Modulation of sleep/wake patterns by gephyrin phosphorylation status.

Sleep/wake cycles intricately shape physiological activities including cognitive brain functions, yet the precise molecular orchestrators of sleep remain elusive. Notably, the clinical impact of benzodiazepine drugs underscores the pivotal role of GABAergic neurotransmission in sleep regulation. However, the specific contributions of distinct GABAA receptor subtypes and their principal scaffolding protein, gephyrin, in sleep dynamics remain unclear. The evolving role of synaptic phospho-proteome alterations at excitatory and inhibitory synapses suggests a potential avenue for modulating gephyrin and, consequently, GABAARs for sleep through on-demand kinase recruitment. Our study unveils the distinctive roles of two prevalent GABAA receptor subtypes, α1- and α2-GABAARs, in influencing sleep duration and electrical sleep activity. Notably, the absence of α1-GABAARs emerges as central in sleep regulation, manifesting significant alterations in both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during dark or active phases, accompanied by altered electroencephalogram (EEG) patterns across various frequencies. Gephyrin proteomics analysis reveals sleep/wake-dependent interactions with a repertoire of known and novel kinases. Crucially, we identify the regulation of gephyrin interaction with ERK1/2, and phosphorylations at serines 268 and 270 are dictated by sleep/wake cycles. Employing AAV-eGFP-gephyrin or its phospho-null variant (S268A/S270A), we disrupt sleep either globally or locally to demonstrate gephyrin phosphorylation as a sleep regulator. In summary, our findings support the local cortical sleep hypothesis and we unveil a molecular mechanism operating at GABAergic synapses, providing critical insights into the intricate regulation of sleep.

Trophic factor BDNF inhibits GABAergic signaling by facilitating dendritic enrichment of SUMO E3 ligase PIAS3 and altering gephyrin scaffold.

Posttranslational addition of a small ubiquitin-like modifier (SUMO) moiety (SUMOylation) has been implicated in pathologies such as brain ischemia, diabetic peripheral neuropathy, and neurodegeneration. However, nuclear enrichment of SUMO pathway proteins has made it difficult to ascertain how ion channels, proteins that are typically localized to and function at the plasma membrane, and mitochondria are SUMOylated. Here, we report that the trophic factor, brain-derived neurotrophic factor (BDNF) regulates SUMO proteins both spatially and temporally in neurons. We show that BDNF signaling via the receptor tropomyosin-related kinase B facilitates nuclear exodus of SUMO proteins and subsequent enrichment within dendrites. Of the various SUMO E3 ligases, we found that PIAS-3 dendrite enrichment in response to BDNF signaling specifically modulates subsequent ERK1/2 kinase pathway signaling. In addition, we found the PIAS-3 RING and Ser/Thr domains, albeit in opposing manners, functionally inhibit GABA-mediated inhibition. Finally, using oxygen-glucose deprivation as an in vitro model for ischemia, we show that BDNF-tropomyosin-related kinase B signaling negatively impairs clustering of the main scaffolding protein at GABAergic postsynapse, gephyrin, whereby reducing GABAergic neurotransmission postischemia. SUMOylation-defective gephyrin K148R/K724R mutant transgene expression reversed these ischemia-induced changes in gephyrin cluster density. Taken together, these data suggest that BDNF signaling facilitates the temporal relocation of nuclear-enriched SUMO proteins to dendrites to influence postsynaptic protein SUMOylation.

Measuring mRNA translation in neuronal processes and somata by tRNA-FRET.

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

Dopamine-Dependent QR2 Pathway Activation in CA1 Interneurons Enhances Novel Memory Formation.

