NAD+ supplementation rejuvenates aged gut adult stem cells

The tissue decline due to aging is associated with the deterioration of adult stem cell function. Here we show the number and proliferative activity of intestinal stem cells (ISCs) but not Paneth cells decline during aging, as does ISC function assessed ex vivo. Levels of SIRT1 and activity of mTORC1 also decline with aging. The treatment with the NAD(+) precursor nicotinamide riboside (NR) rejuvenates ISCs from aged mice and reverses an impaired ability to repair gut damage. The effect of NR is blocked by the mTORC1 inhibitor rapamycin or the SIRT1 inhibitor EX527. These findings demonstrate that small molecules affecting the NAD/SIRT1/mTORC1 axis may guide a translational path for maintenance of the intestine during aging.     

Augmentation of cellular NAD+ by NQO1 enzymatic action improves age‐related hearing impairment

Age‐related hearing loss (ARHL) is a major neurodegenerative disorder and the leading cause of communication deficit in the elderly population, which remains largely untreated. The development of ARHL is a multifactorial event that includes both intrinsic and extrinsic factors. Recent studies suggest that NAD⁺/NADH ratio may play a critical role in cellular senescence by regulating sirtuins, PARP‐1, and PGC‐1α. Nonetheless, the beneficial effect of direct modulation of cellular NAD⁺ levels on aging and age‐related diseases has not been studied, and the underlying mechanisms remain obscure. Herein, we investigated the effect of β‐lapachone (β‐lap), a known plant‐derived metabolite that modulates cellular NAD⁺ by conversion of NADH to NAD⁺ via the enzymatic action of NADH: quinone oxidoreductase 1 (NQO1) on ARHL in C57BL/6 mice. We elucidated that the reduction of cellular NAD⁺ during the aging process was an important contributor for ARHL; it facilitated oxidative stress and pro‐inflammatory responses in the cochlear tissue through regulating sirtuins that alter various signaling pathways, such as NF‐κB, p53, and IDH2. However, augmentation of NAD⁺ by β‐lap effectively prevented ARHL and accompanying deleterious effects through reducing inflammation and oxidative stress, sustaining mitochondrial function, and promoting mitochondrial biogenesis in rodents. These results suggest that direct regulation of cellular NAD⁺ levels by pharmacological agents may be a tangible therapeutic option for treating various age‐related diseases, including ARHL.     

Degradation of cochlear Connexin26 accelerate the development of age-related hearing loss

The GJB2 gene, encoding Connexin26 (Cx26), is one of the most common causes of inherited deafness. Clinically, mutations in GJB2 cause congenital deafness or late-onset progressive hearing loss. Recently, it has been reported that Cx26 haploid deficiency accelerates the development of age-related hearing loss (ARHL). However, the roles of cochlear Cx26 in the hearing function of aged animals remain unclear. In this study, we revealed that the Cx26 expression was significantly reduced in the cochleae of aged mice, and further explored the underlying molecular mechanism for Cx26 degradation. Immunofluorescence co-localization results showed that Cx26 was internalized and degraded by lysosomes, which might be one of the important ways for Cx26 degradation in the cochlea of aged mice. Currently, whether the degradation of Cx26 in the cochlea leads directly to ARHL, as well as the mechanism of Cx26 degradation-related hearing loss are still unclear. To address these questions, we generated mice with Cx26 knockout in the adult cochlea as a model for the natural degradation of Cx26. Auditory brainstem response (ABR) results showed that Cx26 knockout mice exhibited high-frequency hearing loss, which gradually progressed over time. Pathological examination also revealed the degeneration of hair cells and spiral ganglions, which is similar to the phenotype of ARHL. In summary, our findings suggest that degradation of Cx26 in the cochlea accelerates the occurrence of ARHL, which may be a novel mechanism of ARHL.     

NAD+ and its precursors in human longevity

Aging is a complex issue due to its nature in progressive physiological and functional decay. As better medicine, technology, and living conditions became accessible to many people, the longevity of human beings increased during the past centuries. Recent research established vital roles for NAD⁺ and its precursors in protecting and maintaining the redox homeostasis in cells, which might be applicable therapeutically to prevent cell degeneration. Notably, the contribution of NAD⁺ metabolites to lifespan extension in model systems indicates that the potential beneficial effects of NAD⁺ precursors. In this mini review, by introducing the background of NAD⁺-consuming enzymes in “caloric restriction”, we focus on NAD⁺ and its precursors in diet, with further emphasis on its association with health and diseases. We also provide insights in future utilization of NAD⁺ and its precursors as nutrition supplement for lifespan extension.     

