Our results indicate that SIRT1 plays an important role in cellular mechanisms underlying learning and memory in mice. Here we show that SIRT1 localizes in the nuclei of pyramidal and granule neurons of the hippocampus, a structure critically involved in cognitive processes. The lack of SIRT1 leads to significant behavioral alterations in different classical paradigms assessing formation of immediate, associative and spatial memories. Although SIRT1-KO mice exhibit normal exploratory behavior in the open field and discriminatory ability in the novel object recognition test, a significant deficit in immediate memory was revealed by decreased spontaneous alternation in the Y-maze. However, hippocampal-dependent memories showed the most dramatic impairment. The negligible fear behavior exhibited by SIRT1-KO mice when assessed for contextual associative memory, compared to the striking ability of WT mice to discriminate between training and novel contexts tested at either short-term or long-term, together with the impaired spatial abilities of mutant animals in the Barnes maze, indicate the important role of SIRT1 in hippocampus-dependent cognitive learning. Although SIRT1-KO mice are less spontaneously active than WT mice (Boily et al., 2008
), we found that activity levels displayed during each cognitive test were similar or even higher in SIRT1-KO mice as compared to WT mice perhaps because these tests were carried out during the inactive (light) period. In addition, despite the delayed and incomplete eyelid opening in knockout animals (McBurney et al., 2003
), our tests of pupillary reflexes, visual perception and cues, together with the normal capacity of object identification and discrimination in the NORT, indicated that their visual functions were unaltered. In accordance, eye size, lens and retina were similarly conserved in both WT and SIRT1 knockout mice. Thus, our data suggest that neither the lethargic phenotype nor the eyelid defect is likely to account for the impaired behavioral performance in SIRT1-KO mice.
In agreement with our behavioral results, LTP, which is widely considered to represent a cellular mechanism for the formation of specific types of memory, including spatial and contextual learning (McHugh et al., 1996
; Chen and Tonegawa, 1997
), was impaired in SIRT1-KO mice. In contrast, STP as well as basic electrophysiological properties of synaptic transmission, including presynaptic mechanisms and ionotropic receptors functions were unaltered in SIRT1-deficient mice, suggesting that SIRT1 acts downstream of glutamate receptors to regulate LTP formation/consolidation. Based on our data, SIRT1 deletion does not impair LTP induction but instead impairs the maximum expression of LTP. One possibility is that SIRT1 participates in the regulation of AMPA receptor trafficking.
Despite the fact that SIRT1-KO mice have reduced body and organ sizes, including brain size, which is 20% smaller than that of WT mice (Boily et al., 2008
), we did not find alterations in gross brain architecture. In addition, normal dentritic spine density and morphology of CA1 pyramidal neurons, as well as normal expression of synapsin and synaptophysin in SIRT1-KO mice, suggest that neither micro-anatomical differences in spine structure or alterations in levels of synaptic proteins could account for the synaptic plasticity deficits found in SIRT1-KO. However, the significant decreases in branching, length and complexity of neuronal dendritic arborizations of granule cells in the dentate gyrus of SIRT1-KO mice could explain some of the phenotypic differences with WT animals. Furthermore, the decrease dendritic complexity observed in SIRT1-KO mice may be related to their small brain size, similarly to other genes disruptions such as IGF-1 (Niblock et al., 2000
; Beck et al., 1995
), BDNF (Gorski et al., 2003
; Patterson et al., 1996
) or MeCP2 (Zhou et al., 2006
), in which brain size closely correlates with dendritic conformation.
It is well documented that changes in the levels of histone acetylation modify synaptic plasticity and learning abilities (Alarcon et al., 2004
; Levenson et al., 2004a
; Fischer et al., 2007
). Regulation of chromatin remodeling and gene expression through histone acetylation is critical in memory consolidation (Levenson et al., 2004b
). Specific inhibitors of Class I, II and IV HDACs, such as TSA and sodium butyrate, have been shown to induce dendritic sprouting, increase the number of synapses, and reverse LTP and memory deficits observed under various conditions (Alarcon et al., 2004
; Fischer et al., 2007
). In contrast, our data show that a lack of SIRT1, a NAD+
-dependent HDAC (Class III), leads to memory and synaptic plasticity impairment, indicating that both types of histone deacetylases play essential roles in cognition, although their activities differentially impact molecular and cellular process underlying learning and memory. These results indicate that more information is required regarding the identity and functions of the targets of these different classes of deacetylases.
Although the differences in the microarray between SIRT1-KO and WT hippocampus were small, we identified deregulation in the expression of genes involved in synaptic function, membrane fusion, myelination, and lipid and amino acid metabolism. However, to draw conclusions on the connection between these changes in gene expression and the memory impairments observed in SIRT1-KO mice, it is necessary to test further the role of reduced expression of a number of these genes in the behavior phenotypes of the SIRT1-KO mice.
