Sleep Homeostasis and General Anesthesia: Are Fruit Flies Well Rested after Emergence from Propofol?

BACKGROUND: Shared neurophysiologic features between sleep and anesthetic-induced hypnosis indicate a potential overlap in neuronal circuitry underlying both states. Previous studies in rodents indicate that preexisting sleep debt discharges under propofol anesthesia. The authors explored the hypothesis that propofol anesthesia also dispels sleep pressure in the fruit fly. To the authors' knowledge, this constitutes the first time propofol has been tested in the genetically tractable model, Drosophila melanogaster. METHODS: Daily sleep was measured in Drosophila by using a standard locomotor activity assay. Propofol was administered by transferring flies onto food containing various doses of propofol or equivalent concentrations of vehicle. High-performance liquid chromatography was used to measure the tissue concentrations of ingested propofol. To determine whether propofol anesthesia substitutes for natural sleep, the flies were subjected to 10-h sleep deprivation (SD), followed by 6-h propofol exposure, and monitored for subsequent sleep. RESULTS: Oral propofol treatment causes anesthesia in flies as indicated by a dose-dependent reduction in locomotor activity (n = 11 to 41 flies from each group) and increased arousal threshold (n = 79 to 137). Recovery sleep in flies fed propofol after SD was delayed until after flies had emerged from anesthesia (n = 30 to 48). SD was also associated with a significant increase in mortality in propofol-fed flies (n = 44 to 46). CONCLUSIONS: Together, these data indicate that fruit flies are effectively anesthetized by ingestion of propofol and suggest that homologous molecular and neuronal targets of propofol are conserved in Drosophila. However, behavioral measurements indicate that propofol anesthesia does not satisfy the homeostatic need for sleep and may compromise the restorative properties of sleep.

Early and late chaperone intervention therapy boosts XBP1s and ADAM10, restores proteostasis, and rescues learning in Alzheimer's Disease mice.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder that is pervasive among the aging population. Two distinct phenotypes of AD are deficits in cognition and proteostasis, including chronic activation of the unfolded protein response (UPR) and aberrant Aß production. It is unknown if restoring proteostasis by reducing chronic and aberrant UPR activation in AD can improve pathology and cognition. Here, we present data using an APP knock-in mouse model of AD and several protein chaperone supplementation paradigms, including a late-stage intervention. We show that supplementing protein chaperones systemically and locally in the hippocampus reduces PERK signaling and increases XBP1s, which is associated with increased ADAM10 and decreased Aß42. Importantly, chaperone treatment improves cognition which is correlated with increased CREB phosphorylation and BDNF. Together, this data suggests that chaperone treatment restores proteostasis in a mouse model of AD and that this restoration is associated with improved cognition and reduced pathology. One-sentence summary: Chaperone therapy in a mouse model of Alzheimer's disease improves cognition by reducing chronic UPR activity.

Biology of Stress Responses in Aging.

On April 28th, 2022, a group of scientific leaders gathered virtually to discuss molecular and cellular mechanisms of responses to stress. Conditions of acute, high-intensity stress are well documented to induce a series of adaptive responses that aim to promote survival until the stress has dissipated and then guide recovery. However, high-intensity or persistent stress that goes beyond the cell's compensatory capacity are countered with resilience strategies that are not completely understood. These adaptative strategies, which are an essential component of the study of aging biology, were the theme of the meeting. Specific topics discussed included mechanisms of proteostasis, such as the unfolded protein response (UPR) and the integrated stress response (ISR), as well as mitochondrial stress and lysosomal stress responses. Attention was also given to regulatory mechanisms and associated biological processes linked to age-related conditions, such as muscle loss and regeneration, cancer, senescence, sleep quality, and degenerative disease, with a general focus on the relevance of stress responses to frailty. We summarize the concepts and potential future directions that emerged from the discussion and highlight their relevance to the study of aging and age-related chronic diseases.

Reducing ER stress with chaperone therapy reverses sleep fragmentation and cognitive decline in aged mice.

