NMN promotes osteogenesis by regulating mesenchymal stromal cells via the SIRT1 pathway in aged bone marrowV

    Mesenchymal stromal cells (MSCs) can differentiate to various cell types including osteoblasts, chondrocytes, and adipocytes. This cellular flexibility contributes to widespread clinical use of MSCs in tissue repair. However, challenges remain in efficient cellular expansion of MSCs for stem cell therapy. Current MSC culture methods have resulted in reduced self-renewal of MSCs and compromised therapeutic outcomes. This study identifies that nicotinamide mononucleotide (NMN), a key natural NAD+ intermediate, effectively encourages MSC expansion in vitro and in vivo. The in vitro expanded MSCs had heightened osteogenesis, but reduced adipogenesis. Furthermore, NMN supplementation stimulated osteogenesis of endogenous MSCs, and protected bone from aging and irradiation induced damage in mice. Mechanistically, we found that NMN treatment upregulated SIRT1. Genetically overexpressing SIRT1 in MSCs by using Prx1 cre; ColA1flox-stop-flox-SIRT1 mice promoted osteogenesis and reduced adipogenesis in aged mice. Overall, our data demonstrate that NMN promoted MSC self-renewal with strengthened osteogenesis and reduced adipogenesis via upregulating SIRT1 in aged mice.

    Aging is predicted to be an increasingly serious health and financial problem worldwide. Age-related disorders, such as tumor, metabolic disease, memory deterioration, and immunologic degeneration, are associated with declined regenerative capacity in rapidly dividing stem cells. Nicotinamide mononucleotide (NMN), a key NAD+ intermediate which decreases with age in mammals, is an efficient therapy against age-associated diseases. NMN administration alleviates age-related type 2 diabetes, ischemia-reperfusion injury, and Alzheimer’s disease. However, the underlying mechanism of NMN’s protective effect is still unknown. In this study, we explored NMN’s role in combating age-related disorders via regulating mesenchymal stromal cells (MSCs). MSCs are nonhematopoietic multipotent stem cells with regeneration capacity. Loss in number or functionality of MSCs with age profoundly limits tissue regeneration, However, most current MSC culture methods limits self-renewal potential and functionality of MSCs, leading to compromised therapeutic outcomes.Herein, we have investigated the effects and underlying mechanism of NMN on the expansion and differentiation of mouse MSCs in vitro and in vivo. We have found that NMN promotes MSC self-renewal during in vitro culture and in mice. We have further demonstrated that NMN activates Sirtuin1 (SIRT1), which is an NAD+-dependent deacetylase. NMN increases osteogenesis and reduces adipogenesis of MSCs via upregulating SIRT1 in aged mice.

Results and Discussion

     NMN accumulation in FHB-resistant barley cultivars

     We previously identified many barley cultivars that exhibited an FHB-resistant phenotype. Two 2-row cultivars (U389; Maja and U121; Sirius O-525) showed high FHB resistance compared with susceptible cultivars (Fig. S1a,b). We also performed a comparative analysis of the metabolite profiles of these FHB-resistant and FHB-susceptible (T615; Turkey 45) 2-row cultivars. NMN was found to be enriched in the uninoculated barley spikes of the two resistant cultivars compared with the susceptible cultivar (Fig. S1c). At 2 days post inoculation, the NMN contents had not significantly changed in any of the three cultivars (data not shown). NMN is a precursor of NAD, which functions as a cofactor for many enzymes involved in the catalysis of various metabolic reactions. A similar accumulation pattern was not observed for other NAD-related metabolites (NaMN: nicotinic acid mononucleotide, NA: nicotinate, NIC: nicotinamide; data not shown). Unfortunately, NAD(H) and NADP(H) were not quantified in this system. Zhang & Mou reported that extracellular NAD induced PR gene expression and resistance against the bacterial pathogen P. syringae in Arabidopsis thaliana. These results suggest that NMN is also involved in plant disease resistance against Fusarium species. It has also been reported that the amount of NAD significantly increased in barley leaves inoculated with the biotrophic fungal pathogen Erysiphe graminis, the causal organism of powdery mildew. These findings suggest that NAD biosynthesis is likely involved in disease resistance against a broad range of phytopathogens in barley. The NAD(H) and NADP(H) pools function as coenzymes for the various redox reactions. NMN is a precursor of not only NAD(H) and NADP(H) but also antimicrobial pyridine alkaloids. These facts imply that NMN controls plant disease more effectively than NAD or NADP.As stated above, a high concentration (>1 mM) of extracellular NAD activates defence signalling events, including the induction of pathogenesis-related 1 (PR1) expression. To examine whether NMN can activate defence signalling in Arabidopsis plants, we investigated the expression of the SA-responsive PR1 gene, the SA biosynthesis gene ICS1, and the JA/ET-responsive plant defensin 1.2a (PDF1.2a) gene by RT-qPCR (Fig. S2aS2bS2c). Plants were treated with NMN at a concentration of 0.3 mM, which was sprayed onto the surface of rosette leaves. The leaves were then harvested at different time points (0, 6, 24, and 48 h) after spraying. The mRNA levels of the PR1 gene increased at 6 h after NMN treatment and then decreased (Fig. S2a). Correspondingly, ICS1 gene expression was also transiently increased by NMN treatment (Fig. S2b). On the other hand, NMN treatment did not induce expression of the PDF1.2a gene (Fig. S2c). These results suggest that NMN transiently activates the SA-dependent signalling pathway in Arabidopsis leaves.

Author :Jie Song, Jing Li, Fangji Yang, Gang Ning, Limin Zhen, Lina Wu, Yongyuan Zheng, Qi Zhang, Dongjun Lin, Chan Xie & Liang Peng 

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