Main findings
Previous studies have shown that histone methylation is involved in the regulation of learning and memory signalling pathways.18 Histone demethylase encoding gene UTX is a key pathogenic risk gene of KS,19 highlighting its important role in the nervous system, especially in cognitive function. Here, we found that UTX cKO mice displayed remote fear memory deficits, indicating the critical role of UTX in the regulation of learning and memory.
UTX is a pathogenic risk gene for KS,19 a condition characterised by congenital developmental abnormalities and mental disorders. UTX specifically removes methyl groups from histone 3 dimethylated or trimethylated at lysine 27 (H3K27me2/3 methyl group), promoting gene transcription. Given the role of histone 3 methylation and demethylation in modulating the expression of memory-related genes,20 we speculate that impaired UTX expression may result in abnormal histone 3 methylation and induce memory deficit. To investigate the role of UTX in learning and memory, we generated a UTX cKO mouse model. Our data support the hypothesis that UTX regulates methylation-induced memory deficit and indicate that UTX deletion downregulates calmodulin transcription by disrupting H3K27me3 demethylation.
Interestingly, UTX cKO mice presented a significant memory impairment at 4 weeks after aversive stimulus (ie, the electric shocks during fear conditioning training), while the contextual memory tested at 24 hours after training remained unaffected. This may be attributed to the dynamic nature of memory storage, where long-term memories do not form immediately after learning but develop with time,21 22 and de novo gene expression governed by the epigenome necessary for memory stabilisation requires at least 24 hours.23 Additionally, the experience of fear can induce persistent activity-specific transcriptional alterations that last for weeks.24
Long-term synaptic plasticity, a synaptic network in which the number or type of synaptic connections changes in response to learning, is an important mechanism underlying the formation and storage of memory.25 This process, which involves the expression of plasticity-related proteins required to construct and sustain long-lasting synaptic alterations, typically takes time ranging from hours to days.26 In our study, UTX cKO mice presented a cognitive dysfunction only at 4 weeks but not 24 hours after training, possibly due to the disrupted synaptic connection and neuron morphology.
Hippocampal activity-dependent LTP is proposed as a major cellular mechanism of learning and memory. Indeed, it has long been suggested that LTP impairment results in memory deficit via calmodulin-CaMKII signalling pathway.27 Specifically, CaMKII phosphorylation which occurs at Thr286 is activated by calcium-bound calmodulin through a direct binding mechanism that is critical for LTP induction and the memory process.28 In the current study, we observed that LTP was impaired in UTX cKO mice. Furthermore, we found that the expression level of calmodulin was downregulated through disruption of H3K27me3 binding with the promoter region of calmodulin, which directly led to decreased CaMKII phosphorylation at Thr286 site in UTX cKO mouse, suggesting that UTX plays a critical role in maintaining LTP via calmodulin-CaMKII pathway.
Phosphorylation of glutamate receptors, including AMPA and NMDA receptors, is considered to underlie the transmission regulation in LTP and is mediated by CaMKII.29 Here, we examined the phosphorylation of AMPA and NMDA receptors, and found that both were decreased in UTX cKO mice. Interestingly, our result showed that the sEPSC frequency was also decreased in UTX cKO mice, contrary to previous studies reporting that decreased phosphorylation of AMPA and NMDA receptors led to reduced sEPSC amplitude instead of frequency. This highlights the needs for further studies to understand the complex role of UTX in synaptic transmission. Overall, UTX deficit in mouse results in decreased expression of calmodulin, leading to impaired CaMKII phosphorylation, therefore decreasing the phosphorylation of AMPA and NMDA receptors, and ultimately causing LTP abnormality and remote memory deficits. Importantly, all these effects could be rescued with desipramine treatment, further supporting our finding that UTX deficiency results in LTP impairment via modulating the calmodulin-CaMKII pathway.
In summary, KS is a rare developmental disorder and its pathogenesis remains elusive. In this study, we explored how the deficiency of UTX, a primary pathogenic risk gene of KS, leads to plasticity and memory deficits. Through RNA-seq analysis, we examined several critical signalling pathways in UTX cKO mice, including the calmodulin-CaMKII pathway. The rescue effect of desipramine treatment on synaptic and cognitive deficits observed in this study may provide a potential therapeutic target for KS. In addition to the calmodulin-CaMKII pathway, our study identified several other pathways known to be involved in memory regulation in UTX cKO mice, including the dopaminergic pathway, Ras signalling pathway and cAMP signalling pathway.30 Whether these pathways are involved in the synaptic plasticity and memory deficits observed in UTX cKO mice need to be further studied.