Msp1/ATAD1 in Protein Quality Control and Regulation of Synaptic Activities.

Mitochondrial function depends on the efficient import of proteins synthesized in the cytosol. When cells experience stress, the efficiency and faithfulness of the mitochondrial protein import machinery are compromised, leading to homeostatic imbalances and damage to the organelle. Yeast Msp1 (mitochondrial sorting of proteins 1) and mammalian ATAD1 (ATPase family AAA domain-containing 1) are orthologous AAA proteins that, fueled by ATP hydrolysis, recognize and extract mislocalized membrane proteins from the outer mitochondrial membrane. Msp1 also extracts proteins that have become stuck in the import channel. The extracted proteins are targeted for proteasome-dependent degradation or, in the case of mistargeted tail-anchored proteins, are given another chance to be routed correctly. In addition, ATAD1 is implicated in the regulation of synaptic plasticity, mediating the release of neurotransmitter receptors from postsynaptic scaffolds to allow their trafficking. Here we discuss how structural and functional specialization imparts the unique properties that allow Msp1/ATAD1 ATPases to fulfill these diverse functions and also highlight outstanding questions in the field.

Synaptic memory and CaMKII.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and long-term potentiation (LTP) were discovered within a decade of each other and have been inextricably intertwined ever since. However, like many marriages, it has had its up and downs. Based on the unique biochemical properties of CaMKII, it was proposed as a memory molecule before any physiological linkage was made to LTP. However, as reviewed here, the convincing linkage of CaMKII to synaptic physiology and behavior took many decades. New technologies were critical in this journey, including in vitro brain slices, mouse genetics, single-cell molecular genetics, pharmacological reagents, protein structure, and two-photon microscopy, as were new investigators attracted by the exciting challenge. This review tracks this journey and assesses the state of this marriage 40 years on. The collective literature impels us to propose a relatively simple model for synaptic memory involving the following steps that drive the process: 1) Ca2+ entry through N-methyl-d-aspartate (NMDA) receptors activates CaMKII. 2) CaMKII undergoes autophosphorylation resulting in constitutive, Ca2+-independent activity and exposure of a binding site for the NMDA receptor subunit GluN2B. 3) Active CaMKII translocates to the postsynaptic density (PSD) and binds to the cytoplasmic C-tail of GluN2B. 4) The CaMKII-GluN2B complex initiates a structural rearrangement of the PSD that may involve liquid-liquid phase separation. 5) This rearrangement involves the PSD-95 scaffolding protein, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), and their transmembrane AMPAR-regulatory protein (TARP) auxiliary subunits, resulting in an accumulation of AMPARs in the PSD that underlies synaptic potentiation. 6) The stability of the modified PSD is maintained by the stability of the CaMKII-GluN2B complex. 7) By a process of subunit exchange or interholoenzyme phosphorylation CaMKII maintains synaptic potentiation in the face of CaMKII protein turnover. There are many other important proteins that participate in enlargement of the synaptic spine or modulation of the steps that drive and maintain the potentiation. In this review we critically discuss the data underlying each of the steps. As will become clear, some of these steps are more firmly grounded than others, and we provide suggestions as to how the evidence supporting these steps can be strengthened or, based on the new data, be replaced. Although the journey has been a long one, the prospect of having a detailed cellular and molecular understanding of learning and memory is at hand.

Structure and desensitization of AMPA receptor complexes with type II TARP γ5 and GSG1L.

AMPA receptors (AMPARs) mediate the majority of excitatory neurotransmission. Their surface expression, trafficking, gating, and pharmacology are regulated by auxiliary subunits. Of the two types of TARP auxiliary subunits, type I TARPs assume activating roles, while type II TARPs serve suppressive functions. We present cryo-EM structures of GluA2 AMPAR in complex with type II TARP γ5, which reduces steady-state currents, increases single-channel conductance, and slows recovery from desensitization. Regulation of AMPAR function depends on its ligand-binding domain (LBD) interaction with the γ5 head domain. GluA2-γ5 complex shows maximum stoichiometry of two TARPs per AMPAR tetramer, being different from type I TARPs but reminiscent of the auxiliary subunit GSG1L. Desensitization of both GluA2-GSG1L and GluA2-γ5 complexes is accompanied by rupture of LBD dimer interface, while GluA2-γ5 but not GluA2-GSG1L LBD dimers remain two-fold symmetric. Different structural architectures and desensitization mechanisms of complexes with auxiliary subunits endow AMPARs with broad functional capabilities.

Tau target identification reveals NSF-dependent effects on AMPA receptor trafficking and memory formation.