The expression of PHB2 in the cochlea: Possible relation to age-related hearing loss

Age-related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly, but its mechanism remains unclear. Scaffold protein prohibitin 2 (PHB2) has been widely involved in aging and neurodegeneration. However, the role of PHB2 in ARHL is undeciphered to date. To investigate the expression pattern and the role of PHB2 in ARHL, we used C57BL/6 mice and HEI-OC1 cell line as models. In our study, we have found PHB2 exists in the cochlea and is expressed in hair cells, spiral ganglion neurons, and HEI-OC1 cells. In mice with ARHL, mitophagy is reduced and correspondingly the expression level of PHB2 is decreased. Moreover, after H₂O₂ treatment the mitophagy is activated and the PHB2 expression is increased. These findings indicate that PHB2 may exert an important role in ARHL through mitophagy. Findings from this study will be helpful for elucidating the mechanism underlying the ARHL and for providing a new target for ARHL treatment.     

Fingerstick blood assay maps real-world NAD+ disparity across gender and age

Nicotinamide adenine dinucleotide (NAD⁺) level has been associated with various age-related diseases and its pharmacological modulation emerges as a potential approach for aging intervention. But human NAD⁺ landscape exhibits large heterogeneity. The lack of rapid, low-cost assays limits the establishment of whole-blood NAD⁺ baseline and the development of personalized therapies, especially for those with poor responses towards conventional NAD⁺ supplementations. Here, we developed an automated NAD⁺ analyzer for the rapid measurement of NAD⁺ with 5 μL of capillary blood using recombinant bioluminescent sensor protein and automated optical reader. The minimal invasiveness of the assay allowed a frequent and decentralized mapping of real-world NAD⁺ dynamics. We showed that aerobic sport and NMN supplementation increased whole-blood NAD⁺ and that male on average has higher NAD⁺ than female before the age of 50. We further revealed the long-term stability of human NAD⁺ baseline over 100 days and identified major real-world NAD⁺-modulating behaviors.     

Excessive processing and acetylation of OPA1 aggravate age-related hearing loss via the dysregulation of mitochondrial dynamics

The pathogenesis of age-related hearing loss (ARHL) remains unclear. OPA1 is the sole fusion protein currently known to be situated in the inner mitochondrial membrane, which is pivotal for maintaining normal mitochondrial function. While it has already been demonstrated that mutations in OPA1 may lead to hereditary deafness, its involvement in the occurrence and development of ARHL has not been previously explored. In our study, we constructed D-gal-induced senescent HEI-OC1 cells and the cochlea of C57BL/6J mice with a mutated SUMOylation site of SIRT3 using CRISPR/Cas9 technology. We found enhanced L-OPA1 processing mediated by activated OMA1, and increased OPA1 acetylation resulting from reductions in SIRT3 levels in senescent HEI-OC1 cells. Consequently, the fusion function of OPA1 was inhibited, leading to mitochondrial fission and pyroptosis in hair cells, ultimately exacerbating the aging process of hair cells. Our results suggest that the dysregulation of mitochondrial dynamics in cochlear hair cells in aged mice can be ameliorated by activating the SIRT3/OPA1 signaling. This has the potential to alleviate the senescence of cochlear hair cells and reduce hearing loss in mice. Our study highlights the significant roles played by the quantities of long and short chains and the acetylation activity of OPA1 in the occurrence and development of ARHL. This finding offers new perspectives and potential targets for the prevention and treatment of ARHL.     

NAD+ supplementation prevents STING‐induced senescence in ataxia telangiectasia by improving mitophagy

Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A‐T). Loss of mitochondrial function can drive age‐related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence‐associated secretory phenotype (SASP) occur in A‐T patient fibroblasts, and in ATM‐deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD⁺ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1‐dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm^−/− mice. Our findings suggest a central role for mitochondrial dysfunction‐induced senescence in A‐T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.     

Fine mapping of Ahl3 affecting both age-related and noise-induced hearing loss

A region in the vicinity of D17Mit119 on mouse chromosome 17 harbors a susceptibility gene, designated as Ahl3, to age-related hearing loss (AHL). We produced congenic lines of C57BL/6 background that substituted regions around D17Mit119 with MSM-derived ones, and examined auditory brainstem response (ABR) thresholds for their hearing capacity at 6 and 12months of age. Three congenic lines carrying the approximately 14-Mb region between D17Mit274 and D17Mit183 retained normal hearing at 12months of age whereas two congenic lines not carrying this region tended to lose hearing at that age. We also investigated noise-induced hearing loss (NIHL) in congenic lines at 1, 7 and 14days after exposure to the noise of 100dB for 1h. Most congenic mice carrying the 14-Mb region did not exhibit permanent threshold shift (PTS) whereas mice not carrying this region exhibited a strong tendency of PTS, indicating the role of Ahl3 in susceptibility to NIHL. These results indicate that Ahl3 exists within the 14-Mb region and affects not only AHL but also NIHL.     

