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

Role of Homer Proteins in the Maintenance of Sleep-Wake States

Sleep is an evolutionarily conserved process that is linked to diurnal cycles and normal daytime wakefulness. Healthy sleep and wakefulness are integral to a healthy lifestyle; this occurs when an organism is able to maintain long bouts of both sleep and wake. Homer proteins, which function as adaptors for group 1 metabotropic glutamate receptors, have been implicated in genetic studies of sleep in both Drosophila and mouse. Drosophila express a single Homer gene product that is upregulated during sleep. By contrast, vertebrates express Homer as both constitutive and immediate early gene (H1a) forms, and H1a is up-regulated during wakefulness. Genetic deletion of Homer in Drosophila results in fragmented sleep and in failure to sustain long bouts of sleep, even under increased sleep drive. However, deletion of Homer1a in mouse results in failure to sustain long bouts of wakefulness. Further evidence for the role of Homer1a in the maintenance of wake comes from the CREB alpha delta mutant mouse, which displays a reduced wake phenotype similar to the Homer1a knockout and fails to up-regulate Homer1a upon sleep loss. Homer1a is a gene whose expression is induced by CREB. Sustained behaviors of the sleep/wake cycle are created by molecular pathways that are distinct from those for arousal or short bouts, and implicate an evolutionarily-conserved role for Homer in sustaining these behaviors

Potential of Proteomics as a Bioanalytic Technique for Quantifying Sleepiness

Currently, there are no simple ways to assess the degree of sleep loss in individual subjects. Proteins constitute greater than 98% of all molecules in the cell. They participate in physiological interactions, explain all of posttranslational modifications that occur in cellular micro-environments and thus are potential candidates for identification of a biomarker of sleepiness. A variety of proteonomic techniques are available which are being used in a current sleep deprivation study of monozygotic and dizygotic twin pairs we are performing

Structure-function studies on alpha1-antichymotrypsin: A.~Effect of P1 mutation on specificity. B.~Identification of a novel DNA-binding motif. C.~Stopped-flow kinetic analysis of formation of the chymotrypsin-ACT complex

$\alpha$1-Antichymotrypsin (ACT) is a serine protease inhibitor found in serum. ACT is a major acute phase reactant, its serum levels rise rapidly in response to inflammatory states. ACT is unique among serine protease inhibitors in its ability to bind DNA. Further it has also been localized in the nuclei of certain tumor cells. The physiological relevance of ACT-DNA interaction or nuclear localization is not known. Site specific mutagenesis has been used to produce a variant of ACT in which the leucine at the P1 position has been changed to methionine (L358M-rACT). L358M-rACT has a specificity of inhibitory activity towards serine proteases closely similar to that of native ACT. Using site specific mutagenesis and chemical modification, we have identified two domains within the protein that mediate the potentially important DNA binding function. ACT has a trilysine sequence (residues 210-212) falling within a solvent exposed loop (Baumann et al., 1991; Wei et al., 1994). Mutation of all three lysines to either glutamates or threonines abolishes ACT binding to DNA. Partial replacement of the lysines by threonines results in reduced DNA binding affinity. Limited acetylation of ACT with acetic anhydride leads to a loss of DNA binding. DNA protects ACT from acetylation. A combination of CNBr digestion, peptide separation, and peptide sequencing allows identification of a C terminus peptide (residues 390-398), containing two lysines at positions 391 and 396 respectively. Mutation of K396 and K391 yields a variant K396T/K391T-rACT that has very little affinity for DNA. The $\varepsilon$-amines of lysines 210-212 are 8-15 A across a shallow cleft from the $\varepsilon$-amines in K391 and K396, and together these two elements form a plausible DNA binding domain, albeit one that is unusual among DNA binding proteins. The interaction between ACT and chymotrypsin has been investigated using fluorescence stopped flow spectrophotometry. We have established that the formation of the stable enzyme-inhibitor complex proceeds minimally via a two step mechanism, in which a weak intermediate is converted to a more stable complex by an isomerization step

Neuroenhancement and the Developing Brain: Commentary on the <i>AIMS Neuroscience</i> Special Issue on “Neuroenhancers”

The use of pharmaceutical neuroenhancers to improve cognitive function poses unique neurobiological concerns as stimulants are being widely prescribed to adolescents and young adults with increasing prevalence. In the following commentary on the papers by Hoffman et al [1] and Cheung and Pierre [2] in the special issue on Neuroenhancers, we discuss the need to consider the effects of stimulant use in healthy adolescents. We review some of the data that has emerged on the neurobiological and behavioral effects of adolescent neuroenhancement, and conclude that special consideration should be taken to characterize the consequences of neuroenhancement use in the developing brain. Studies focused specifically on adolescent vulnerabilities to neuroenhancement are necessary because the brain undergoes dynamics changes that are unique to this period of development, which differentiates it from the healthy adult response to neuroenhancer exposure. Moving forward, scientists and physicians should take careful consideration to examine the long-term neurological consequences of neuroenhancers so that the therapeutic benefits that might be gained from neuroenhancement are not shadowed by negative consequences to public health in the future

