NMN supplementation extended lifespan of Ndufs4-KO mice

We observed a significant decline in NAD+ levels and lowered NAD+/NADH ratio in the brain of the Ndufs4-KO mice compared to that of age-matched wildtype (WT) mice (Fig. 1A,B, Supplementary Fig. 1). Similar to what was observed in the heart18,19, the NAD+ redox imbalance was associated with protein hyperacetylation in Ndufs4-KO brain (Fig. 1C). To counteract the decline of NAD+ levels in Ndufs4-KO mice, we sought to increase NAD+ pool by targeting the NAD+ salvage pathway (Supplementary Fig. 2A). We delivered NAD+ precursor, nicotinamide mononucleotide (NMN), or vehicle via intraperitoneal injection, to Ndufs4-KO mice starting from postnatal day 21 (P-21) throughout their lifetime (Supplementary Fig. 2B). NMN treatment extended the survival of Ndufs4-KO mice by approximately 2-fold (Fig. 1D; median lifespan 110 days vs 60 days). However, the benefits of NMN appeared to be partial, as the growth of body weight of Ndufs4-KO mice remained stunted after NMN treatment (Fig. 1E).

Figure 1 Targeting altered NAD+ metabolism of Ndufs4-KO mice to extend lifespan. Brain tissues of age-matched (P-65 to -75) WT and Ndufs4-KO mice were collected. (A) NAD+ levels (B) NAD+/NADH ratio and (C) protein acetylation (LysAc) levels were measured. SDHA was used as loading control. N = 4–5. Unpaired 2-tailed t-tests were used. (D) Survival curves of WT and Ndufs4-KO mice with indicated treatments. N = 10–12. (E) Body weight plot of indicated mice. *P < 0.05 versus WT or WT-VEH. #P < 0.05 versus KO-VEH. Log-rank test was used. Full blot images are presented in Supplementary Fig. 7. Full size image

Interventions targeting the NAD+ salvage pathway failed to impact brain NAD+ level of Ndufs4-KO mice

Despite prolonged survival, NMN did not elevate NAD+ levels or NAD+/NADH ratio in the brain of Ndufs4-KO mice harvested at P-50 (Fig. 2A,B, Supplementary Fig. 2C). Levels of NADH and protein acetylation in Ndufs4-KO brains remained high after NMN treatment (Fig. 2C,D). Similarly, protein acetylation levels in heart and liver of Ndufs4-KO mice remained high after NMN treatment (Supplementary Fig. 3A,B). NMN slightly elevated NAD+ levels and NAD+/NADH ratio in Ndufs4-KO hearts but had no effect in Ndufs4-KO liver (Supplementary Fig. 3C,D). Lactate levels in the brain tissue of Ndufs4-KO mice were elevated and not affected by NMN treatment (Fig. 2E). These data suggested that this regimen did not provide metabolic benefits to Ndufs4-KO brain but may have partial benefits to peripheral tissues. We speculated that this treatment regimen (Supplementary Fig. 2B) did not sufficiently deliver NMN to the brain. To test this hypothesis, we increased NMN dosing frequency to daily injections for seven consecutive days in WT mice (Supplementary Fig. 3E). Although this regimen increased NAD+ levels in the heart and liver, it still failed to increase brain NAD+ levels (Supplementary Fig. 3F). Using 1H NMR spectroscopy22 as an alternative method to measure NAD+ levels, we still did not observe elevations of NAD+ levels in the brain (Supplementary Fig. 3G).

Figure 2 NMN or P7C3 failed to impact NAD+ metabolism in Ndufs4-KO brain. Brain tissues of indicated mice were collected at P-50. (A) NAD+ levels, (B) NAD+/NADH ratio (C) NADH levels of brain from mice as indicated were measured. (D) Protein acetylation levels and (E) lactate levels in brain from mice as indicated were quantified. N = 5. SDHA was used as loading control. *P < 0.05 versus WT-VEH. One-way ANOVA with Newman-Keuls multiple comparison test was used. Full blot images are presented in Supplementary Fig. 7. WT or Ndufs4-KO mice were treated with P7C3 daily starting from P-21. (F) Survival curves of WT and Ndufs4-KO mice treated with VEH or P7C3 were plotted. Log-rank test was used. NAD+ pools (NAD+ plus NADH levels) of brain tissues from (G) WT or (H) Ndufs4-KO mice after P7C3 treatment were measured. N = 3. P < 0.05 versus VEH; #P < 0.05 versus KO-VEH. Unpaired 2-tailed t-tests were used. (I) Table summarizing median lifespan and clasping occurrence of Ndufs4-KO mice with vehicle or P7C3 treatment. N = 10. #P < 0.05 versus KO-VEH. NS: not statistically significant versus KO-VEH. Full size image

