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. 2008 Oct 7;105(40):15599-604.
doi: 10.1073/pnas.0800612105. Epub 2008 Sep 30.

Histone deacetylase SIRT1 modulates neuronal differentiation by its nuclear translocation

Shin Hisahara  1 , Susumu Chiba, Hiroyuki Matsumoto, Masaya Tanno, Hideshi Yagi, Shun Shimohama, Makoto Sato, Yoshiyuki Horio
Affiliations

Histone deacetylase SIRT1 modulates neuronal differentiation by its nuclear translocation

Shin Hisahara et al. Proc Natl Acad Sci U S A. .

Abstract

Neural precursor cells (NPCs) differentiate into neurons, astrocytes, and oligodendrocytes in response to intrinsic and extrinsic changes. Notch signals maintain undifferentiated NPCs, but the mechanisms underlying the neuronal differentiation are largely unknown. We show that SIRT1, an NAD(+)-dependent histone deacetylase, modulates neuronal differentiation. SIRT1 was found in the cytoplasm of embryonic and adult NPCs and was transiently localized in the nucleus in response to differentiation stimulus. SIRT1 started to translocate into the nucleus within 10 min after the transfer of NPCs into differentiation conditions, stayed in the nucleus, and then gradually retranslocated to the cytoplasm after several hours. The number of neurospheres that generated Tuj1(+) neurons was significantly decreased by pharmacological inhibitors of SIRT1, dominant-negative SIRT1 and SIRT1-siRNA, whereas overexpression of SIRT1, but not that of cytoplasm-localized mutant SIRT1, enhanced neuronal differentiation and decreased Hes1 expression. Expression of SIRT1-siRNA impaired neuronal differentiation and migration of NPCs into the cortical plate in the embryonic brain. Nuclear receptor corepressor (N-CoR), which has been reported to bind SIRT1, promoted neuronal differentiation and synergistically increased the number of Tuj1(+) neurons with SIRT1, and both bound the Hes1 promoter region in differentiating NPCs. Hes1 transactivation by Notch1 was inhibited by SIRT1 and/or N-CoR. Our study indicated that SIRT1 is a player of repressing Notch1-Hes1 signaling pathway, and its transient translocation into the nucleus may have a role in the differentiation of NPCs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Nuclear translocation of SIRT1 in NPCs. (A) Immunostaining of E14.5 mouse brain with anti-SIRT1 (red) and anti-nestin (green) antibodies. VZ, ventricular zone; V, ventriculus. (Scale bar: 20 μm.) (B) SIRT1 immunostaining of E16.5 mouse brain. Nuclei are shown in red as a pseudocolor. Some cells express nuclear SIRT1 (arrows). (Scale bar: 50 μm.) (C) Immunostaining of an undifferentiated neurosphere. (Scale bar: 20 μm.) (D) Dissociated neurosphere cells were transferred into differentiation conditions, fixed immediately (0 h), 3 h, and 6 h after the transfer and then immunostained. (Scale bars: 20 μm and 10 μm, Inset.) (E) Cytoplasmic (C) and nuclear (N) fractions were prepared from cells before (0 h) or 3 h after transfer to differentiation conditions and then subjected to Western blot analysis. GAPDH (G) and lamin (L) are cytoplasmic and nuclear markers, respectively. S, SIRT1. (F and G) Time-lapse image analyses of SIRT1-EGFP (F) and SIRT1mtNLS-EGFP (G). Nuclei are shown in red. In G, a cluster of three cells, two of which express SIRT1mtNLS-EGFP, is shown. (Scale bars: 10 μm.)
Fig. 2.
Fig. 2.
Inhibition of SIRT1 impairs neuronal differentiation. (A) Neurospheres were cultured in the absence (C) or presence of SIRT1 inhibitors (SP, splitomicin; NA, nicotinamide) and stained with anti-Tuj1 (neurons, red), anti-GFAP (astrocytes, blue), and anti-O4 (oligodendrocytes, green) antibodies. (Scale bar: 50 μm.) (B) The number of neurospheres containing Tuj1+ (N), GFAP+ (A), and/or O4+ cells (O) was counted. *, P < 0.05; **, P < 0.01. (C) NPCs were electroporated with control vector (V) or SIRT1H355Y (DN) and immunostained after differentiation. (Scale bar: 50 μm.) (D and F) The number of spheres containing Tuj1+ (N) and/or GFAP+ (A) cells was counted. **, P < 0.01. (E) NPCs were untreated (C) or infected with lentivirus vector (V) or SIRT1-siRNA lentivirus (siRNA) and immunostained after differentiation. (Scale bar: 50 μm.) (G) E14 brain was electroporated with EGFP (E), SIRT1-EGFP with EGFP (S + E), or SIRT1-siRNA with EGFP (siRNA + E) and examined at E17. Arrowheads indicate tangential thickness of the brain section. P, pia; V, ventriculus. (Scale bars: Upper, 100 μm; Lower, 20 μm.) (H) NeuN immunostaining of E17 brains. Black arrows indicate the localization of the sections. White arrows indicate EGFP+/NeuN+ cells. (Scale bars: 50 μm.)
Fig. 3.
Fig. 3.
SIRT1 and N-CoR promotes neuronal differentiation. (A) NPCs were untreated (C) or transfected with vector (V), SIRT1 (S) or SIRT1mtNLS (mtNLS) and then immunostained with anti-Tuj1 (red) and anti-GFAP (blue) antibodies after differentiation. (Scale bar: 50 μm.) (B) The number of spheres containing Tuj1+ cells was counted. More than 20 visual fields were counted for each experiment. **, P < 0.01. (C) NPCs were cultured in the differentiation conditions for 3 h and then cultured in MHM medium containing EGF and bFGF for 5 days (3 h). For control, NPCs were cultured in the differentiation conditions for 5 days (5 d). Cells were immunostained with an anti-Tuj1 antibody. (Scale bars: 50 μm.) (D) Tuj1+ cells were counted. (E) GST-SIRT1 binds N-CoR from COS cells and fetal brain. (F and G) Immunoprecipitation of N-CoR with anti-SIRT1 antibody. (F) N-CoR in COS cells is coimmunoprecipitated with SIRT1. (G) N-CoR from E16.5 embryonic brain cells cultured in differentiation conditions for 3 h is immunoprecipitated by anti-SIRT1 antibody. (H and I) SIRT1 and N-CoR induce neuronal differentiation. NPCs transfected with the indicated plasmids (V, control vector; S, SIRT1; N, N-CoR; DN, SIRT1H355Y) were analyzed. (Scale bar: 50 μm.) (I) The number of spheres containing Tuj1+ cells was counted. **, P < 0.01.
Fig. 4.
Fig. 4.
Suppression of Hes1 by SIRT1. (A) Effects of SIRT1 (S), N-CoR (N), and SIRT1H355Y (DN) on the Hes1 promoter activity induced by Notch-ICD (Notch). (B) Effects of SIRT1 (S), N-CoR (N), and SIRT1H355Y (DN) on the 12XCSL1 promoter activity induced by Notch-ICD (Notch). (C) Binding of SIRT1 and N-CoR on the Hes1 and Hes5 promoter regions in differentiating NPCs. (D) SIRT1 suppresses Hes1 expression. NPCs electroporated with SIRT1-EGFP (S) or SIRT1mtNLS-EGFP (mtNLS) were cultured for 24 h in differentiation conditions and then immunostained with an anti-Hes1 antibody. (Scale bars: 20 μm.)
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