In an effort to advance the understanding on the mechanisms of action through which HDAC4 regulates LTM, the focus of this study was to identify upstream regulators or downstream targets of the molecular pathway in which HDAC4 acts. As HDAC4 is a demonstrated transcriptional regulator (Miska et a., 1999; Grozinger and Schreiber, 2000; Huang et al., 2000; Kao et al., 2000; Vega et al., 2004; Li et al., 2012; Sando et al., 2012) and since it is able to shuttle between the nucleus and the cytoplasm, a transcriptome analysis was performed to detect whether in Drosophila neuronal nuclei, HDAC4 mediates transcriptional changes. The experimental set up was designed in order determine whether the LTM impairment observed in flies overexpressing HDAC4 in all neurons of the brain (Fitzsimons et al., 2013) could be related to transcriptional changes induced by HDAC4OE. Very few changes in transcription were detected.
A limitation of this approach, could reside in the fact that sequencing was performed on cDNA of RNA isolated from whole heads rather than from the brain tissue specifically, likely resulting in a decreased sensitivity to detect global changes. The analysis was however able to detect significant differential transcriptional changes in 28 genes compared to wild-type samples, including both HDAC4 and the white gene. A further explanation that could account for the minimal effect on transcription is that HDAC4 is predominantly cytoplasmic and localises in a small subset of nuclei, most of which are in the Kenyon cells of the mushroom body (P. S. Freymuth personal communication).
Isolation of those neuronal nuclei would be a valuable approach to limit the transcriptional profiling to the single nuclei of the mushroom body, distinguished from the rest of the cells by expression of a tag such as green fluorescent protein (GFP) (Deal and Henikoff., 2010). A strategy named INTACT (isolation of nuclei tagged in specific cell types) could be adopted to genetically tag nuclei in the brain and purify them from the total pool through affinity isolation.Isolated nuclei can be used for applications such as gene expression analysis and chromatin immunoprecipitation. Weake and colleagues described a protocol specific for Drosophila, in which the GAL4/UAS binary expression system is used to genetically label the nuclei of cells of interest with a nuclear envelope-
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localized EGFP tag. The EGFP-tagged nuclei can then be isolated using antibodies against GFP that are coupled to magnetic beads (Ma and Weake, 2014).
Despite the above mentioned limitations, these results were in line with microarray studies in which manipulation of HDAC4 did not yield significant changes in global gene expression in the mouse hippocampus (Kim et al., 2012). However, capability of HDAC4 to regulate transcription was shown in a study by Sando and colleagues (2012) who reported that expression of a nuclear restricted form of HDAC4 in mouse primary cortical neurons resulted in differential transcript abundance of a subset of genes essential for synaptic function, meaning that HDAC4 is capable of regulating transcription when present into the nucleus. Of the genes found to be transcriptionally regulated by HDAC4 in this study, only a few were significantly expressed in the brain, including Activity regulated cytoskeleton associated protein 1 (Arc1),Kekkon 2 and Laminin A.
Kekkon 2 is expressed in neurons and it is a member of a class of genes highly conserved from invertebrates to humans (MacLaren et al., 2004) that encodes transmembrane proteins with six leucine-rich repeats and a single immunoglobulin loop (LIG family) (Musacchio and Perrimon, 1996). Kekkon 2 was identified in a microarray screen for genes whose transcriptional activity is regulated by neuronal excitability (Guan et al., 2005). It was found that Kekkon 2 is enriched in axons and synaptic terminal of the Drosophila larvae and that its absolute levels are required to regulate synaptic varicosity number, as both increased and decreased levels of expression of this gene caused a reduction in the number of boutons at the neuromuscular junction, suggesting a role for Kekkon 2 in synaptic plasticity (Guan et al., 2005). Interestingly, a conserved Kekkon 2- like mammalian member of the LIG family named AMIGO/Alivin was shown to be subjected to activity-dependent transcriptional regulation and to promote neurite outgrowth (Kuja-Panula et al., 2003), suggesting the possibility of a conserved role in activity-dependent modification of synaptic connectivity (MacLaren et al., 2004).
Laminin is a glycoprotein complex of the extracellular matrix, which has been shown to promote neurite outgrowth in vitro (Kuhn et al., 1995) and further studies showed that extracellular matrix containing Laminin A is required for the normal pathfinding by the ocellar pioneer axons in Drosophila (García-Alonso et al., 1996). Laminin A localises at the synaptic clefts and its overexpression in the postsynaptic region results in decreased growth of the neuromuscular junction (Tsai et al., 2012).
Arc1 is one of three Drosophila homologs of mammalian activity-regulated cytoskeleton-associated protein (ARC)
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In mammals ARC is an immediate-early gene153
essential for synaptic plasticity and possible implications in onset of Alzheimer’s disease have been recently suggested in mouse model of Alzheimer’s disease, where intraneuronal amyloid-β expression in the CA3 region of the hippocampus correlated with increased neuronal activation leading to impairments in synaptic function and memory processes (Morin et al., 2016). However, in Drosophila, an Arc null mutant did not display synaptic plasticity defects at the larval neuromuscular junction nor impairments in memory formation, although behavioural data were limited to immediate memory recall (zero to two minutes after training) and to two hour STM of courtship conditioning. LTM was not evaluated (Mattaliano et al., 2007). Interestingly, Arc expression is positively regulated by Mef2 in neurons (Flavell et al., 2006) and an interaction between HDAC4 and MEF2 is known to occur in neuronal nuclei with HDAC4 repressing MEF2 transcriptional activity (Li et al., 2012) suggesting that decreased levels of Arc1 in the fly head may result from negative regulation of Mef2 activity by HDAC4.
In summary, these genes are potential HDAC4 targets and further investigation on the interaction between these genes and HDAC4 and their roles in brain development and memory formation, may shed light on the molecular pathway through which HDAC4 regulates such processes.