Cerebral ischemia–reperfusion (I/R) injury is a leading cause of neurological disability and is characterized by mitochondrial dysfunction and oxidative stress. Although depletion of nicotinamide adenine dinucleotide (NAD+) is a hallmark of ischemic injury, therapeutic strategies aimed at NAD+ replenishment have shown limited efficacy. Whether impaired mitochondrial NAD+ import contributes to neuronal vulnerability after I/R remains poorly understood.
Results:
We found that cerebral I/R disrupts the balance of NAD+ distribution between the cytoplasm and mitochondria in the cortex due to upregulated expression of SLC25A51. Augmenting SLC25A51 expression restored mitochondrial NAD+ pools, improved mitochondrial respiratory function, reduced oxidative lipid damage, and attenuated neuronal injury. In contrast, SLC25A51 deficiency exacerbated mitochondrial dysfunction and heightened susceptibility to I/R stress. These effects occurred independently of global NAD+ biosynthesis, indicating that mitochondrial NAD+ transport rather than NAD+ availability per se is a critical determinant of neuronal survival.
Innovation:
This study reveals the subcellular distribution change of NAD+-mediated by SLC25A51 and its neuroprotective effects via modulating mitochondrial function after cerebral I/R injury.
Conclusion:
This study identifies defective mitochondrial NAD+ import as a previously underrecognized mechanism of cerebral I/R injury. By establishing SLC25A51-dependent NAD+ trafficking as a key regulator of mitochondrial redox balance and neuronal resilience, our findings shift the therapeutic paradigm from NAD+ supplementation to restoration of subcellular NAD+ distribution, highlighting mitochondrial NAD+ transport as a promising target for ischemic brain injury. Antioxid. Redox Signal. 00, 000–000.
AdzikaGK, Velázquez AponteRA, BaurJA. Mitochondrial NAD(+) transporter SLC25A51 linked to human aortic disease. Nat Cardiovasc Res2025;4(3):256–258.
2.
AyoubIA, LeeEJ, OgilvyCS, et al.Nicotinamide reduces infarction up to two hours after the onset of permanent focal cerebral ischemia in Wistar rats. Neurosci Lett1999;259(1):21–24.
3.
BaiL, YangZX, MaPF, et al.Overexpression of SLC25A51 promotes hepatocellular carcinoma progression by driving aerobic glycolysis through activation of SIRT5. Free Radic Biol Med2022;182:11–22.
4.
CaoJ, ChenX, ChenL, et al.DHODH-mediated mitochondrial redox homeostasis: A novel ferroptosis regulator and promising therapeutic target. Redox Biol2025;85:103788.
5.
CarbonellT, RamaR. Iron, oxidative stress and early neurological deterioration in ischemic stroke. Curr Med Chem2007;14(8):857–874.
6.
CovarrubiasAJ, PerroneR, GrozioA, et al.NAD (+) metabolism and its roles in cellular processes during ageing. Nat Rev Mol Cell Biol2021;22(2):119–141.
7.
CuiY, ZhangZ, LvM, et al.Chromatin target of protein arginine methyltransferases alleviates cerebral ischemia/reperfusion-induced injury by regulating RNA alternative splicing. iScience2024;27(1):108688.
8.
FiskumG, DanilovCA, MehrabianZ, et al.Postischemic oxidative stress promotes mitochondrial metabolic failure in neurons and astrocytes. Ann N Y Acad Sci2008;1147:129–138.
9.
GaoN, ZhangW, QianY, et al.SERPINE1 drives ferroptosis in acute respiratory distress syndrome by disrupting mitochondrial NAD+ homeostasis and suppressing Sirt3 activity. Redox Biol2026:104146.
10.
GloverHL, SchreinerA, DewsonG, et al.Mitochondria and cell death. Nat Cell Biol2024;26(9):1434–1446.
11.
GuoY, LiangN, ZhangX, et al.Mitochondrial GPX4 acetylation is involved in cadmium-induced renal cell ferroptosis. Redox Biol2024;73:103179.
12.
HeG, ChenZ, LiJ, et al.Analysis of potential key ferroptosis genes in the pathogenesis of ankylosing spondylitis by bioinformatics. Medicine Advances2023;1(3):219–233.
13.
HerpichF, RinconF. Management of acute ischemic stroke. Crit Care Med2020;48(11):1654–1663.
14.
HøylandLE, VanLindenMR, NiereM, et al.Subcellular NAD (+) pools are interconnected and buffered by mitochondrial NAD (+). Nat Metab2024;6(12):2319–2337.
15.