As the aging population grows, the need to understand age-related changes in health is vital. Two prominent behavioral changes that occur with age are disrupted sleep and impaired cognition. Sleep disruptions lead to perturbations in proteostasis and endoplasmic reticulum (ER) stress in mice. Further, consolidated sleep and protein synthesis are necessary for memory formation. With age, the molecular mechanisms that relieve cellular stress and ensure proper protein folding become less efficient. It is unclear if a causal relationship links proteostasis, sleep quality, and cognition in aging. Here, we used a mouse model of aging to determine if supplementing chaperone levels reduces ER stress and improves sleep quality and memory. We administered the chemical chaperone 4-phenyl butyrate (PBA) to aged and young mice, and monitored sleep and cognitive behavior. We found that chaperone treatment consolidates sleep and wake, and improves learning in aged mice. These data correlate with reduced ER stress in the cortex and hippocampus of aged mice. Chaperone treatment increased p-CREB, which is involved in memory formation and synaptic plasticity, in hippocampi of chaperone-treated aged mice. Hippocampal overexpression of the endogenous chaperone, binding immunoglobulin protein (BiP), improved cognition, reduced ER stress, and increased p-CREB in aged mice, suggesting that supplementing BiP levels are sufficient to restore some cognitive function. Together, these results indicate that restoring proteostasis improves sleep and cognition in a wild-type mouse model of aging. The implications of these results could have an impact on the development of therapies to improve health span across the aging population.

Homer1a regulates Shank3 expression and underlies behavioral vulnerability to stress in a model of Phelan-McDermid syndrome.

Mutations of SHANK3 cause Phelan-McDermid syndrome (PMS), and these individuals can exhibit sensitivity to stress, resulting in behavioral deterioration. Here, we examine the interaction of stress with genotype using a mouse model with face validity to PMS. In Shank3ΔC/+ mice, swim stress produces an altered transcriptomic response in pyramidal neurons that impacts genes and pathways involved in synaptic function, signaling, and protein turnover. Homer1a, which is part of the Shank3-mGluR-N-methyl-D-aspartate (NMDA) receptor complex, is super-induced and is implicated in the stress response because stress-induced social deficits in Shank3ΔC/+ mice are mitigated in Shank3ΔC/+;Homer1a-/- mice. Several lines of evidence demonstrate that Shank3 expression is regulated by Homer1a in competition with crosslinking forms of Homer, and consistent with this model, Shank3 expression and function that are reduced in Shank3ΔC/+ mice are rescued in Shank3ΔC/+;Homer1a-/- mice. Studies highlight the interaction between stress and genetics and focus attention on activity-dependent changes that may contribute to pathogenesis.

Conservation of sleep: insights from non-mammalian model systems.

The past 10 years have seen new approaches to elucidating genetic pathways regulating sleep. The emerging theme is that sleep-like states are conserved in evolution, with similar signaling pathways playing a role in animals as distantly related as flies and humans. We review the evidence for the presence of sleep states in non-mammalian species including zebrafish (Danio rerio), fruitflies (Drosophila melanogaster) and roundworms (Caenorhabditis elegans). We describe conserved sleep-regulatory molecular pathways with a focus on cAMP and epidermal growth factor signaling; neurotransmitters with conserved effects on sleep and wake regulation, including dopamine and GABA; and a conserved molecular response to sleep deprivation involving the chaperone protein BiP/GRP78.

Changes in components of energy regulation in mouse cortex with increases in wakefulness.