NAD precursors cycle between host tissues and the gut microbiome

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Effect of vitamin C depletion on age-related hearing loss in SMP30/GNL knockout mice

Using senescence marker protein 30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), we examined whether modulating VC level affects age-related hearing loss (AHL). KO and wild-type (WT) C57BL/6 mice were given water containing 1.5 g/L VC [VC(+)] or 37.5 mg/L VC [VC(−)]. At 10 months of age, KO VC(−) mice showed significant reduction in VC level in the inner ear, plasma, and liver, increase in auditory brainstem response (ABR) thresholds, and decrease in the number of spiral ganglion cells compared to WT VC(−), WT VC(+), and KO VC(+) mice. There were no differences in VC level in the inner ear, ABR thresholds, or the number of spiral ganglion cells among WT VC(−), WT VC(+), and KO VC(+) mice. These findings suggest that VC depletion can accelerate AHL but that supplementing VC may not increase VC level in the inner ear or slow AHL in mice.     

Quantitative Analysis of NAD Synthesis-Breakdown Fluxes

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Ahl3, a third locus on mouse chromosome 17 affecting age-related hearing loss

Genetic variation in humans probably plays a role in determining the range of individual susceptibility to age-related hearing loss (AHL), but no contributing loci have been identified because of the difficulties of dissecting complex traits in humans. This paper reports mapping of an AHL locus using a panel of consomic mice between C57BL/6J (B6) and MSM strains, which covered more than a half of chromosome sets. B6 strain exhibited AHL beginning at 10 months of age whereas MSM strain, derived from Japanese wild mice, had normal hearing throughout life. Individuals in the panel were examined with auditory brainstem response (ABR) at various months of age, revealing that one particular strain (B6-Chr17(MSM)) substituting the chromosome 17 with the MSM-derived one showed a prominent resistance, having still good hearing at 18 months of age. Subsequent mapping using 89 individuals in the cross between B6-Chr17(MSM) and B6 was performed, which showed a significant association of ABR thresholds with loci in the vicinity of D17Mit119. These results show a novel AHL-resistant locus, designated as Ahl3, on the chromosome 17.     

Generation,Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5′-nucleotidases (5′-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5′-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5′-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other''s NAD supply by providing alternative precursors.     

Liver parenchyma heterogeneity in the response to extracellular NAD+

The perfused rat liver responds intensely to NAD+ infusion (20-100 microM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption are some of the effects that were observed. The aim of the present work was to investigate the distribution of the response to extracellular NAD+ along the hepatic acinus. The bivascularly perfused rat liver was used. Various combinations of perfusion directions (antegrade and retrograde) and infusion routes (portal vein, hepatic vein and hepatic artery) were used in order to supply NAD+ to different regions of the liver parenchyma, also taking advantage of the fact that its extracellular transformation generates steep concentration gradients. Oxygen uptake was stimulated by NAD+ in retrograde perfusion (irrespective of the infusion route) and transiently inhibited in antegrade perfusion. This indicates that the signal causing oxygen uptake inhibition is generated in the periportal area. The signal responsible for oxygen uptake stimulation is homogenously distributed. Stimulation of glucose release was more intense when NAD+ was infused into the portal vein or into the hepatic artery, indicating that stimulation of glycogenolysis predominates in the periportal area. The increases in perfusion pressure were more pronounced when the periportal area was supplied with NAD+ suggesting that the vasoconstrictive elements responding to NAD+ predominate in this region. The response to extracellular NAD+ is thus unequally distributed in the liver. As a paracrine agent, NAD+ is likely to be released locally. It can be concluded that its effects will be different depending on the area where it is released.     

A novel P2RX2 mutation in an Italian family affected by autosomal dominant nonsyndromic hearing loss

Hereditary hearing loss (HHL) is a common disorder accounting for at least 60% of prelingual deafness. It is characterized by a large genetic heterogeneity, and despite the presence of a major gene, still there is a need to search for new causative mutations/genes. Very recently, a mutation within ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) gene (P2RX2) at DNFA41 locus has been reported leading to a bilateral and symmetrical sensorineural non-syndromic autosomal dominant HHL in two Chinese families. We performed a linkage analysis in a large Italian family with a dominant pattern of inheritance showing a significant 3.31 LOD score in a 2 Mb region overlapping with the DNFA41 locus. Molecular analyses of P2RX2 identified a novel missense mutation (p.Gly353Arg) affecting a residue highly conserved across species. Visual inspection of the protein structure as obtained from comparative modeling suggests that substitution of the small glycine residue with a charged bulky residue such as an arginine that is close to the ‘neck’ of the region responsible for ion channel gating should have a high energetic cost and should lead to a severely destabilization of the fold. The identification of a second most likely causative mutation in P2RX2 gene further supports the possible role of this gene in causing autosomal dominant HHL.