Metabolic control of adult neural stem cell activity by Fasn-dependent lipogenesis

Abstract

Mechanisms controlling the proliferative activity of neural stem and progenitor cells (NSPCs) have a pivotal role to ensure life-long neurogenesis in the mammalian brain. How metabolic programs are coupled with NSPC activity remains unknown. Here we show that fatty acid synthase (Fasn), the key enzyme of de novo lipogenesis, is highly active in adult NSPCs and that conditional deletion of Fasn in mouse NSPCs impairs adult neurogenesis. The rate of de novo lipid synthesis and subsequent proliferation of NSPCs is regulated by Spot14, a gene previously implicated in lipid metabolism, that we found to be selectively expressed in low proliferating adult NSPCs. Spot14 reduces the availability of malonyl-CoA, which is an essential substrate for Fasn to fuel lipogenesis. Thus, we identify here a functional coupling between the regulation of lipid metabolism and adult NSPC proliferation.

Figure 1

NSPCs are in a distinct metabolic state and require Fasn- dependent lipogenesis for proliferation. a, In situ hybridization shows expression of Fasn within the neurogenic dentate niche. Fasn protein is expressed in nestin-GFP1 cells. Insets show high-power views of Fasn- expressing cells with 49,6-diamidino-2-phenylindole (DAPI; grey). GCL, granule-cell layer. b, Metabolomics show a distinct metabolic state of proliferating dentate gyrus and SVZ NSPCs (mDG prol and mSVZ prol) compared to proliferating Schwann cells (Schwann prol), non-proliferating Schwann cells (Schwann nprol), and differentiated progeny of hippocampal and SVZ NSPCs (mDG diff and mSVZ diff). Note the close clustering of distinct cell populations. c, Pharmacological inhibition of Fasn using orlistat leads to a dose-dependent reduction of hippocampal NSPC proliferation. BrdU, 5-bromodeoxyuridine. d, Genetic deletion of Fasn decreases proliferation of adult hippocampal NSPCs. Con, control; Cre, transduction of Cre-expressing virus to delete Fasn. Left panel, cells that were transduced with a red fluorescent protein (RFP)-expressing control virus and Cre-GFP-expressing retrovirus, and pulsed with BrdU. e, Conditional Fasn deletion in NSPCs reduces hippocampal neurogenesis. Shown are YFP-expressing cells in controls and mutant mice (Fasn-cKO). f, Quantification of YFP-labelled cells in the dentate gyrus (DG) 5 days and 40 days after tamoxifen (TAM)-induced recombination. g, Phenotyping of recombined, YFP-labelled cells reveals that conditional Fasn deletion reduces the number of radial glia-like NSPCs expressing Sox2 and Gfap 40 days after tamoxifen and leads to a substantial decrease in differentiated YFP-labelled cells. h, Quantification of phenotypes after conditional Fasn deletion. Error bars represent mean s.e.m. Scale bars represent 100 μm (a top panel, e), 40 μm (a bottom panel), 50 μm (c, d) and 20 μm(g).*P, 0.05

Figure 2

Spot14 is expressed in adult hippocampal NSPCs. a, In situ hybridization shows restricted expression of Spot14 in the SGZ of the dentate gyrus (top panel). Enhanced GFP expression under the regulatory elements of the Spot14 gene labels cells in the SGZ (bottom panel). b, Spot14-GFP co-labels with NSPC markers Sox2 and nestin. Arrows, GFP and Sox2 co-labelled cells; arrowheads, GFP and nestin co-labelled radial glia-like processes (bottom-left panel). A fraction of Spot14-GFP cells divide. Arrow, a Ki67-positive, Spot14-GFP co-labelled cell (bottom-right panel). c, YFP expression in radial and non-radial NSPCs labelled by Sox2 and Gfap 10 days after tamoxifen (TAM) injections in Spot14-CreERT2 mice. One month after tamoxifen injections, YFP-expressing cells co-label with Dcx, and 3 months after tamoxifen injections these cells co-label with the mature neuronal marker NeuN (bottom-right panel). Note also that radial NSPCs were labelled with YFP 3 months after tamoxifen injections (bottom-left panel). DAPI is shown in grey. d, Quantification of cellular phenotypes generated 10 days, 1 month and 3 months after tamoxifen injections in Spot14-CreERT2 mice. Astro, astrocytes. Scale bars represent 100 μm (a) and 20 μm (b, c).

Figure 3

Spot14 regulates proliferative activity of adult NSPCs.a, Hippocampal Spot14+ cells are less proliferative than Spot14- NSPCs.b, Time-lapse imaging of single hippocampal NSPCs cultured in hydrogel- based microwells reveals reduced proliferation of Spot14+ cells compared to Spot14- NSPCs on a single-cell level. Shown are snapshots every 9 h for Spot14- cells (top panels) and Spot14+ (bottom panels), and the quantification of the number of cell divisions over 81 h. c, Retroviral overexpression of Spot14 (Spot14OE) reduces proliferation of hippocampal NSPCs. d, shRNA-mediated knockdown of Spot14 (Spot14 shRNA) leads to a shift from radial towards non- radial Spot141 NSPCs compared to a non-targeting, shRNA-expressing lentivirus (Con shRNA). Arrows, non-radial NSPCs; arrowheads, radial NSPCs. Error bars represent mean s.e.m. Scale bars represent 40 μm (a) and 100 μm (b, d). *P, 0.05.

Figure 4

Spot14 modulates malonyl-CoA levels and de novo lipogenesis. a, Fasn protein is expressed in Spot14+ cells. Insets show high-power views of a Spot14-GFP and Fasn co-labelled cell. b, Mig12 is expressed in adult NSPCs. c, shRNA-mediated knockdown of Mig12 in vivo (Mig12 shRNA, right panel) reduces proliferation of hippocampal NSPCs. d, Retroviral overexpression of Spot14 reduces malonyl-CoA levels in NSPCs. dpm, disintegrations per minute; pmol, picomol. e, Radioactive tracing using 14C-acetate and 14C- glucose shows a decrease in de novo lipogenesis after Spot14 overexpression. f, Lipid separation by thin-layer chromatography after 14C-acetate labelling shows a reduction in de novo synthesized free fatty acids, TAGs (triacylglycerols) and polar lipids in Spot14-overexpressing hippocampal NSPCs. g, Scheme showing the proposed mechanism: high-proliferating NSPCs require Fasn-dependent lipogenesis. Spot14 downregulates NSPC proliferation by reducing the Fasn-dependent generation of new complex fatty acids (FAs). Error bars represent mean 6 s.e.m. Scale bars represent 40 μm (a), 20 μm (b) and 50 μm (c). *P, 0.05.