The formation of memory for a novel experience is a critical cognitive capacity. The ability to form novel memories is sensitive to age-related pathologies and disease, to which prolonged metabolic stress is a major contributing factor. Presently, we describe a dopamine-dependent redox modulation pathway within the hippocampus of male mice that promotes memory consolidation. Namely, following novel information acquisition, quinone reductase 2 (QR2) is suppressed by miRNA-182 (miR-182) in the CA1 region of the hippocampus via dopamine D1 receptor (D1R) activation, a process largely facilitated by locus coeruleus activity. This pathway activation reduces ROS generated by QR2 enzymatic activity, a process that alters the intrinsic properties of CA1 interneurons 3 h following learning, in a form of oxidative eustress. Interestingly, novel experience decreases QR2 expression predominately in inhibitory interneurons. Additionally, we find that in aged animals this newly described QR2 pathway is chronically under activated, resulting in miR-182 underexpression and QR2 overexpression. This leads to accumulative oxidative stress, which can be seen in CA1 via increased levels of oxidized, inactivated potassium channel Kv2.1, which undergoes disulfide bridge oligomerization. This newly described interneuron-specific molecular pathway lies alongside the known mRNA translation-dependent processes necessary for long-term memory formation, entrained by dopamine in CA1. It is a process crucial for the distinguishing features of novel memory, and points to a promising new target for memory enhancement in aging and age-dependent diseases.SIGNIFICANCE STATEMENT One way in which evolution dictates which sensory information will stabilize as an internal representation, relies on information novelty. Dopamine is a central neuromodulator involved in this process in the mammalian hippocampus. Here, we describe for the first time a dopamine D1 receptor-dependent quinone reductase 2 pathway in interneurons. This is a targeted redox event necessary to delineate a novel experience to a robust long-term internal representation. Activation of this pathway alone can explain the effect novelty has on "flashbulb" memories, and it can become dysfunctional with age and diseases, such as Alzheimer's disease.

Local Inhibition of PERK Enhances Memory and Reverses Age-Related Deterioration of Cognitive and Neuronal Properties.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of four known kinases that respond to cellular stress by deactivating the eukaryotic initiation factor 2 α (eIF2α) or other signal transduction cascades. Recently, both eIF2α and its kinases were found to play a role in normal and pathological brain function. Here, we show that reduction of either the amount or the activity of PERK, specifically in the CA1 region of the hippocampus in young adult male mice, enhances neuronal excitability and improves cognitive function. In addition, this manipulation rescues the age-dependent cellular phenotype of reduced excitability and memory decline. Specifically, the reduction of PERK expression in the CA1 region of the hippocampus of middle-aged male mice using a viral vector rejuvenates hippocampal function and improves hippocampal-dependent learning. These results delineate a mechanism for behavior and neuronal aging and position PERK as a promising therapeutic target for age-dependent brain malfunction.SIGNIFICANCE STATEMENT We found that local reduced protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) expression or activity in the hippocampus enhances neuronal excitability and cognitive function in young normal mice, that old CA1 pyramidal cells have reduced excitability and increased PERK expression that can be rescued by reducing PERK expression in the hippocampus, and that reducing PERK expression in the hippocampus of middle-aged mice enhances hippocampal-dependent learning and memory and restores it to normal performance levels of young mice. These findings uncover an entirely new biological link among PERK, neuronal intrinsic properties, aging, and cognitive function. Moreover, our findings propose a new way to fight mild cognitive impairment and aging-related cognitive deterioration.

Restoring GABAB receptor expression in the ventral tegmental area of methamphetamine addicted mice inhibits locomotor sensitization and drug seeking behavior.