HuX, BaoY, LiM, et al.The role of ferroptosis and its mechanism in ischemic stroke. Exp Neurol2024;372:114630.
16.
HuangQ, SunM, LiM, et al.Combination of NAD (+) and NADPH offers greater neuroprotection in ischemic stroke models by relieving metabolic stress. Mol Neurobiol2018;55(7):6063–6075.
17.
KlaidmanL, MoralesM, KemS, et al.Nicotinamide offers multiple protective mechanisms in stroke as a precursor for NAD+, as a PARP inhibitor and by partial restoration of mitochondrial function. Pharmacology2003;69(3):150–157.
18.
KlimovaN, FearnowA, LongA, et al.NAD (+) precursor modulates post-ischemic mitochondrial fragmentation and reactive oxygen species generation via SIRT3 dependent mechanisms. Exp Neurol2020;325:113144.
19.
KoryN, Uit de BosJ, van der RijtS, et al.MCART1/SLC25A51 is required for mitochondrial NAD transport. Sci Adv2020;6(43):eabe5310.
20.
LautrupS, SinclairDA, MattsonMP, et al.NAD (+) in brain aging and neurodegenerative disorders. Cell Metab2019;30(4):630–655.
21.
LiY, BieJ, ZhaoL, et al.SLC25A51 promotes tumor growth through sustaining mitochondria acetylation homeostasis and proline biogenesis. Cell Death Differ2023;30(8):1916–1930.
22.
LinL, WangX, YuZ. Ischemia-reperfusion Injury in the Brain: Mechanisms and Potential Therapeutic Strategies. Biochem Pharmacol (Los Angel)2016;5(4):213.
23.
LiuL, LiY, CaoD, et al.SIRT3 inhibits gallbladder cancer by induction of AKT-dependent ferroptosis and blockade of epithelial-mesenchymal transition. Cancer Lett2021;510:93–104.
24.
LiuX, XieC, WangY, et al.Ferritinophagy and ferroptosis in cerebral ischemia reperfusion injury. Neurochem Res2024;49(8):1965–1979.
25.
LiuY, LuS, WuLL, et al.The diversified role of mitochondria in ferroptosis in cancer. Cell Death Dis2023a;14(8):519.
26.
LiuY, WanY, JiangY, et al.GPX4: The hub of lipid oxidation, ferroptosis, disease and treatment. Biochim Biophys Acta Rev Cancer2023b;1878(3):188890.
27.
LuMJ, BusquetsJ, ImpedovoV, et al.SLC25A51 decouples the mitochondrial NAD (+)/NADH ratio to control proliferation of AML cells. Cell Metab2024;36(4):808–821.e6.
28.
LuongoTS, EllerJM, LuMJ, et al.SLC25A51 is a mammalian mitochondrial NAD (+) transporter. Nature2020;588(7836):174–179.
29.
LvM, DuanZ, TanJ, et al.PHGDH-mediated serine synthesis in astrocytes supports neuroinflammation by sustaining NADH level to promote histone acetylation. Cell Death Dis2025;16(1):397.
30.
Martínez-ReyesI, ChandelNS. Mitochondrial TCA cycle metabolites control physiology and disease. Nat Commun2020;11(1):102.
31.
MoralesM, XueX. Targeting iron metabolism in cancer therapy. Theranostics2021;11(17):8412–8429.
32.
NarneP, PhanithiPB. Role of NAD (+) and FAD in ischemic stroke pathophysiology: An epigenetic nexus and expanding therapeutic repertoire. Cell Mol Neurobiol2023;43(5):1719–1768.
33.
OhSJ, IkedaM, IdeT, et al.Mitochondrial event as an ultimate step in ferroptosis. Cell Death Discov2022;8(1):414.
34.
OuyangY, BottAJ, RutterJ. Maestro of the SereNADe: SLC25A51 orchestrates mitochondrial NAD. Trends Biochem Sci2021;46(5):348–350.
35.
OwensK, ParkJH, SchuhR, et al.Mitochondrial dysfunction and NAD (+) metabolism alterations in the pathophysiology of acute brain injury. Transl Stroke Res2013;4(6):618–634.
36.
PanconesiR, WidmerJ, CarvalhoMF, et al.Mitochondria and ischemia reperfusion injury. Curr Opin Organ Transplant2022;27(5):434–445.
37.
QinN, LiuR, DengR, et al.Modulation of mitochondrial dysfunction: Mechanisms and strategies for the use of natural products to treat stroke. Neural Regen Res2026;21(6):2215–2226.
38.
