The PDGF receptor alpha signal regulates the senescence of neural stem cells and their niche via the close functional correlation with NAD

概要

〔Purpose〕
　The aging is the most important risk factor for the decline of physiological function and the development of many neurological diseases in the elderly, threatening healthy life expectancy. The aging-associated changes of adult neural stem cells (NSCs) have been assumed to be crucially implicated in the brain aging, and the inhibition of NSC senescence is attracting attention as a antiaging strategy of the neural tissue. The decline of an oxidized form of nicotinamide adenine dinucleotide (NAD+; thereafter referred to as NAD) is a hopeful potent therapeutic target for antiaging, because the decline of NAD was closely related with the aging of neurological tissue. However, the underlying mechanisms of the function of NAD and the age-related NAD reduction are needed to be further explored to establish efficient antiaging therapeutic strategy. The expression of platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) was abundant during perinatal periods and were decreased with age. Accumulated data have suggested the role of PDGFRα in the proliferation of adult NSCs. Therefore, I sought to explore the role of PDGFRα to regulate NSC aging in implication with NAD for investigating further efficient antiaging therapeutics.

〔Methods and results〕
　I conducted in vivo and invitro studies following the conditional Pdgfra inactivation strategy. In vivo analyses, postnatal day (P)-15-old Nestin-nlsCre+/−; Pdgfraflox/flox (Nes-KO) mice, and control Pdgfraflox/flox mice (Flox mice) were used to isolate the outside wall of lateral ventricle (owLV) that includes NSC niche for metabolome and real-time PCR (RT-PCR). For the in vitro analyses, I used NSCs isolated and cultured from the SVZ of P1-old CAGG-CreER+/-;Pdgfraflox/flox mice (CAGG-iKO mice), CAGG-CreER+/-; Pdgfraflox/flox; Rosa26 R26R -H2B-mCherry/+ (CAGG-iKO-MC), and Flox mice. Pdgfra inactivation was induced by 4-hydroxytamoxifen, thereafter these NSCs were subjected for the analyses. Metabolome, RT-PCR, western blotting, and histological analyses were performed in day in vitro (DIV) 14; migration assay in DIV17, and proliferation assay in DIV21. To confirm the mechanism, rescue experiments were done in which, Pdgfra inactivated NSCs were treated with N- acetyl cysteine (NAC; antioxidant), and β-nicotinamide mononucleotide (NMN; a precursor of NAD) in CAGG-iKO to approve the functional correlation between Pdgfra inactivation and NAD metabolism at DIV14 and DIV21 (Rescue 1, and 2 experiments, respectively).
　In vivo study concern to the owLV, RT-PCR analyses showed that the levels of the gene implicated to senescence, mitochondrial dysfunction, oxidative stress-overload, DNA damage were increased in Nes-KO than in Flox mice. In compared with Flox mice, the metabolome analyses detected mitochondrial dysfunction with decreased levels of ATP, NAD, and NADH, and the oxidative stress overload with decreased arginine and elevated O-Acetyl-ADP-Ribose, a product after sirtuins consume NAD to counteract oxidative stress. Thus, the NSC-specific Pdgfra inactivation in owLV partly recapitulated the senescence of neural phenotype.
　In vitro study, the efficient Pdgfra inactivation was proved by the decreased levels of Pdgfra in RT-PCR, and by the decreased Pdgfra and immunofluorescent staining with complementary mCherry expression as a marker for DNA recombination in the cultured CAGG-iKO-MC NSCs. As compared with control Flox NSCs, the stemness and self-renewal was decreased that was accompanied by the increased senescence indicator expression in CAGG-iKO NSCs. Metabolome and RT-PCR indicated mitochondrial dysfunction and oxidative stress overload with decreased NAD/NADH ratio, antioxidant related metabolites, and with the increased gene expressions implicated to mitophagy, and redox-reactions. Along with this, decreased glycolysis and energy production, increased oxidative stress-induced DNA damage, enhanced PARP1 activity, Cd38 expression, and decreased Nampt synthesis were detected. It was suggested that these identified feedforward cycle of NAD metabolism was implicated in the accelerate senescence in CAGG-iKO NSCs.
　In the rescue experiments by NAC and NMN; depending on the experimental condition, NMN NAC partly replenished stemness and self-renewal of CAGG-iKO NSCs, while the rescue effects by single NAC treatment were not detected. These indicated the implication of the decreased levels of NAD, at least partly, in the accelerated senescence after Pdgfra inactivation.

〔Conclusion〕
　After all, the present both in vivo and in vitro studies, for the first time, showed the crucial implication of PDGFRα in the antiaging of owLV and NSCs. The Pdgfra inactivation induced severe metabolic disturbances in NAD-related metabolism and altered gene expressions including those implicated in mitochondrial dysfunction, oxidative stress overload with accompanying DNA damages, and the increased expressions of many senescence markers. Replenishment of declined NAD by NMN partly supplemented the dysfunction after Pdgfra inactivation. Therefore, at least partly, NAD mediated the senescence of NSCs after Pdgfra inactivation. PDGFs reportedly exert diverse effects that were similar with those regulated by NAD metabolism. The further studies would be required to uncover and apply the intimate functional correlation between PDGFRa signal and NAD-related metabolism in prevention of NSC aging.