We next sought to stimulate the NAD+ salvage pathway by activating its key enzyme, nicotinamide phosphoribosyltransferase (NAMPT), using P7C323 (Supplementary Fig. 2A). P7C3 has been shown to be a proneurogenic agent that protects mice from traumatic brain injury24. Daily delivery of P7C3 to Ndufs4-KO mice from P-21 led to a moderate lifespan extension (Fig. 2F, Supplementary Fig. 3H), which was less prominent compared to NMN treatment (median lifespan: 80 vs 110 days). Although P7C3 moderately increased NAD+ levels in the brain of WT mice, it failed to do so in the brain of Ndufs4-KO mice (Fig. 2G,H). The inability to elevate NAD+ by P7C3 was not due to the lack of NAMPT protein in Ndufs4-KO mice, as NAMPT protein levels did not change (Supplementary Fig. 3I). We also assessed the progression of neurodegeneration using the incidence of forelimb clasping as a neuro-behavior marker25. Ndufs4-KO mice showed early clasping phenotype with median occurrence around 46 days as previously observed25. P7C3 treatment did not delay the median clasping occurrence in Ndufs4-KO mice (Fig. 2I). These data collectively suggest that it is particularly challenging to boost the NAD+ levels in the brain of Ndufs4-KO mice. This prevented us from addressing the efficacy of increasing brain NAD+ level as a therapy for LS.

NMN attenuated NAD+ redox imbalance, protein hyperacetylation and suppressed lactate levels of Ndufs4-KO skeletal muscle

We next examined the non-neurological impacts of NMN supplementation on Ndufs4-KO mice to account for the lifespan extension (Fig. 1D). As in LS patients, serum lactate levels were elevated in Ndufs4-KO mice compared to WT mice (Fig. 3A). NMN treatment suppressed serum lactic acidosis in Ndufs4-KO mice (Fig. 3A), suggesting an improvement of systemic metabolism by the treatment. NMN suppressed lactate levels in skeletal and cardiac muscles of Ndufs4-KO mice (Fig. 3B, Supplementary Fig. 4A). In parallel to the attenuated lactic acidosis, the NAD+ pool was expanded in the skeletal muscle and the heart (Fig. 3C and Supplementary Fig. 3C). NAD+ redox balance in skeletal muscle and heart of Ndufs4-KO mice was partially restored by NMN despite a higher level of NADH (Fig. 3C–E, Supplementary Fig. 3C). Normalization of tissue NAD+/NADH ratio would reduce the conversion of pyruvate to lactate by lactate dehydrogenase (LDHA), contributing to the lowered tissue and serum lactate levels (Fig. 3B). Attenuated NAD+ redox imbalance by NMN lowered protein hyperacetylation in skeletal muscle of Ndufs4-KO mice (Fig. 3F). NMN treatment also improved cardiac function, as measured by murine echocardiography, in Ndufs4-KO mice (Supplementary Fig. 4B). These results suggest that the lifespan extension of Ndufs4-KO mice by NMN is attributable to improved metabolism and function in peripheral tissues, independent of the brain.