QuW, RaltoKM, QinT, et al.NAD (+) precursor nutritional supplements sensitize the brain to future ischemic events. J Cereb Blood Flow Metab2023;43(2_suppl):37–48.
39.
SahuMP, NikkiläO, LågasS, et al.Culturing primary neurons from rat hippocampus and cortex. Neuronal Signal2019;3(2):Ns20180207.
40.
SakakibaraY, MithaAP, OgilvyCS, et al.Post-treatment with nicotinamide (vitamin B (3)) reduces the infarct volume following permanent focal cerebral ischemia in female Sprague-Dawley and Wistar rats. Neurosci Lett2000;281(2–3):111–114.
41.
SchöndorfDC, IvanyukD, BadenP, et al.The NAD+ precursor nicotinamide riboside rescues mitochondrial defects and neuronal loss in iPSC and fly models of Parkinson’s disease. Cell Rep2018;23(10):2976–2988.
42.
SelimMH, RatanRR. The role of iron neurotoxicity in ischemic stroke. Ageing Res Rev2004;3(3):345–353.
43.
ShademanB, AvciCB, KaramadV, et al.The role of mitochondrial biogenesis in ischemic stroke. J Integr Neurosci2023;22(4):88.
44.
StockwellBR, Friedmann AngeliJP, BayirH, et al.Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease. Cell2017;171(2):273–285.
45.
TanQ, ZhangX, LiS, et al.DMT1 differentially regulates mitochondrial complex activities to reduce glutathione loss and mitigate ferroptosis. Free Radic Biol Med2023;207:32–44.
46.
TuW, WangL, Special Writing Group of China Stroke Surveillance Report. China stroke surveillance report 2021. Mil Med Res2023;10(1):33.
47.
TuoQ, LeiP. Ferroptosis in ischemic stroke: Animal models and mechanisms. Zool Res2024;45(6):1235–1248.
48.
TuoQ, LiuY, XiangZ, et al.Thrombin induces ACSL4-dependent ferroptosis during cerebral ischemia/reperfusion. Signal Transduct Target Ther2022;7(1):59.
49.
WangQ, GuoY, ChenS, et al.Histidine triad nucleotide-binding protein 2 attenuates metabolic dysfunction-associated steatotic liver disease through NAD (+)-dependent sirtuin-3 activation. Exp Mol Med2025;57(5):990–1004.
50.
WangX, LY, SunX, et al.A highly efficient siRNA transfection method in primary cultured cortical neurons. Bio Protoc2026;16(2):e5567.
51.
WangX, MaoG, LiQ, et al.Neuroprotection of NAD (+) and NBP against ischemia/reperfusion brain injury is associated with restoration of sirtuin-regulated metabolic homeostasis. Front Pharmacol2023;14:1096533.
52.
XuX, DuanZ, ZhouX, et al.SFXN1 reduction alleviates cerebral ischemia-reperfusion injury by promoting neuronal survival and reducing neuroinflammation. CNS Neurosci Ther2025;31(5):e70457.
53.
YanHF, ZouT, TuoQZ, et al.Ferroptosis: Mechanisms and links with diseases. Signal Transduct Target Ther2021;6(1):49.
54.
YingW. NAD+ and NADH in ischemic brain injury. Front Biosci2008;13:1141–1151.
55.
YuanY, WeiB, ZhuW, et al.Systolic pressure target after endovascular thrombectomy in acute large-vessel occlusion ischemic stroke patients: Comment on ENCHANTED2/MT. Brain-X2023;1(1):e4.
56.
YuanY, ZhangD, WangJ, et al.N4-acetylcytidine modification of LINC02802 promotes non-small cell lung cancer progression by modulating mitochondrial NAD+/NADH ratio. Int J Biol Sci2025;21(11):4908–4926.
57.
ZemirliN, MorelE, MolinoD. Mitochondrial dynamics in basal and stressful conditions. Int J Mol Sci2018;19(2):564.
58.
ZhangJ, TangY, ZhangS, et al.Mitochondrial NAD (+) deficiency in vascular smooth muscle impairs collagen III turnover to trigger thoracic and abdominal aortic aneurysm. Nat Cardiovasc Res2025;4(3):275–292.
59.
ZhangM, LiuQ, MengH, et al.Ischemia-reperfusion injury: Molecular mechanisms and therapeutic targets. Signal Transduct Target Ther2024;9(1):12.
60.
ZhuY, LiuJ, ParkJ, et al.Subcellular compartmentalization of NAD (+) and its role in cancer: A sereNADe of metabolic melodies. Pharmacol Ther2019;200:27–41.
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