STUDY OBJECTIVES: Increases in ATP production machinery have been described in brain after 3 h of sleep deprivation. Whether this is sustained with longer durations of extended wakefulness is unknown. We hypothesized that energy depletion could be a mechanism leading to difficulty maintaining wakefulness and assessed changes in components of the electron transport chain. DESIGN: Protein levels of key subunits of complexes IV and V of the electron transport chain (COXI, COXIV, ATP5B) and uncoupling protein 2 (UCP2) in isolated mitochondria by Westerns in mouse cerebral cortex after 3 and 12 h of sleep deprivation were compared to that in control mice. Activity of complex IV enzyme and relevant transcription factors-Nrf1, Nrf2 (Gabp), and phosphorylation of AMP-dependent kinase (AMPK)-were also assessed. PARTICIPANTS: 8-10 week old C57BL/6J male mice (n = 91). INTERVENTIONS: 3, 6, and 12 h of sleep deprivation. MEASUREMENTS AND RESULTS: After both 3 and 12 h of sleep deprivation, complex IV proteins and enzyme activity were significantly increased. The complex V catalytic subunit was significantly increased after 12 h of sleep deprivation only. Increased levels of UCP2 protein after 12 h of sleep deprivation suggests that there might be alterations in the ATP/AMP ratio as wakefulness is extended. That phosphorylation of AMPK is increased after 6 h of sleep deprivation supports this assertion. The increase in Nrf1 and Nrf2 (Gabp) mRNA after 6 h of sleep deprivation provides a mechanism by which there is up-regulation of key proteins. CONCLUSIONS: There are complex dynamic changes in brain energy regulation with extended wakefulness.

The energy hypothesis of sleep revisited.

One of the proposed functions of sleep is to replenish energy stores in the brain that have been depleted during wakefulness. Benington and Heller formulated a version of the energy hypothesis of sleep in terms of the metabolites adenosine and glycogen. They postulated that during wakefulness, adenosine increases and astrocytic glycogen decreases reflecting the increased energetic demand of wakefulness. We review recent studies on adenosine and glycogen stimulated by this hypothesis. We also discuss other evidence that wakefulness is an energetic challenge to the brain including the unfolded protein response, the electron transport chain, NPAS2, AMP-activated protein kinase, the astrocyte-neuron lactate shuttle, production of reactive oxygen species and uncoupling proteins. We believe the available evidence supports the notion that wakefulness is an energetic challenge to the brain, and that sleep restores energy balance in the brain, although the mechanisms by which this is accomplished are considerably more complex than envisaged by Benington and Heller.

Sleep and Cellular Stress.

Sleep is a universal phenomenon occurring in all species studied thus far. Sleep loss results in adverse physiological effects at both the organismal and cellular levels suggesting an adaptive role for sleep in the maintenance of overall health. This review examines the bidirectional relationship between sleep and cellular stress. Cellular stress in this review refers to a shift in cellular homeostasis in response to an external stressor. Studies that illustrate the fact that sleep loss induces cellular stress and those that provide evidence that cellular stress in turn promotes sleep will be discussed.

Genetic disruption of the putative binding site for Homer on DmGluRA reduces sleep in Drosophila.

Homer proteins mediate plasticity and signaling at the postsynaptic density of neurons and are necessary for sleep and synaptic remodeling during sleep. The goal of this study was to investigate the mechanisms of sleep regulation by Homer signaling. Using the Drosophila animal model, we demonstrate that knockdown of Homer specifically in the brain reduces sleep and that Drosophila Homer binds to the sole Drosophila mGluR, known as DmGluRA. This is the first evidence that DmGluRA, which bears greatest homology to group II mammalian metabotropic glutamate receptors (mGluRs), shares functional homology with group I mGluRs which couple to Homer proteins in mammals. As sleep is associated with the physical dissociation of Homer and mGluRs proteins at the synapse, we sought to determine the functional necessity of Homer × DmGluRA interaction in sleep regulation. Using the CRISPR/Cas9 gene editing system, we generated a targeted amino acid replacement of the putative binding site for Homer on DmGluRA to prevent Homer and DmGluRA protein binding. We found that loss of the conserved proline-rich PPXXF sequence on DmGluRA reduces Homer/DmGluRA associations and significantly reduces sleep amount. Thus, we identify a conserved mechanism of synaptic plasticity in Drosophila and demonstrate that the interaction of Homer with DmGluRA is necessary to promote sleep.

Acute Sleep Loss Upregulates the Synaptic Scaffolding Protein, Homer1a, in Non-canonical Sleep/Wake Brain Regions, Claustrum, Piriform and Cingulate Cortices.

