Numerous studies have consistently shown a remarkably large change in the transcriptome across sleep/wake states. Our preliminary evidence based on RNAseq technology suggests more than 2800 genes are differentially expressed in recovery sleep in response to four hours of sleep deprivation, compared to control conditions. These DEG's are enriched for genes related to synaptic function and for targets of a transcription factor, myocyte enhancing factor 2 (MEF2C), that controls synaptic strength. A leading proposal for the function of sleep posits an overall buildup of cortical synaptic strength during waking experience and a decrease of synaptic strength during ensuing sleep. Expression of the active form of Mef2 decreases synaptic strength. Our preliminary evidence suggests that four hours of sleep deprivation increases the MEF2 (the active form) to pMEF2 (the repressive form) ratio. This has lead us to the hypothesis that sleep- need modulates the activity of the transcription factor, MEF2C, to alter the expression of downstream mRNA to reduce glutamate synapse strength on forebrain cortical glutamate neurons. We plan to test this hypothesis by first establishing the differential transcriptome expression across behavioral state conditions in wildtype mice and then to compare it across these same conditions to the expression in conditional Mef2c knockout mutants (the ko will be restricted to glutamate forebrain neurons). We plan to characterize a sleep need/resolution phenotype in the Mef2c mutant using three indices of sleep need. Mutants and wildtype controls will be examined under baseline and sleep deprivation (SD) conditions. Our preliminary evidence shows that the homeostatic sleep response is lost in the mutants. The phosphorylation state of MEF2 determines its activity so we will assess the phosphorylation state of MEF2 in correlation with behavioral state condition. Additionally, since high noradrenergic activity can cause cleavage of the N-terminal of HDAC4 to inhibit MEF2 transcriptional activity, we will assess this N-terminal product across sleep wake states as well. We will characterize sleep-related, Mef2 dependent structural and functional changes of synapses, including morphologically defined spine number, dendritic structure and mEPSC frequency and amplitude recorded from layer 2-3 & 5-6 pyramidal neurons, in frontal cortical lobe (the anterior cingulate) in vitro. This proposal will provide one of the first comprehensive RNAseq based analyses across sleep/wake states that we be a useful resource for investigators to aid in the investigation and understanding of the large transcriptomic change that takes place in response to prolonged waking. It can provide an essential starting point for the identification of sleep related cell autonomous signaling cascades and molecular targets, important to brain health.
Disrupted sleep is associated with all major psychiatric disorders, metabolic syndrome and neurodegeneration consistent with a sleep?related pathological role(s). However, our lack of understanding of sleep?related neurobiology has been problematic for the characterization of these striking associations in terms of underlying neurobiological mechanisms. This project will provide a foundation for the understanding of how sleep elicited cell autonomous molecular and synaptic mechanisms can be related to these CNS pathologies.
