mitochondrial NAD + transporter [103,104]. A notable exception to this notion may be represented by red blood cells, which show a marked dependence on NMNAT3 for NAD + formation [105,106]. ...
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.
... Most mitochondrial transporters belong to the solute carrier family 25 (SLC25), which are typically embedded in the mitochondrial inner membrane and play critical roles in supplying this organelle with essential metabolic intermediates and coenzymes. Recently, three independent research groups discovered that SLC25A51 serves as a mammalian NAD + transporter [107][108][109], revealing how mitochondria are supplied with this key cofactor that fuels most cellular redox reactions. NAD + functions as both an electron acceptor (in its oxidized form) and donor (in its reduced form) [107][108][109] and is an important signaling molecule in numerous cellular processes. ...
... Recently, three independent research groups discovered that SLC25A51 serves as a mammalian NAD + transporter [107][108][109], revealing how mitochondria are supplied with this key cofactor that fuels most cellular redox reactions. NAD + functions as both an electron acceptor (in its oxidized form) and donor (in its reduced form) [107][108][109] and is an important signaling molecule in numerous cellular processes. The identification of SLC25A51/MCART1 as the primary mitochondrial NAD + transporter represents a significant advancement in understanding cellular metabolism. ...
Mitochondrial structure often determines the function of these highly dynamic, multifunctional, eukaryotic organelles, which are essential for maintaining cellular health. The dynamic nature of mitochondria is apparent in descriptions of different mitochondrial shapes [e.g., donuts, megamitochondria (MGs), and nanotunnels] and crista dynamics. This review explores the significance of dynamic alterations in mitochondrial morphology and regulators of mitochondrial and cristae shape. We focus on studies across tissue types and also describe new microscopy techniques for detecting mitochondrial morphologies both in vivo and in vitro that can improve understanding of mitochondrial structure. We highlight the potential therapeutic benefits of regulating mitochondrial morphology and discuss prospective avenues to restore mitochondrial bioenergetics to manage diseases related to mitochondrial dysfunction.