Repeated exposure to psychostimulants such as methamphetamine (METH) induces neuronal adaptations in the mesocorticolimbic dopamine system, including the ventral tegmental area (VTA). These changes lead to persistently enhanced neuronal activity causing increased dopamine release and addictive phenotypes. A factor contributing to increased dopaminergic activity in this system appears to be reduced GABAB receptor-mediated neuronal inhibition in the VTA. Dephosphorylation of serine 783 (Ser783) of the GABAB2 subunit by protein phosphatase 2A (PP2A) appears to trigger the downregulation GABAB receptors in psychostimulant-addicted rodents. Therefore, preventing the interaction of GABAB receptors with PP2A using an interfering peptide is a promising strategy to restore GABAB receptor-mediated neuronal inhibition. We have previously developed an interfering peptide (PP2A-Pep) that inhibits the GABAB receptors/PP2A interaction and thereby restores receptor expression under pathological conditions. Here, we tested the hypothesis that restoration of GABAB receptor expression in the VTA of METH addicted mice reduce addictive phenotypes. We found that the expression of GABAB receptors was significantly reduced in the VTA and nucleus accumbens but not in the hippocampus and somatosensory cortex of METH-addicted mice. Infusion of PP2A-Pep into the VTA of METH-addicted mice restored GABAB receptor expression in the VTA and inhibited METH-induced locomotor sensitization as assessed in the open field test. Moreover, administration of PP2A-Pep into the VTA also reduced drug seeking behavior in the conditioned place preference test. These observations underscore the importance of VTA GABAB receptors in controlling addictive phenotypes. Furthermore, this study illustrates the value of interfering peptides targeting diseases-related protein-protein interactions as an alternative approach for a potential development of selective therapeutic interventions.

The gephyrin scaffold modulates cortical layer 2/3 pyramidal neuron responsiveness to single whisker stimulation.

Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) of gephyrin alter GABAA receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABAA receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.

Targeting the interaction of GABAB receptors with CaMKII with an interfering peptide restores receptor expression after cerebral ischemia and inhibits progressive neuronal death in mouse brain cells and slices.

Cerebral ischemia is the leading cause for long-term disability and mortality in adults due to massive neuronal death. Currently, there is no pharmacological treatment available to limit progressive neuronal death after stroke. A major mechanism causing ischemia-induced neuronal death is the excessive release of glutamate and the associated overexcitation of neurons (excitotoxicity). Normally, GABAB receptors control neuronal excitability in the brain via prolonged inhibition. However, excitotoxic conditions rapidly downregulate GABAB receptors via a CaMKII-mediated mechanism and thereby diminish adequate inhibition that could counteract neuronal overexcitation and neuronal death. To prevent the deleterious downregulation of GABAB receptors, we developed a cell-penetrating synthetic peptide (R1-Pep) that inhibits the interaction of GABAB receptors with CaMKII. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions restored cell surface expression and function of GABAB receptors. R1-Pep did not affect CaMKII expression or activity but prevented its T286 autophosphorylation that renders it autonomously and persistently active. Moreover, R1-Pep counteracted the aberrant downregulation of G protein-coupled inwardly rectifying K+ channels and the upregulation of N-type voltage-gated Ca2+ channels, the main effectors of GABAB receptors. The restoration of GABAB receptors activated the Akt survival pathway and inhibited excitotoxic neuronal death with a wide time window in cultured neurons. Restoration of GABAB receptors and neuroprotective activity of R1-Pep was verified by using brain slices prepared from mice after middle cerebral artery occlusion (MCAO). Treatment with R1-Pep restored normal GABAB receptor expression and GABA receptor-mediated K+ channel currents. This reduced MCAO-induced neuronal excitability and inhibited neuronal death. These results support the hypothesis that restoration of GABAB receptor expression under excitatory conditions provides neuroprotection and might be the basis for the development of a selective intervention to inhibit progressive neuronal death after ischemic stroke.

Protein phosphatase 2A regulation of GABA B receptors normalizes ischemia-induced aberrant receptor trafficking and provides neuroprotection.