Homer proteins are a component of the post-synaptic density of neurons that are necessary for the maintenance and consolidation of behavioral state. The dominant negative protein homer1a is rapidly increased by neuronal activity and sleep loss. Homer1a knockout mice with globally absent homer1a have reduced ability to sustain wakefulness during the active period. It is not known whether homer1a is required globally or in very specific brain regions or neurons for its role in maintaining wake. In this study, we examined the expression of homer1a, an immediate early gene involved in intracellular signaling cascades, in mice subjected to extended wakefulness. We found that mice displayed increased expression of homer1a in the claustrum, a brain region thought to be involved in consciousness, as well as the cingulate and piriform cortices compared to non-sleep deprived mice. In situ hybridization (ISH) studies also indicate that homer1a is not induced in the known wake promoting regions with sleep deprivation, but is instead upregulated primarily in the claustrum and piriform cortex. Examination of homer1a expression levels with recovery sleep after sleep deprivation indicate that baseline homer1a expression levels were restored. Further, we have identified that homer1a is upregulated in excitatory neurons of the claustrum suggesting that homer1a promotes wakefulness through activating excitatory neurons. This work identifies regions previously unknown to be involved in sleep regulation that respond to acute sleep deprivation or enhanced waking.

Evolutionarily Conserved Regulation of Sleep by the Protein Translational Regulator PERK.

Sleep is a cross-species phenomenon whose evolutionary and biological function remain poorly understood. Clinical and animal studies suggest that sleep disturbance is significantly associated with disruptions in protein homeostasis-or proteostasis-in the brain, but the mechanism of this link has not been explored. In the cell, the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway modulates proteostasis by transiently inhibiting protein synthesis in response to proteostatic stress. In this study, we examined the role of the PERK pathway in sleep regulation and provide the first evidence that PERK signaling is required to regulate normal sleep in both vertebrates and invertebrates. We show that pharmacological inhibition of PERK reduces sleep in both Drosophila and zebrafish, indicating an evolutionarily conserved requirement for PERK in sleep. Genetic knockdown of PERK activity also reduces sleep in Drosophila, whereas PERK overexpression induces sleep. Finally, we demonstrate that changes in PERK signaling directly impact wake-promoting neuropeptide expression, revealing a mechanism through which proteostatic pathways can affect sleep and wake behavior. Taken together, these results demonstrate that protein synthesis pathways like PERK could represent a general mechanism of sleep and wake regulation and provide greater insight into the relationship between sleep and proteostasis.

Eif-2a protects brainstem motoneurons in a murine model of sleep apnea.

Obstructive sleep apnea is associated with neural injury and dysfunction. Hypoxia/reoxygenation exposures, modeling sleep apnea, injure select populations of neurons, including hypoglossal motoneurons. The mechanisms underlying this motoneuron injury are not understood. We hypothesize that endoplasmic reticulum injury contributes to motoneuron demise. Hypoxia/reoxygenation exposures across 8 weeks in adult mice upregulated the unfolded protein response as evidenced by increased phosphorylation of PERK [PKR-like endoplasmic reticulum (ER) kinase] in facial and hypoglossal motoneurons and persistent upregulation of CCAAT/enhancer-binding protein-homologous protein (CHOP)/growth arrest and DNA damage-inducible protein (GADD153) with nuclear translocation. Long-term hypoxia/reoxygenation also resulted in cleavage and nuclear translocation of caspase-7 and caspase-3 in hypoglossal and facial motoneurons. In contrast, occulomotor and trigeminal motoneurons showed persistent phosphorylation of eIF-2a across hypoxia/reoxygenation, without activations of CHOP/GADD153 or either caspase. Ultrastructural analysis of rough ER in hypoglossal motoneurons revealed hypoxia/reoxygenation-induced luminal swelling and ribosomal detachment. Protection of eIF-2alpha phosphorylation with systemically administered salubrinal throughout hypoxia/reoxygenation exposure prevented CHOP/GADD153 activation in susceptible motoneurons. Collectively, this work provides evidence that long-term exposure to hypoxia/reoxygenation events, modeling sleep apnea, results in significant endoplasmic reticulum injury in select upper airway motoneurons. Augmentation of eIF-2a phosphorylation minimizes motoneuronal injury in this model. It is anticipated that obstructive sleep apnea results in endoplasmic reticulum injury involving motoneurons, whereas a critical balance of phosphorylated eIF-2a should minimize motoneuronal injury in obstructive sleep apnea.

