Aarti Jagannath

Sleep deprivation (SD) triggers characteristic changes in the cortical electroencephalogram (EEG) during waking and sleep, as well as upregulating molecular signatures of neuronal activity, synaptic plasticity, and endoplasmic reticulum (ER) stress. This includes the induction of the unfolded protein response (UPR), the primary marker and adaptive mechanism of ER stress. Yet how cortical neuronal activity during SD contributes to the induction of these molecular signatures remains unknown. Here we investigate whether manipulating cortical activity during SD with a physiological stimulation paradigm (flickering light stimulation, FLS) affects the induction of the UPR. As expected, SD alone upregulated activity-dependent and UPR-related genes in cortex and led to increased EEG slow-wave activity (SWA) during subsequent NREM sleep, an electrophysiological signature of sleep need. Delivering FLS during SD, however, prevented the upregulation of activity- and UPR-related genes while inducing an additional increase in SWA during subsequent sleep. Further analysis of neuronal firing revealed that FLS induced brief neuronal silent (OFF) periods in awake animals during stimulation, thereby shifting cortical activity towards a brain state resembling sleep. These findings suggest that physiological manipulation of cortical activity during waking is sufficient to dissociate molecular signatures of ER stress from electrophysiological markers of sleep pressure. We propose that global sleep-wake history and cortical network dynamics differentially regulate activity-dependent transcription and the accumulation of sleep pressure.

Disrupted sleep and circadian rhythms are among the most common features of diabetes, and are known to raise the risk of developing the disease. Whether the relationship also runs the other way, with diabetes itself disrupting the clock, has remained unresolved. Using an inducible mouse model of diabetes, in which hyperglycaemia can be induced and then reversed within the same animal, we show that elevated blood glucose is a reversible cause of circadian disruption. Raising blood glucose reduced the amplitude of circadian rest-activity rhythms by 50%, in proportion to the degree of hyperglycaemia, and shortened the free-running period of the clock. Restoring normoglycaemia reversed both effects. Diabetic animals had a delayed phase of activity onset, i.e. later chronotype, and re-entrained more slowly after a simulated jet lag. However, phase shifting to nocturnal light pulses was intact, suggesting normal photic responses. Brain glucose and O-GlcNAcylation, a nutrient-sensitive protein modification, were both elevated in diabetic animals, showing that peripheral hyperglycaemia reaches the brain and alters its metabolic state, and providing a molecular readout of that exposure. Together, these findings establish that a peripheral metabolic state is a reversible determinant of circadian rest-activity behaviour, completing a bidirectional link between blood glucose and the clock and arguing that the two must be treated together rather than in isolation. Graphical

Lithium is the gold standard mood stabiliser used to treat cycling mania and depression in bipolar disorder. Despite seven decades of clinical use, the mechanisms of its mood stabilisation are incompletely understood, fundamentally limiting development of improved alternatives. Two established lithium targets, glycogen synthase kinase 3β (GSK3β) and inositol monophosphatase, both modulate phosphorylation, suggesting lithium may exert broad effects on neuronal phosphorylation networks. We performed a discovery-phase in vitro screen of 140 kinases at 10mM LiCl and demonstrated that lithium inhibits 17 kinases beyond GSK3β. We therefore used untargeted quantitative phosphoproteomics to create a comprehensive map of lithium's phosphorylation signature in mouse synaptoneurosomes collected at dawn and dusk, matching peaks in phosphorylation driven by the sleep/wake cycle. Genes encoding lithium-sensitive phosphoproteins were significantly enriched in bipolar disorder genome-wide association studies, providing independent genomic evidence that these phosphorylation networks are relevant to bipolar pathophysiology. We further refined existing models of lithium’s action by showing that GSK3β inhibition is temporally restricted to dawn, indicating cross talk with sleep/wake cycles of phosphorylation. Overall, our data demonstrate that lithium’s pleiotropic effects may result from coordinated multi-kinase network reorganisation rather than single-target inhibition — a principle with direct implications for rational polypharmacology in mood stabiliser development.

The two-process model posits that sleep-wake timing results from the integration of circadian (Process C) and homeostatic (Process S) signals, but how these processes converge at a molecular level remains unknown. Here we show that Salt-Inducible Kinase 1 (SIK1) gates circadian control of wake timing through direct phosphorylation of suprachiasmatic nucleus (SCN) presynaptic networks. Using quantitative phosphoproteomics of the SCN combined with kinase-substrate validation (KALIP), we show that SIK1 controls a presynaptic signalling network that maintains SCN network synchrony. Loss of SIK1 catalytic activity disrupts a SIK1→PKCγ→Ca²⁺ cascade, enabling rapid behavioural shifts in sleep/wake timing under circadian misalignment. Critically, behavioural shifts precede transcriptional re-alignment of clock gene expression, establishing phosphorylation-driven SCN network dynamics as a determinant of circadian wake timing. Since the loss of SIK1 activity alters Process C without disrupting Process S, we could isolate the molecular signatures of circadian wake signals in the cortex. Cortical phosphoproteomics showed that circadian wake signals engage the same CaMKII-dependent synaptic phosphorylation substrates previously identified as markers of homeostatic sleep pressure, establishing convergent 'wake substrates' where Process C and Process S interface. Collectively, these findings identify the molecular mechanisms through which the circadian clock controls wake timing and their convergence with homeostatic sleep regulation.