One major factor regulating the strength of GABA B receptor signaling and thereby neuronal excitability is the dynamic control of their cell surface expression. GABA B receptors are constitutively internalized and recycled back to the plasma membrane to maintain a stable number of receptors at cell surface for appropriate signaling. Protein phosphatase 2A (PP2A) dependent dephosphorylation of serine 783 (S783) in the GABA B2 subunit is a key event for downregulating GABA B receptor cell surface expression particularly under conditions associated with excitotoxicity. Here, we investigated the role of PP2A in regulating GABA B receptor cell surface expression under physiological and excitotoxic conditions. For this purpose, we developed an interfering peptide (PP2A-Pep) that inhibits the interaction of GABA B receptors with PP2A. Using cultured cortical neurons, we found that PP2A downregulates GABA B receptor cell surface expression by inhibiting recycling of the receptors and thereby promoting degradation of the receptors. Inhibition of the GABA B receptor/PP2A interaction by PP2A-Pep in cultured cortical neurons restored GABA B receptor cell surface expression after excitotoxic stress and inhibited progressing neuronal death even when added 48 h after the insult. To explore the therapeutic potential of PP2A-Pep, we further analyzed effect of PP2A-Pep in the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia. Incubation of brain slices prepared from MCAO-treated mice with PP2A-Pep restored normal GABA B receptor expression and GABA B receptor-mediated inhibition, reduced ischemic-induced overexcitability of neurons, and prevented neuronal death in the ischemic penumbra. This data illustrates the crucial role of regulating GABA B receptor phosphorylation by PP2A for controlling neuronal inhibition and excitability. The results further suggest that interfering with the GABA B receptor/PP2A interaction is a promising strategy for the development of specific therapeutic interventions to treat neurological diseases associated with a disturbed excitation/inhibition balance and downregulation of GABA B receptors.

Calreticulin mutations affect its chaperone function and perturb the glycoproteome.

Calreticulin (CALR) is an endoplasmic reticulum (ER)-retained chaperone that assists glycoproteins in obtaining their structure. CALR mutations occur in patients with myeloproliferative neoplasms (MPNs), and the ER retention of CALR mutants (CALR MUT) is reduced due to a lacking KDEL sequence. Here, we investigate the impact of CALR mutations on protein structure and protein levels in MPNs by subjecting primary patient samples and CALR-mutated cell lines to limited proteolysis-coupled mass spectrometry (LiP-MS). Especially glycoproteins are differentially expressed and undergo profound structural alterations in granulocytes and cell lines with homozygous, but not with heterozygous, CALR mutations. Furthermore, homozygous CALR mutations and loss of CALR equally perturb glycoprotein integrity, suggesting that loss-of-function attributes of mutated CALR chaperones (CALR MUT) lead to glycoprotein maturation defects. Finally, by investigating the misfolding of the CALR glycoprotein client myeloperoxidase (MPO), we provide molecular proof of protein misfolding in the presence of homozygous CALR mutations.

Sustained Baclofen-Induced Activation of GABA B Receptors After Cerebral Ischemia Restores Receptor Expression and Function and Limits Progressing Loss of Neurons.

One important function of GABA B receptors is the control of neuronal activity to prevent overexcitation and thereby excitotoxic death, which is a hallmark of cerebral ischemia. Consequently, sustained activation of GABA B receptors with the selective agonist baclofen provides neuroprotection in in vitro and in vivo models of cerebral ischemia. However, excitotoxic conditions severely downregulate the receptors, which would compromise the neuroprotective effectiveness of baclofen. On the other hand, recent work suggests that sustained activation of GABA B receptors stabilizes receptor expression. Therefore, we addressed the question whether sustained activation of GABA B receptors reduces downregulation of the receptor under excitotoxic conditions and thereby preserves GABA B receptor-mediated inhibition. In cultured neurons subjected to oxygen and glucose deprivation (OGD), to mimic cerebral ischemia, GABA B receptors were severely downregulated. Treatment of the cultures with baclofen after OGD restored GABA B receptor expression and reduced loss of neurons. Restoration of GABA B receptors was due to enhanced fast recycling of the receptors, which reduced OGD-induced sorting of the receptors to lysosomal degradation. Utilizing the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia, we verified the severe downregulation of GABA B receptors in the affected cortex and a partial restoration of the receptors after systemic injection of baclofen. Restored receptor expression recovered GABA B receptor-mediated currents, normalized the enhanced neuronal excitability observed after MCAO and limited progressive loss of neurons. These results suggest that baclofen-induced restoration of GABA B receptors provides the basis for the neuroprotective activity of baclofen after an ischemic insult. Since GABA B receptors regulate multiple beneficial pathways, they are promising targets for a neuroprotective strategy in acute cerebral ischemia.