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. 2011 Dec 18;18(1):159-65.
doi: 10.1038/nm.2559.

Sirt1 mediates neuroprotection from mutant huntingtin by activation of the TORC1 and CREB transcriptional pathway

Hyunkyung Jeong  1 , Dena E Cohen, Libin Cui, Andrea Supinski, Jeffrey N Savas, Joseph R Mazzulli, John R Yates 3rd, Laura Bordone, Leonard Guarente, Dimitri Krainc
Affiliations

Sirt1 mediates neuroprotection from mutant huntingtin by activation of the TORC1 and CREB transcriptional pathway

Hyunkyung Jeong et al. Nat Med. .

Abstract

Sirt1, a NAD-dependent protein deacetylase, has emerged as a key regulator of mammalian transcription in response to cellular metabolic status and stress. Here we show that Sirt1 has a neuroprotective role in models of Huntington's disease, an inherited neurodegenerative disorder caused by a glutamine repeat expansion in huntingtin protein (HTT). Brain-specific knockout of Sirt1 results in exacerbation of brain pathology in a mouse model of Huntington's disease, whereas overexpression of Sirt1 improves survival, neuropathology and the expression of brain-derived neurotrophic factor (BDNF) in Huntington's disease mice. We show that Sirt1 deacetylase activity directly targets neurons to mediate neuroprotection from mutant HTT. The neuroprotective effect of Sirt1 requires the presence of CREB-regulated transcription coactivator 1 (TORC1), a brain-specific modulator of CREB activity. We show that under normal conditions, Sirt1 deacetylates and activates TORC1 by promoting its dephosphorylation and its interaction with CREB. We identified BDNF as a key target of Sirt1 and TORC1 transcriptional activity in both normal and Huntington's disease neurons. Mutant HTT interferes with the TORC1-CREB interaction to repress BDNF transcription, and Sirt1 rescues this defect in vitro and in vivo. These studies suggest a key role for Sirt1 in transcriptional networks in both the normal and Huntington's disease brain and offer an opportunity for therapeutic development.

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Figures

Figure 1
Figure 1. Ablation of neuronal Sirt1 exacerbates and over-expression of Sirt1 ameliorates phenotypes in R6/2 mouse
(a) Latency to fall from accelerating rotarod at 10 weeks of age. n = 4 - 10 per group. *P < 0.05 for HD vs. BSKO-HD by ANOVA. (b) Striatal volumes at 20 weeks of age. n = 4 per group. *P < 0.01 by ANOVA. (c) Striatal neuronal volumes at 20 weeks of age. n = 4 per group. * P < 0.05 by t-test. (d) Aggregates of HTT in striata at 20 weeks of age. ** P < 0.01 (n=6 per group) (e) Survival of male HD (closed square) and Sirt1-KI-HD (open square) mice. n = 26 - 27 per group. P < 0.001 by log-rank test. (f) Striatal volume at 100 d of age. n = 5 – 6 per group. * P < 0.05 by ANOVA. (g) Striatal neuronal volumes at 100 d of age. n = 5 - 6 per group. *P < 0.05 by t-test. (h) Aggregates of HTT in striata at 100 d of age. ** P < 0.01 (n=7 per group).
Figure 2
Figure 2. Deaceylase activity of Sirt1 protects cortical neurons from mutant HTT Toxicity
(a) Upper panels: neurofilament (NF) staining in primary cortical neurons infected with indicated lentivirus. Lower panels: double staining of mt HTT- and Sirt1-infected neurons. Scale bar, 300 µm. (b) In-cell western (ICW) for NF and Draq5 + Sapphire 700 staining of cortical neurons infected with indicated lentivirus. *** P < 0.001 for mt HTT vs. WT HTT, *** P < 0.001 for mt HTT + Sirt1 vs. mt HTT. (c) BNDF mRNA levels in mouse cortex at 100 d of age. *P < 0.05 for HD vs Sirt1-KI-HD by ANOVA. * P < 0.05 for Sirt1-KI vs. WT by ANOVA (n=4 per group). (d) BDNF mRNA levels in mouse cortex at 3 months of age. n = 5 per group. *P < 0.05 by t-test. (e) Toxicity in primary cortical neurons from Sirt1 KO mice or WT littermates. * P < 0.05 for white bars, KO vs WT; *** P < 0.001 for black bars, KO vs WT; *** P < 0.001 for white bars, KO + BDNF vs KO; ** P < 0.01 for black bars, KO + BDNF vs KO by t-test.
Figure 3
Figure 3. Sirt1 deacetylates and activates TORC1
(a) BDNF promoter IV activity in N2a cells cotransfected with indicated plasmids at 24 h posttransfection. ** P < 0.01 vs. vector + pEGFP, ***P < 0.001 vs. vector + pEGFP by t-test. (b, c) Coimmunoprecipitations in N2a cells with indicated plasmids. (d) TORC1 immunoprecipitation and acetylation in N2a cells assessed by antibody to acetyl-lysine (AcK) (bottom panel). Western blots of TORC1 in input samples (top panel) and IP samples (middle panel). (e) Western blot of TORC1 KR or KQ mutants in N2a cells transfected with indicated TORC1 mutants. Long and short exposures are shown. (f) Left: western blot of TORC1 with or without in vitro treatment of alkaline phosphatase (APase). Right: Western blot of TORC1 and p-TORC1 and coimmunoprecipitation assay of CREB and TORC1 in N2a cells transfected with indicated plasmids. TORC1 position marked by arrows. (g) Dephosphorylation of p-TORC1 by forskolin (FSK) in the presence of Sirt1 knockdown in primary cortical neurons. Phospho-TORC1 marked with arrow heads. * P < 0.05 by t-test.
Figure 4
Figure 4. Sirt1 rescues mutant HTT-mediated interference with TORC1 activity
(a) Immunostaining of HTT, TORC1 and DAPI in primary neurons infected with lentiviral WT HTT (upper panels) or mt HTT (lower panels). *** P < 0.001 by t-test. Scale bar, 50 µm. (b) Chromatin immunoprecipitation (ChIP) of TORC1 on BDNF in cortex of WT or HD mice. **P < 0.01 vs WT, n = 6. (c) Coimmunoprecipitations assay of indicated plasmids in N2a cells. (d) BDNF promoter IV activities in N2a cells cotransfected with indicated plasmids at 48 hooo posttransfection. ** P < 0.01. (e) NF staining intensity in primary cortical neurons infected with indicated lentivirus. * P < 0.05 by t-test. *** P < 0.001 by t-test. (f) Toxicity was assessed as in (e). * P < 0.05, *** P < 0.001 by t-test.
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