The regulation of insulin/IGF-1 signaling and IRS2/ERK1/2 pathway may also contribute to the effect of SIRT1 in mouse cognition. SIRT1 upregulates IGF-1 by either derepressing IGFBP-1 (Lemieux et al., 2005
) or deacetylating insulin receptor substrate-2 (IRS-2) (Zhang, 2007
; Li et al., 2008
). In turn, reduction of IGF-1 signaling can decrease the downstream mitogen-activated protein kinase (MAPK), ERK1/2 and PI3 kinase, which are important for various brain functions (Huang et al., 2008
; Zhang, 2007
). Mice with reduced IGF-1 levels have impaired spatial learning and this effect is partially reversed by IGF-1 replenishment (Trejo et al., 2007
). In agreement with this, brain specific SIRT1 mutant mice show somatotropic axis disruption and a marked IGF-1 reduction (Cohen et al., 2009
). Also, MAPK activation is required for several forms of LTP and for spatial learning and fear conditioning (Selcher et al., 1999
). In addition, activation of ERK1/2 has been shown to be associated with LTP induction as well as with learning and memory (Trifilieff et al., 2007
). We previously showed that SIRT1 inhibition reduces ERK1/2 activity in part through the inactivation of IRS-2 and that ERK1/2 phoshorylation decreases in hippocampus of SIRT1-KO mice (Li et al., 2008
). Interestingly, we found that several genes regulated by SIRT1 in hippocampus involved in myelination or lipid metabolism are also regulated by IGF-I, IRS2 and/or ERK1/2 (Table S3
). Thus, SIRT1 may function as a coordinator of multiple proteins/enzymes involved in IGF-I signaling, ranging from IGF-I binding proteins, to key components of the IGF-IR signaling pathway involved in learning and memory, such as MAPK and ERK1/2.
In contrast to the SIRT1-KO mice, increased brain levels of SIRT1 in NeSTO mice did not affect LTP, immediate, spatial or associative memory, although these mice exhibited increased synaptic excitability, possibly due to changes in some properties of AMPA receptors. Normal synaptic plasticity together with unaltered associative learning in NeSTO mice lead us to propose that the high levels of SIRT1 protein (~16-fold increase than WT) reached in the hippocampus of the transgenic mice may interfere with the potential effects of SIRT1 overexpression. Thus, it is possible that LTP and cognitive abilities might become unresponsive to the excessive amounts of SIRT1 in NeSTO mouse brain. In agreement with this idea, previous studies have shown that only low to moderate levels of SIRT1 overexpression confer beneficial effects in mouse heart (up to 7.5 fold) (Alcendor et al., 2007
), intestine (Firestein et al., 2008
) and bone marrow lymphocyte progenitors (Oberdoerffer et al., 2008
) (3- to 4-fold in both cases). Alternatively, it is possible that the excess protein did not assemble into the appropriate protein complexes, or that an NAD+
decrease may have compensated for increased SIRT1 levels. Also, it is important to stress that our NeSTO animals, which showed no alterations in LTP and memory, were analyzed at a relatively young age; it is therefore possible that the effects of SIRT1 overexpression in cognition would become evident during the course of aging. Interestingly, a recent study shows that old but not young mice overexpressing SIRT1 exhibited differences in NORT tested 24 h after training (Kakefuda et al., 2009
). Performing learning tests in older NeSTO animals together with ongoing studies using SIRT1 agonists in transgenic mice should provide more insights regarding the effect of SIRT1 overexpression on cognition.
Memory and synaptic plasticity are highly vulnerable to decline with aging. Calorie restriction (CR) is a dietary regimen that attenuates age-dependent degenerative processes, as well as both cognitive deficits and synaptic plasticity (Weindruch et al., 1986
; Roth et al., 1995
; Fontan-Lozano et al., 2007
; Pearson et al., 2008
). SIRT1 is a potential mediator of the beneficial effects conferred by CR (Cohen et al., 2004
; Guarente, 2005
), and it was recently shown that CR does not increase lifespan of SIRT1 KO mice (Boily et al., 2008
; Li et al., 2008
). Thus, our findings raise the question of the potential role of this protein in CR-mediated improvement of cognitive performance. However, here we found that the deficit in synaptic plasticity caused by the lack of SIRT1 is independent of NMDA receptors, opposite to the NMDAR-dependent synaptic plasticity facilitation by CR (Fontan-Lozano et al., 2007
) and that high levels of SIRT1 in brain do not alter synaptic plasticity.
In summary, our results indicate that the NAD+-dependent deacetylase, SIRT1, is critical for maintaining normal acquisition and consolidation of short-term and long–term hippocampus-dependent memories and synaptic plasticity, without modifying basal synaptic properties, dendritic spine structure of CA1 neurons or synaptic proteins levels. An important decrease in neuronal dendritic tree arborization, branch length and complexity may account for some of the deficits observed in SIRT1-KO mice. Also, our work suggests that deficiency in the ERK1/2-MAPK pathway in combination with altered expression of genes involved in synaptic functions and myelination are some of the mechanisms through which SIRT1 may regulate memory, learning and synaptic plasticity in hippocampus. Additional studies should provide further insights into the unknown molecular mechanisms through which SIRT1 regulates normal mouse cognitive functions.