Recent molecular, cellular, and behavioral studies indicate that pharmacological inhibition of HDACs can ameliorate cognitive deficits produced by ageing or neurodegeneration (Guan et al., 2009
; Peleg et al., 2010
; Fischer et al., 2010
; Gräff et al., 2012
). Harnessing the therapeutic potential of HDAC inhibitors linked to cognitive enhancement requires knowledge of the specific HDAC family member(s) involved, and the downstream transcriptional pathways regulated by these HDACs that may be involved in memory. With the long-term goal of determining if these HDAC-regulated pathways can be targeted for therapeutic benefit in the context of disorders of cognition, we have sought to identify improved small-molecule probes of HDACs relevant to neuroplasticity. shows potential mechanisms by which one such probe molecule, crebinostat, could have these effects by enhancing CREB-dependent transcription (see Introduction and Results for details).
Potential models of regulation of CREB-dependent transcription, synaptic function, and memory by HDACs
Crebinostat was selected from amongst a library of over 1,200 small molecules designed to target the HDAC family (Tang et al., 2011
) on the basis of its strong activity in the primary CREB reporter gene assay. Consistent with its strong cellular activity, crebinostant was found to potently inhibit the class I HDACs 1, 2, 3, and class IIb HDAC6, and to a lesser extent the class I HDAC8 (), suggesting that one or more of these isoforms repress CREB-dependent transcription. Indeed, HDAC1 has been reported to promote CREB dephosphorylation through the recruitment of PP1 (Canettieri et al., 2003
). In addition, HDAC2 has recently been demonstrated to repress transcription of the CREB target genes Egr1
in the brain (Guan et al., 2009
). Finally, epistasis experiments in C. elegans
indicate that HDAC3 can oppose CREB-mediated neuroprotection (Bates et al., 2006
). While our data with crebinostat do not support a role for class IIa HDACs, it has been reported that HDAC4 can repress CREB-dependent transcription (Bolger & Yao, 2005
). Moreover, we were not able to study the effects of crebinostat on the enzymatic activity of HDACs 10 and 11 in vitro
due to current limitations in our ability to develop robust biochemical assays for these isoforms, and thus the role of these two HDACs is uncertain. Further work will be required to fully elucidate which HDAC isoforms repress CREB-dependent transcription, and whether different isoforms play distinct roles in a cell-type specific or signaling-dependent manner. It will also be important to determine whether class I HDAC isoform-selective inhibitors can enhance CREB-dependent transcription and cognition.
Our results demonstrating that crebinostat treatment increases the number of synapsin I positive punctae, reflective of the assembly and clustering of pre-synaptic vesicles along Map2 staining dendrites, are consistent with those described by our group recently for a structurally related HDAC inhibitor, synapsinostat, which we showed could enhance synaptogenesis in a microfluidics-based co-culture assay (Shi et al., 2012). However, data reported here indicate that crebinostat is capable of either enhancing the formation of synapses, or preventing their loss, or both, and both the pre-synaptic and post-synaptic contributions to this process occur in primary cultured mouse neurons tested here, in contrast to the artificial conditions of the co-culture assay.
Our transcript profiles generated from primary mouse neurons treated with crebinostat allowed genome-wide assessment of the effects of a memory enhancing probe on the regulation of gene expression. As anticipated from early studies on the effects of HDAC inhibitors on CREB-mediated transcription (Fass et al., 2003
), as well as systematic studies of neural-activity dependent changes in gene expression (Benito et al., 2011
), we observed both upregulation and downregulation of gene expression. The analyses performed here, particularly the intersection with global datasets of promoter occupancy by CREB through chromatin immunoprecipitation experiments, and synaptome constituent identification proteomic studies, represent one of the first efforts to generate a complete picture of the synaptic regulatory protein interaction network modulated by activation of CREB target genes by an HDAC inhibitor that is known to enhance cognition in mice. We found that crebinostat upregulated the expression of several experimentally validated CREB target genes () that have been reported to play a role in cognition, synaptic function, or neuronal development. For example, one of the networks of genes regulated in our expression profiling in response to crebinostat was that of the Ras GTPase pathway. Dysregulation of Ras-MAPK signaling has been implicated in multiple human genetic disorders that include cognitive deficits, including neurofibromatosis, Noonan syndrome, Costello syndrome, LEOPARD syndrome, CFC syndrome, and Legius syndrome (reviewed in van Bokhoven, 2011
). Thus, beyond Rubinstein-Taybi syndrome, these findings point to a potential broader application of HDAC inhibitors in treating intellectual disabilities, although the specific consequence of each disease causing mutation on downstream signaling and how these altered neuronal cell states respond to HDAC inhibitor treatment will need to be carefully investigated.
It is possible that there are additional CREB target genes in our list of transcripts upregulated by crebinostat (Supplementary Table 1
)—these genes might be identified in future CREB ChIP-seq studies performed on brain tissue. It will be important to identify the full set of CREB target genes that are regulated in the brain by crebinostat, and to determine which of these contribute to the cellular events that mediate its effect on synapse density and enhancement of memory. It will also be important to fully understand how the proteins encoded by these CREB target genes interact to form networks that modulate synaptic physiology in the brain (). A full understanding of these networks will allow us to identify critical modules that drive and control network activity—these modules may be ideal targets for effective therapeutic intervention in disorders of cognition and altered neuroplasticity.
While not addressed here, it will be important to consider the regulation of acetylation of non-histone substrates as part of gaining a comprehensive understanding of the role of HDACs in mediating cognition and neuroplasticity. For example, Yeh et al. (2004)
implicated acetylation of the transcription factor NF-kappaB in memory formation, although the precise HDACs involved in mediating this effect and its relevance to different types of memory and forms of neuroplasticity remain unclear. More recently, quantitative proteomics studies performed in non-neuronal cell lines have identified a total of 1,750 acetylated proteins and 3,600 individual lysine acetylation sites in these proteins (Choudhary et al., 2009
), many of which were sensitive to HDAC inhibitors such as SAHA. Taken together, these findings point to the need in the future better understand the possible contribution of non-histone substrates to the short-term and long lasting effects of HDAC inhibitors such as crebinostat on neuroplasticity. Indeed, such studies are ongoing in our lab.
While there are attractive features of crebinostat as a probe of chromatin-mediated neuroplasticity and memory, there are limitations to the in vivo
pharmacological properties of crebinostat as revealed by the analysis of the brain-to-plasma Cmax
ratio of 0.02, exposure (AUClast
) ratio of 0.02, and T1/2
in brain of ~43 min following IP administration of crebinostat (Supplementary Figure 4
). Now that these limitations are known, future optimization of the pharmacological properties of this series of memory enhancers will aim to increase overall brain exposure and the brain:plasma ratio. While ultimately the optimal exposure profile for HDAC inhibitors for memory enhancement remains to be rigorously determined, and may vary for different behavioral paradigms, we speculate that the pulsatile nature of crebinostat exposure in the brain may be advantageous for the following reasons: 1) a pulsatile type of exposure may mimic aspects of salient cognitive experiences that occur in short duration yet induce long-lasting memories; 2) pulsatile exposure has been reported to be optimal in inducing neuroprotection (Langley et al., 2008
); and 3) pulsatile exposure may minimize undesired effects of long-term HDAC inhibition. An interesting challenge for future studies will be to understand and delineate, amongst different classes of HDAC inhibitors, with different binding kinetics and residence times on different HDAC targets, which parameters determine the optimal pharmacokinetic profiles for HDAC inhibition leading to memory enhancement and other behavioral effects.