Sirt1 overexpression in neurons promotes neurite outgrowth and cell survival through inhibition of the mTOR signaling

Abstract

The mammalian nicotinamide-adenine dinucleotide (NAD)-dependent deacetylase Sirt1 impacts different processes involved in the maintenance of brain integrity and in the pathogenic pathways associated with several neurodegenerative disorders, including Alzheimer's disease. Here we used human Sirt1 transgenic mice to demonstrate that neuron-specific Sirt1 overexpression promoted neurite outgrowth and improved cell viability under normal and nutrient-limiting conditions in primary culture systems and that Sirt1-overexpressing neurons exhibited higher tolerance to cell death or degeneration induced by amyloid-β1-42 oligomers. Coincidentally, we found that enhanced Sirt1 expression in neurons downregulated the mammalian target of rapamycin (mTOR) protein levels and its phosphorylation without changes in its mRNA levels, which was accompanied by concomitant inhibition of the mTOR downstream signaling activity as revealed by decreased p70S6 kinase (p70S6K) phosphorylation at Thr389. Consistently with this, using a Sirt1 siRNA transfection approach, we observed that reduction of endogenous mouse Sirt1 led to increased levels of mTOR and phosphorylation of itself and p70S6K as well as impaired cell survival and neurite outgrowth in wild-type mouse primary neurons, corroborating a suppressing effect of mTOR by Sirt1. Correspondingly, the mTOR inhibitor rapamycin markedly improved neuronal cell survival in response to nutrient deprivation and significantly enhanced neurite outgrowth in wild-type mouse neurons. The protective effect of rapamycin was extended to neurons even with Sirt1 siRNA knockdown that displayed developmental abnormalities compared with siRNA control-treated cells. Collectively, our findings suggest that Sirt1 may act to promote growth and survival of neurons in the central nervous system via its negative modulation of mTOR signaling.

MicroRNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration

“…S5B), suggesting that SIRT1 has the ability to promote axon growth. In support of this, previous studies have shown that overexpression of SIRT1 can drastically promote axon growth of embryonic cortical neurons (Guo et al 2011;Li et al 2013), which do not up-regulate SIRT1 automatically in culture.…”

“…Among several candidates, we selected SIRT1 as a potential target of miR-138 because SIRT1 has been shown to control axon growth and degeneration (Araki et al 2004;Guo et al 2011) and to be highly expressed in mouse DRGs (Sakamoto et al 2004). To validate that SIRT1 expression is regulated by miR-138, we made a luciferase reporter construct by inserting the full-length mouse SIRT1 39 untranslated region (UTR) containing the predicted miR-138 target site and flanking sequences into the 39 of a Renilla luciferase (R-luc) reporter gene (Fig.…”

Neuritic complexity of hippocampal neurons depends on WIP‐mediated mTORC1 and Abl family kinases activities

“…With the exception of a previous work in which mTOR and S6K activity was also inversely correlated with the level of neuritic complexity (Guo et al. 2011), the relationship observed between the activity of these pathways and the level of cellular morphological complexity differs from most of published data. These differences may be due to the studied stage of neuron development, as most of the published experiments have been carried out with more mature neurons than those studied in this work (Woodring et al.…”

Cross-talk between Sirtuin and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in the Regulation of S6 Kinase 1 (S6K1) Phosphorylation

“…After washing with buffer ST, beads were washed with 1ϫ S6K1 kinase reaction buffer without ␤-mercaptoethanol (20 mM Hepes, pH 7.2, 10 mM MgCl 2 , 1 mg/ml BSA). S6K1 kinase assay was performed in 1ϫ S6K1 kinase reaction buffer with 2 mM ␤-mercaptoethanol, 2 g of GST-S6, 0.5 mM cold ATP, and 5 Ci of [␥- 32 P]ATP for 10 min at 30°C. The reactions were terminated by denaturation in SDS sample buffer at 100°C for 5 min, and the reaction samples were separated by SDS-PAGE and transferred to PVDF membrane.…”