Lithium is the gold standard mood stabiliser used to treat cycling mania and depression in bipolar disorder. Despite seven decades of clinical use, the mechanisms of its mood stabilisation are incompletely understood, fundamentally limiting development of improved alternatives. Two established lithium targets, glycogen synthase kinase 3β (GSK3β) and inositol monophosphatase, both modulate phosphorylation, suggesting lithium may exert broad effects on neuronal phosphorylation networks. We performed a discovery-phase in vitro screen of 140 kinases at 10mM LiCl and demonstrated that lithium inhibits 17 kinases beyond GSK3β. We therefore used untargeted quantitative phosphoproteomics to create a comprehensive map of lithium’s neural phosphorylation signature in lithium-treated mouse synaptoneurosomes. Samples were collected at dawn and dusk to match the peaks in phosphorylation that are induced by the sleep/wake cycle. Pathway analysis revealed convergence on synaptic plasticity, neurotransmitter release, and chemical transmission. Critically, lithium-sensitive phosphoproteins are significantly enriched in bipolar disorder genome-wide association study (GWAS) loci, providing independent genomic evidence that the phosphorylation networks we identified are relevant to bipolar pathophysiology. We identified novel kinase targets and phosphorylation sites not previously associated with lithium’s mechanism of action and tied them to bipolar pathology. We further refined existing models of lithium’s action by showing that GSK3β inhibition is temporally restricted to dawn, indicating cross talk with sleep/wake cycles of phosphorylation. Overall, our data demonstrate that lithium’s pleiotropic effects result from coordinated multi-kinase network reorganisation rather than single-target inhibition — a principle with direct implications for rational polypharmacology in mood stabiliser development.

Historically, the primary method for measuring murine circadian activity in vivo has been monitoring voluntary wheel running. Recently, passive infrared (PIR) motion sensors have emerged as an alternative that is not reliant on voluntary behaviour. While research has examined the differences between the two methods for measuring circadian parameters, little focus has been placed on how these techniques may confound the assessment of therapeutic interventions. Here, we show that wheel running activity is disproportionately affected by daily oral gavage of saline compared to sham gavage treatment. In contrast, PIR-monitored activity indicates little difference between the two treatments. Both PIR and running-wheel-measured activity show a reduction in circadian amplitude and an increase in intradaily variability during both types of gavage, likely reflecting the stress of daily gavage, though the mice showed no weight loss. This finding indicates that pre- and post-intervention comparisons will misattribute gavage effects to the intervention unless appropriate sham and vehicle controls are included. More broadly, the choice of circadian measurement technique fundamentally shapes the interpretation of pharmacological interventions and must be considered in experimental design.

Throughout history, the development of new sleep medicines has been driven by progress in our understanding of the mechanisms underlying sleep. Ancient civilisations used their understanding of the sedative nature of natural herbs and compounds to induce sleep. The discovery of barbiturates and bromides heralded a new era of synthetic sleep medicine in the 19th century. This was followed by the development of benzodiazepines that were used to inhibit signalling throughout the brain by promoting gamma‐amino butyric acid release and thereby produce loss of consciousness. As our understanding of sleep has deepened, newer therapies have more specifically targeted the wake‐inducing neurotransmitter orexin with fewer side effects. Given the newly highlighted role of kinases in sleep/wake regulation, we predict that the next breakthroughs in sleep medicine will likely target these kinases. Given the fundamental role that sleep plays in maintaining brain health through processes such as glymphatic clearance, sleep medicine has therapeutic potential beyond just sleep. Recent evidence suggests that sleep disruptions directly contribute to the build‐up of pathological neuronal proteins in neurodegenerative disorders. Therefore, sleep medicine could improve prognosis in disorders such as these. Great attention must be paid to the mechanism of action of each sleep medicine, however, as sleep medicines which do not fully mimic sleep could actually worsen disease progression.

Sleep deprivation (SD) negatively impacts nearly all brain functions including cognition, memory consolidation and metabolism. However, the gene regulatory networks that underlie these biological effects are not well understood. In order to identify these networks, we conducted a multiomic analysis to analyse how gene expression, chromatin accessibility, enhancer activity and DNA methylation change with acute SD in mice. By studying three brain regions involved in different aspects of sleep - the cortex (CTX), dentate gyrus (DG), and suprachiasmatic nucleus (SCN) - we found that the effects of SD on the multiome varied widely, impacting physiological processes specific to each area, from spine formation in the dentate gyrus to neuropeptide release in the SCN. Our integrated analysis showed that distinct brain region-specific networks of regulatory factors dynamically alter the epigenomic landscape in response to SD to orchestrate transcriptional responses. These findings provide new understanding of the regulatory grammar encoded within the genome, which enables a general physiological signal like SD to produce unique effects in a tissue-specific context.

Multiple studies have documented sex differences in sleep behaviour, however, the molecular determinants of such differences remain unknown. Furthermore, most studies addressing molecular mechanisms have been performed only in males, leaving the current state of knowledge biased towards the male sex. To address this, we studied the differences in the transcriptome of the cerebral cortex of male and female C57Bl/6 J mice after 6 h of sleep deprivation. We found that several genes, including the neurotrophin growth factor Bdnf, immediate early genes Fosb and Fosl2, and the adenylate cyclase Adcy7 are differentially upregulated in males compared to females. We identified the androgen-receptor activating transcription factor EZH2 as the upstream regulatory element specifying sex differences in the sleep deprivation transcriptome. We propose that the pathways downstream of these transcripts, which impact on cellular re-organisation, synaptic signalling, and learning may underpin the differential response to sleep deprivation in the two sexes.