A novel pathway regulates memory and plasticity via SIRT1 and miR-134

Abstract

The NAD-dependent deacetylase Sir2 was initially identified as a mediator of replicative lifespan in budding yeast and was subsequently shown to modulate longevity in worms and flies. Its mammalian homologue, SIRT1, seems to have evolved complex systemic roles in cardiac function, DNA repair and genomic stability. Recent studies suggest a functional relevance of SIRT1 in normal brain physiology and neurological disorders. However, it is unknown if SIRT1 has a role in higher-order brain functions. We report that SIRT1 modulates synaptic plasticity and memory formation via a microRNA-mediated mechanism. Activation of SIRT1 enhances, whereas its loss-of-function impairs, synaptic plasticity. Surprisingly, these effects were mediated via post-transcriptional regulation of cAMP response binding protein (CREB) expression by a brain-specific microRNA, miR-134. SIRT1 normally functions to limit expression of miR-134 via a repressor complex containing the transcription factor YY1, and unchecked miR-134 expression following SIRT1 deficiency results in the downregulated expression of CREB and brain-derived neurotrophic factor (BDNF), thereby impairing synaptic plasticity. These findings demonstrate a new role for SIRT1 in cognition and a previously unknown microRNA-based mechanism by which SIRT1 regulates these processes. Furthermore, these results describe a separate branch of SIRT1 signalling, in which SIRT1 has a direct role in regulating normal brain function in a manner that is disparate from its cell survival functions, demonstrating its value as a potential therapeutic target for the treatment of central nervous system disorders.

Figure 1. SIRT1 loss-of-function impairs memory and synaptic plasticity

(a) Freezing time of SIRT1Δ mice and littermate controls (Cont) 24 hr after contextual fear conditioning training. (b) Shock sensitivities did not differ between control and SIRT1Δ mice. (c) SIRT1Δand control mice were tested for novel object discrimination 24 hrs after training. (d) Pre-test exploration times were equal for each object. (e) Escape latencies of SIRT1Δ and control mice were examined with the Morris water maze hidden platform test. (f) Left panel: the swimming time spent in each quadrant in the probe trial on day 5 (T, target; L, left; O, opposite; R, right). Right panel: representative path tracings of the probe test. (g) LTP measurements were performed in the CA1 region of acute slices from SIRT1Δ mice and controls. (h) SVP immunoreactivity in SIRT1Δ and control hippocampi. (i) Western blots from hippocampal lysates examined SVP in SIRT1Δ and control mice. (j) The density of Golgi-impregnated hippocampal neuronal dendritic spines was measured in SIRT1Δ and control mice. Scale bar: 10 μm. TBS: theta-burst stimulation, SVP: synaptophysin, OD: optical density. *p < 0.05, **p < 0.01, ***p < 0.001. All histograms represent average ± SEM.

Figure 2. BDNF and CREB are downregulated, while miRNA-134 is up-regulated, in SIRT1Δ mice

(a) BDNF in the SIRT1Δ and control mouse hippocampus. Left: mRNA levels. Right: Western blot. (b) Chromatin immunoprecipitation (ChIP) with an anti-CREB antibody was followed by qPCR for BDNF promoters 1, 2, and 4. (c) CREB in the SIRT1Δ and control mouse hippocampus. Left: Western blot. Right: mRNA levels. (d) qPCR of selected miRNAs from hippocampi of SIRT1Δ and control mice. (e) CAD cells were transfected with the plasmids or LNA indicated together with CRE-Luc. (f) Luciferase reporter constructs containing either a wildtype (WT-CREB) or a mutated (mut-CREB) CREB 3’UTR region, were cotransfected with miR-134 or control. (g) CREB protein expression in CAD cells after transfection with the indicated constructs or LNAs. Scr: scrambled, mut: mutant, LNA: locked-nucleic-acid (LNA), CRE-Luc: CREB activity reporter construct. **p < 0.01; ***p < 0.001; NS, not significant.

Figure 3. SIRT1 regulates CREB through miR-134

(a) From SIRT1 ChIP, two genomic regions, R3 and R7, corresponding to base pairs 1418~830 and 3427~2901 upstream of miR-134, respectively, were amplified from the wild type, but not the SIRT1 total knockout (SIRT1KO), brain tissue. (b) miR-134 promoter regions R3 and R7 were quantified from SIRT1 ChIP with qPCR. (c) Reporter constructs containing R3, R5, or R7 regions upstream of a minimal promoter in a luciferase reporter were co-transfected with SIRT1, SIRT1 shRNA, or empty vector. (d) qPCR for miR-134 in CAD cells after transfection with the indicated plasmids. (e) CRE-luciferase reporter assay in CAD cells. (f) ChIP was performed from CAD cells with anti-YY1 and anti-SIRT1 antibodies. (g) Anti-YY1 ChIP followed by qPCR for regions R3 and R7. (h) Luciferase reporter constructs containing R3, R5, or R7 regions were co-transfected with the indicated plasmids. (i) miR-134 levels were measured in CAD cells with qPCR after transfection with the indicated plasmids. (j) Western blotting measured CREB protein levels in CAD cells after transfections with the indicated plasmids. shRNA Cont: scrambled shRNA control. *p < 0.05; **p < 0.01; ***p < 0.001; NS: not significant. All histograms represent average ± SEM.

Figure 4. MiR-134 knockdown rescues the LTP and memory impairments caused by SIRT1 deficiency

(a) LTP was measured in acute hippocampal slices of mice six weeks after injection with Lv-miR-134 or Lv-Scr-miR. (b) Lv-miR-134 and Lv-Scr-miR-injected mice were tested with a contextual fear conditioning task. (c) Following hippocampal injections with LNA-miR-134 or LNA-scr-miR, LTP was measured in SIRT1Δ and control mouse acute hippocampal slices. (d) Freezing behavior in the contextual fear conditioning task was examined in SIRT1Δ and control mice after hippocampal injections of LNA-miR-134 or LNA-scr-miR. (e). Western blot for CREB and BDNF in brain lysate after in vivo miR-134 knockdown. Control scrambled miR lentivirus: Lv-Scr-miR. **p < 0.01, NS: not significant.