Aging impairs the unfolded protein response to sleep deprivation and leads to proapoptotic signaling.

Protein misfolding, accumulation, and aggregation characterize many aging-related diseases. Protein aggregates do not accumulate in unstressed cells primarily because of the existence of competent cellular "quality control" machinery. The endoplasmic reticulum (ER) is a major part of this quality control system. Accumulation of misfolded proteins in the ER causes ER stress and activates a signaling pathway called the unfolded protein response (UPR). The UPR limits protein load by upregulating ER chaperones such as Ig binding protein (BiP)/glucose-regulated protein 78 (GRP78) and by attenuating protein translation through eukaryotic initiation factor 2 alpha (eIF2alpha) phosphorylation. Acute sleep deprivation (6 h) in young mice leads to induction of the UPR with upregulation of BiP/GRP78 and attenuation of protein translation. We demonstrate here that aging impairs this adaptive response to sleep deprivation. Aged mice do not display an increase in BiP expression with acute sleep deprivation. In addition, there is decreased basal expression of BiP/GRP78 in aged mice. There is a decline in eIF2alpha phosphorylation in aged mouse cerebral cortex that is associated with higher levels of GADD34 (growth arrest and DNA damage 34) and proapoptotic proteins such as CCAAT/enhancer-binding protein-homologous protein and activated caspase-12, suggesting that young animals possess an efficient ER adaptive response that declines with aging.

The endoplasmic reticulum stress response and aging.

The endoplasmic reticulum (ER) is a multifunctional organelle which co-ordinates protein folding, lipid biosynthesis, calcium storage and release. Perturbations that disrupt ER homeostasis lead to ER stress and upregulation of a signaling pathway called the unfolded protein response (UPR). The UPR while robust in young animals appears to be compromised with aging; many of the components of the UPR have decreased expression and activity with age. There is also considerable evidence of oxidative damage. There are suggestions that an impaired UPR may contribute to the acceleration of neurodegenerative disorders.

Loss of DmGluRA exacerbates age-related sleep disruption and reduces lifespan.

Declines in sleep amount and quality-characterized by excessive daytime sleepiness and an inability to sleep at night-are common features of aging. Sleep dysfunction is also associated with age-related ailments and diseases, suggesting that sleep is functionally relevant to the aging process. Metabotropic glutamate receptors (mGluRs)-which are critical regulators of neurotransmission and synaptic plasticity-have been implicated in both age-related disease and sleep regulation. Therefore, in this study, we examined the sleep and aging effect of complete genetic loss of mGluR signaling in Drosophila melanogaster. Genetic knockdown of the sole Drosophila mGluR-known as DmGluRA-reduced daytime wakefulness and nighttime sleep, recapitulating age-related sleep changes that occur across species. Furthermore, loss of DmGluRA significantly reduced lifespan and exacerbated age-related sleep loss in older flies. Thus, we identify DmGluRA as a novel regulator of sleep whose loss results in an age-relevant sleep phenotype that is associated with shortened lifespan. This is the first evidence that mGluR signaling regulates sleep/wake in a manner that is relevant to the aging process.

Sleep, Aging, and Cellular Health: Aged-Related Changes in Sleep and Protein Homeostasis Converge in Neurodegenerative Diseases.