Figure 3 NMN supplementation attenuated NAD+ redox imbalance and protein hyperacetylation, and suppressed lactate levels in skeletal muscle of Ndufs4-KO mice. (A,B) Serum and skeletal muscle of indicated mice were collected at P-50 and lactate levels were measured. (C) NAD+ levels, (D) NAD+/NADH ratio and (E) NADH levels of skeletal muscle from mice as indicated were measured. (F) Protein acetylation levels of skeletal muscle were measured by Western blot. N = 5. SDHA was used as loading control. Full blot images are presented in Supplementary Fig. 7. *P < 0.05 versus WT-VEH; #P < 0.05 versus KO- VEH. One-way ANOVA with Newman-Keuls multiple comparison test was used. Full size image

NMN blunted activation of hypoxic signaling in Ndufs4-KO mice

Defective oxidative metabolism in mitochondrial disease promotes anaerobic glycolysis that produces lactate from pyruvate while regenerating NAD+ from NADH. Sustained glycolysis eventually lowers NAD+/NADH ratio. The resultant lactic acidosis and abnormal NAD+ redox state are markers in LS and linked to cardiovascular risk and neurodegeneration26. We found that hypoxia inducible factor 1 alpha (HIF1a), an important transcriptional activator of glycolysis, was elevated in skeletal muscle and brain of Ndufs4-KO mice (Fig. 4A, Supplementary Fig. 5A). The HIF1a downstream protein targets, such as LDHA, were also increased in Ndufs4-KO skeletal muscle and brain (Fig. 4B, Supplementary Fig. 5A). HIF1a can be stabilized by ROS or protein acetylation27,28,29. Although protein acetylation levels of Ndufs4-KO skeletal muscle and brain were elevated (Figs 2D and 3F), we did not detect changes in the acetylation level of HIF1a protein (Fig. 4C, Supplementary Fig. 5B). Instead, hyperacetylation of a key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), was found in the brain of Ndufs4-KO mice (Supplementary Fig. 5C). Acetylation of SOD2 has been shown to inhibit its ROS scavenging activity30. To determine whether a higher level of ROS contributed to HIF1a accumulation in Ndufs4-KO mice, protein nitrotyrosine (NT) levels, a marker of oxidative stress, were measured. Protein nitrotyrosine levels were elevated in Ndufs4-KO brain (Supplementary Fig. 5D), consistent with a prior report of increased ROS accumulation in the brain of Ndufs4-KO mice31. These results collectively suggest that ROS-induced HIF1a accumulation is a likely cause of increased glycolysis in Ndufs4-KO brain.

Figure 4 NMN blunted the activation of hypoxia signaling in Ndufs4-KO muscle via up-regulation of α-ketoglutarate (KG) levels. Protein levels of (A) HIF1a and (B) LDHA of skeletal muscle from mice as indicated were measured by Western blot. SDHA was used as loading control. (C) Acetylation levels of HIF1a and (D) KG levels from skeletal muscle of mice as indicated were quantified. (E) Glutamate dehydrogenase (GDH) catalytic reaction. (F) Acetylation levels of GDH in skeletal muscle from mice treated as indicated were measured by Western blot analysis. N = 4–5. *P < 0.05 versus WT-VEH; #P < 0.05 versus KO-VEH. One-way ANOVA with Newman-Keuls multiple comparison test was used. Full blot images are presented in Supplementary Fig. 7. Full size image

NMN supplementation lowered levels of HIF1a and LDHA proteins in the skeletal muscle but not in the brain of Ndufs4-KO mice (Fig. 4A,B, Supplementary Fig. 5A). This finding is coincident with the expanded NAD+ pool and the lowered lactate levels in skeletal muscle of Ndufs4-KO mice by NMN treatment, but not in the brain (Figs 2–3). These results collectively suggest that hypoxic signaling and glycolysis in Ndufs4-KO mice could be manipulated via NAD+-sensitive mechanisms. We found that alpha-ketoglutarate (KG) levels were elevated in Ndufs4-KO muscle after NMN treatment (Fig. 4D). As KG is a required co-substrate for hydroxylation of HIF1a for degradation, increased KG promotes HIF1a reduction. KG can be produced from glutamate via glutamate dehydrogenase (GDH) reaction, which is coupled with conversion of NAD+ to NADH in the direction of KG production (Fig. 4E). Since NMN elevated NAD+/NADH ratio in Ndufs4-KO skeletal muscle (Fig. 3D), KG production was favored. Furthermore, hyperacetylation of GDH causes a loss of catalytic activity due to conformational change32,33. NMN treatment lowered GDH hyperacetylation in Ndufs4-KO skeletal muscle but not in brain (Fig. 4F, Supplementary Fig. 5E). These data suggest that increasing NAD+ level enhances KG production (Fig. 6), which in turn attenuates hypoxic signaling in Ndufs4-KO mice.