Protein Synthesis during Sleep Consolidates Cortical Plasticity In Vivo

Sleep consolidates experience-dependent brain plasticity, but the precise cellular mechanisms mediating this process are unknown [1]. De novo cortical protein synthesis is one possible mechanism. In support of this hypothesis, sleep is associated with increased brain protein synthesis [2, 3] and transcription of messenger RNAs (mRNAs) involved in protein synthesis regulation [4, 5]. Protein synthesis in turn is critical for memory consolidation and persistent forms of plasticity in vitro and in vivo [6, 7]. However, it is unknown whether cortical protein synthesis in sleep serves similar functions. We investigated the role of protein synthesis in the sleep-dependent consolidation of a classic form of cortical plasticity in vivo (ocular dominance plasticity, ODP; [8, 9]) in the cat visual cortex. We show that intracortical inhibition of mammalian target of rapamycin (mTOR)-dependent protein synthesis during sleep abolishes consolidation but has no effect on plasticity induced during wakefulness. Sleep also promotes phosphorylation of protein synthesis regulators (i.e., 4E-BP1 and eEF2) and the translation (but not transcription) of key plasticity related mRNAs (ARC and BDNF). These findings show that sleep promotes cortical mRNA translation. Interruption of this process has functional consequences, because it abolishes the consolidation of experience in the cortex. [Display omitted] ► mTORC1 inhibition selectively impairs sleep-dependent cortical plasticity ► Sleep promotes translation factor regulation ► ARC and BDNF transcription and translation are divided across wake and sleep ► Transcription occurs in response to waking experience, but translation requires slee

Protein Synthesis during Sleep Consolidates Cortical Plasticity In Vivo

Sleep consolidates experience-dependent brain plasticity, but the precise cellular mechanisms mediating this process are unknown [1]. De novo cortical protein synthesis is one possible mechanism. In support of this hypothesis, sleep is associated with increased brain protein synthesis [2, 3] and transcription of messenger RNAs (mRNAs) involved in protein synthesis regulation [4, 5]. Protein synthesis in turn is critical for memory consolidation and persistent forms of plasticity in vitro and in vivo [6, 7]. However, it is unknown whether cortical protein synthesis in sleep serves similar functions. We investigated the role of protein synthesis in the sleep-dependent consolidation of a classic form of cortical plasticity in vivo (ocular dominance plasticity, ODP; [8, 9]) in the cat visual cortex. We show that intracortical inhibition of mammalian target of rapamycin (mTOR)-dependent protein synthesis during sleep abolishes consolidation but has no effect on plasticity induced during wakefulness. Sleep also promotes phosphorylation of protein synthesis regulators (i.e., 4E-BP1 and eEF2) and the translation (but not transcription) of key plasticity related mRNAs (ARC and BDNF). These findings show that sleep promotes cortical mRNA translation. Interruption of this process has functional consequences, because it abolishes the consolidation of experience in the cortex

Mechanisms of sleep-dependent consolidation of cortical plasticity

Sleep is thought to consolidate changes in synaptic strength, but the underlying mechanisms are unknown. We investigated the cellular events involved in this process in ocular dominance plasticity (ODP) - a canonical form of in vivo cortical plasticity triggered by monocular deprivation (MD) and consolidated by sleep via undetermined, activity-dependent mechanisms. We find that sleep consolidates ODP primarily by strengthening cortical responses to non-deprived eye stimulation. Consolidation is inhibited by reversible, intracortical antagonism of NMDA receptors (NMDARs) or cAMP-dependent protein kinase (PKA) during post-MD sleep. Consolidation is also associated with sleep-dependent increases in the activity of remodeling neurons, and in the phosphorylation of proteins required for potentiation of glutamatergic synapses. These findings demonstrate that synaptic strengthening via NMDAR and PKA activity is a key step in sleep-dependent consolidation of ODP

Mechanisms of Sleep-Dependent Consolidation of Cortical Plasticity

Sleep is thought to consolidate changes in synaptic strength, but the underlying mechanisms are unknown. We investigated the cellular events involved in this process during ocular dominance plasticity (ODP)—a canonical form of in vivo cortical plasticity triggered by monocular deprivation (MD) and consolidated by sleep via undetermined, activity-dependent mechanisms. We find that sleep consolidates ODP primarily by strengthening cortical responses to nondeprived eye stimulation. Consolidation is inhibited by reversible, intracortical antagonism of NMDA receptors (NMDARs) or cAMP-dependent protein kinase (PKA) during post-MD sleep. Consolidation is also associated with sleep-dependent increases in the activity of remodeling neurons and in the phosphorylation of proteins required for potentiation of glutamatergic synapses. These findings demonstrate that synaptic strengthening via NMDAR and PKA activity is a key step in sleep-dependent consolidation of ODP