Many neurodegenerative diseases manifest in an overall aged population, the pathology of which is hallmarked by abnormal protein aggregation. It is known that across aging, sleep quality becomes less efficient and protein homeostatic regulatory mechanisms deteriorate. There is a known relationship between extended wakefulness and poorly consolidated sleep and an increase in cellular stress. In an aged population, when sleep is chronically poor, and proteostatic regulatory mechanisms are less efficient, the cell is inundated with misfolded proteins and suffers a collapse in homeostasis. In this review article, we explore the interplay between aging, sleep quality, and proteostasis and how these processes are implicated in the development and progression of neurodegenerative diseases like Alzheimer's disease (AD). We also present data suggesting that reducing cellular stress and improving proteostasis and sleep quality could serve as potential therapeutic solutions for the prevention or delay in the progression of these diseases.

The leptin sensitizer celastrol reduces age-associated obesity and modulates behavioral rhythms.

The prevalence of obesity increases with age in humans and in rodents. Age-related obesity is characterized by leptin resistance and associated with heightened risk of metabolic disorders. However, the effect of leptin resistance per se has been difficult to disentangle from other effects of aging. Here we demonstrate that celastrol, a natural phytochemical that was previously shown to act as a leptin sensitizer, induces weight loss in aged animals, but not in young controls. Celastrol reduces food intake and lowers fasting glucose without affecting energy expenditure. Unexpectedly, administration of celastrol just before the dark period disrupted circadian rhythms of sleep and activity. This regimen was also associated with loss of lean mass an outcome that would not be desirable in elderly patients. Adjusting the timing of celastrol administration by 12 hr, to the beginning of the light period, avoided interference with circadian rhythms while retaining the reductions in body weight and adiposity. Thus, targeting leptin signaling is an effective strategy to ameliorate age-associated weight gain, and can profoundly impact circadian rhythms.

AMP-activated protein kinase phosphorylation in brain is dependent on method of killing and tissue preparation.

AMP-activated protein kinase (AMPK) is activated when the catalytic alpha subunit is phosphorylated on Thr172 and therefore, phosphorylation of the alpha subunit is used as a measure of activation. However, measurement of alpha subunit of AMPK (alpha-AMPK) phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring alpha-AMPK phosphorylation in the mouse brain, we compared different methods of killing and tissue preparation. We found that freeze/thawing samples after homogenization on ice dramatically increased alpha-AMPK phosphorylation in mice killed by cervical dislocation. Killing of mice by focused microwave irradiation, which rapidly heats the brain and causes enzymatic inactivation, prevented the freeze/thaw-induced increase in alpha-AMPK phosphorylation and similar levels of phosphorylation were observed compared with mice killed with cervical dislocation without freeze/thawing of samples. Sonication of samples in hot 1% sodium dodecyl sulfate blocked the freeze/thaw-induced increase in alpha-AMPK phosphorylation, but phosphorylation was higher in mice killed by cervical dislocation compared with mice killed by focused microwave irradiation. These results demonstrate that alpha-AMPK phosphorylation is dependent on method of killing and tissue preparation and that alpha-AMPK phosphorylation can increase in a manner that does not reflect biological alterations.

Molecular mechanisms of sleep and wakefulness.

Major questions on the biology of sleep include the following: what are the molecular functions of sleep; why can wakefulness only be sustained for defined periods before there is behavioral impairment; what genes contribute to the individual differences in sleep and the response to sleep deprivation? Behavioral criteria to define sleep have facilitated identification of sleep states in a number of different model systems: Drosophila, zebrafish, and Caenorhabditis elegans. Each system has unique strengths. Studies in these model systems are identifying conserved signaling mechanisms regulating sleep that are present in mammals. For example, the PKA-CREB signaling mechanism promotes wakefulness in Drosophila, mice, and C. elegans. Microarray studies indicate that genes whose expression is upregulated during sleep are involved in macromolecule biosynthesis (proteins, lipids [including cholesterol], heme). Thus, a key function of sleep is likely to be macromolecule synthesis. Moreover, in all species studied to date, there is upregulation of the molecular chaperone BiP with extended wakefulness. Sleep deprivation leads to cellular ER stress in brain and the unfolded protein response. Identification of genes regulating sleep has the potential for translational studies to elucidate the genetics of sleep and response to sleep deprivation in humans.