DMKG supplementation increased lifespan, improved neurological phenotype and suppressed hypoxic signaling of Ndufs4-KO mice

We next sought to directly increase KG level in the brain of Ndufs4-KO mice using dimethyl-ketoglutarate (DMKG), a cell permeable form of KG. DMKG was administered to Ndufs4-KO mice intraperitoneally, daily starting from P-21 (Supplementary Fig. 6A). We observed a significant lifespan extension in DMKG-treated Ndufs4-KO mice compared to vehicle-treated Ndufs4-KO mice (Fig. 5A,B). The median lifespan of DMKG-treated Ndufs4-KO mice was similar to NMN-treated mice (100 days in DMKG-treated Ndufs4-KO mice versus 110 days in NMN-treated group).

Figure 5 Supplementation of dimethyl α-ketoglutarate (DMKG) extended lifespan and delays the onset of clasping in Ndufs4-KO mice. (A) Survival curves of WT mice, KO mice treated with vehicle (VEH), NMN or DMKG. N = 10–16. (B) Table summarizing median lifespan and clasping occurrence of Ndufs4-KO mice with vehicle, NMN or DMKG treatments. N = 10–16. Log-rank test was used. (C) Levels of SOD2 protein, SOD2 acetylation, H2Ax phosphorylation (H2Ax-Pi), and protein PAR in brain tissues in DMKG treatment cohort at P-50 were quantified. N = 3–6. Protein levels of (D) HIF1a and (E) LDHA in brain tissues were measured by Western blots. N = 3. Full blot images are presented in Supplementary Fig. 7. *P < 0.05 versus WT-VEH; #P < 0.05 versus KO-VEH. NS: not statistically significant versus KO-VEH. One-way ANOVA with Newman-Keuls multiple comparison test was used. SDHA and actin were used as loading control. Full size image

Using positive forelimb clasping as a readout25, we assessed and compared the effects of NMN and DMKG on the neurological system of Ndufs4-KO mice. It has been shown that neurological symptoms in Ndufs4-KO mice manifested around P-35, which coincided with the body weight peak of the mice (Supplementary Figs 1E and 6B) as previously shown20,25. Survivorship of Ndufs4-KO mice declined after P-35 as the incidence of clasping progressively increased (Fig. 5A, Supplementary Fig. 6B). NMN treatment did not change the incidence of clasping of Ndufs4-KO mice (median clasping occurrence of P-45 versus P-46 in vehicle, Fig. 5B, Supplementary Fig. 6B) as the brain NAD+ pool was not expanded. In contrast, DMKG treatment delayed incidence of clasping in Ndufs4-KO mice with a median clasping occurrence at P-68 (Fig. 5B, Supplementary Fig. 6B). Consistent with the unchanged NAD+ levels in brain of Ndufs4-KO mice (Fig. 2A), NMN treatment did not alter hyperacetylation of SOD2 in Ndufs4-KO brain, and DNA damage, as evidenced by elevated histone 2 A phosphorylation (H2Ax-Pi) remained high (Supplementary Figs 5 and 6C). Although NMN lowered acetylation levels of SOD2 in Ndufs4-KO muscle, protein nitrotyrosine levels (NT) were not altered in Ndufs4-KO muscle or by NMN treatment (Supplementary Fig. 6D). These data suggested that oxidative stress contributed to the impairments of Ndufs4-KO brain, but not in skeletal muscle, and the benefits of NMN treatment were not mediated by relieving oxidative stress. DMKG treatment did not alter SOD2 acetylation and histone 2A phosphorylation in Ndufs4-KO brain (Fig. 5C). However, DMKG elevated protein poly-ADP-ribosylation levels in Ndufs4-KO brain (Fig. 5C, Supplementary Fig. 6E). Importantly, DMKG treatment suppressed hypoxia signaling by lowering HIF1a and LDHA levels in Ndufs4-KO brain without affecting the NAD+ pool of Ndufs4-KO brain (Fig. 5D,E, Supplementary Fig. 6F). These results suggest that DMKG is a viable option to suppress hypoxia signaling independent of the NAD+ salvage pathway in mitochondrial disease.