Microtubule-associated protein tau is a central factor in Alzheimer's disease and other tauopathies. However, the physiological functions of tau are unclear. Here, we used proximity-labelling proteomics to chart tau interactomes in primary neurons and mouse brains in vivo. Tau interactors map onto pathways of cytoskeletal, synaptic vesicle and postsynaptic receptor regulation and show significant enrichment for Parkinson's, Alzheimer's and prion disease. We find that tau interacts with and dose-dependently reduces the activity of N-ethylmaleimide sensitive fusion protein (NSF), a vesicular ATPase essential for AMPA-type glutamate receptor (AMPAR) trafficking. Tau-deficient (tau-/- ) neurons showed mislocalised expression of NSF and enhanced synaptic AMPAR surface levels, reversible through the expression of human tau or inhibition of NSF. Consequently, enhanced AMPAR-mediated associative and object recognition memory in tau-/- mice is suppressed by both hippocampal tau and infusion with an NSF-inhibiting peptide. Pathologic mutant tau from mouse models or Alzheimer's disease significantly enhances NSF inhibition. Our results map neuronal tau interactomes and delineate a functional link of tau with NSF in plasticity-associated AMPAR-trafficking and memory.

Aß peptide enhances GluA1 internalization via lipid rafts in Alzheimer's-related hippocampal LTP dysfunction.

Amyloid ß (Aß) is a central contributor to neuronal damage and cognitive impairment in Alzheimer's disease (AD). Aß disrupts AMPA receptor-mediated synaptic plasticity, a key factor in early AD progression. Numerous studies propose that Aß oligomers hinder synaptic plasticity, particularly long-term potentiation (LTP), by disrupting GluA1 (encoded by GRIA1) function, although the precise mechanism remains unclear. In this study, we demonstrate that Aß mediates the accumulation of GM1 ganglioside in lipid raft domains of cultured cells, and GluA1 exhibits preferential localization in lipid rafts via direct binding to GM1. Aß enhances the raft localization of GluA1 by increasing GM1 in these areas. Additionally, chemical LTP stimulation induces lipid raft-dependent GluA1 internalization in Aß-treated neurons, resulting in reduced cell surface and postsynaptic expression of GluA1. Consistent with this, disrupting lipid rafts and GluA1 localization in rafts rescues Aß-mediated suppression of hippocampal LTP. These findings unveil a novel functional deficit in GluA1 trafficking induced by Aß, providing new insights into the mechanism underlying AD-associated cognitive dysfunction.

Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aß pathology.

Accumulation of amyloid-beta (Aß) can lead to the formation of aggregates that contribute to neurodegeneration in Alzheimer's disease (AD). Despite globally reduced neural activity during AD onset, recent studies have suggested that Aß induces hyperexcitability and seizure-like activity during the early stages of the disease that ultimately exacerbate cognitive decline. However, the underlying mechanism is unknown. Here, we reveal an Aß-induced elevation of postsynaptic density protein 95 (PSD-95) in cultured neurons in vitro and in an in vivo AD model using APP/PS1 mice at 8 weeks of age. Elevation of PSD-95 occurs as a result of reduced ubiquitination caused by Akt-dependent phosphorylation of E3 ubiquitin ligase murine-double-minute 2 (Mdm2). The elevation of PSD-95 is consistent with the facilitation of excitatory synapses and the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors induced by Aß. Inhibition of PSD-95 corrects these Aß-induced synaptic defects and reduces seizure activity in APP/PS1 mice. Our results demonstrate a mechanism underlying elevated seizure activity during early-stage Aß pathology and suggest that PSD-95 could be an early biomarker and novel therapeutic target for AD.

Tuning synaptic strength by regulation of AMPA glutamate receptor localization.

Long-term potentiation (LTP) of excitatory synapses is a leading model to explain the concept of information storage in the brain. Multiple mechanisms contribute to LTP, but central amongst them is an increased sensitivity of the postsynaptic membrane to neurotransmitter release. This sensitivity is predominantly determined by the abundance and localization of AMPA-type glutamate receptors (AMPARs). A combination of AMPAR structural data, super-resolution imaging of excitatory synapses, and an abundance of electrophysiological studies are providing an ever-clearer picture of how AMPARs are recruited and organized at synaptic junctions. Here, we review the latest insights into this process, and discuss how both cytoplasmic and extracellular receptor elements cooperate to tune the AMPAR response at the hippocampal CA1 synapse.

Impairments in endogenous AMPA receptor dynamics correlates with learning deficits in Alzheimer's disease model mice.

AMPA receptors (AMPARs) play a critical role in synaptic plasticity and learning and memory, and dysfunction or dysregulation of AMPARs could lead to various neurological and psychiatric disorders, such as Alzheimer's disease (AD). However, the dynamics and/or longitudinal changes of AMPARs in vivo during AD pathogenesis remain elusive. Here, employing 5xFAD SEP-GluA1 KI mice, we investigated endogenous AMPA receptor dynamics in a whisker deflection-associated Go/No-go learning paradigm. We found a significant increase in synaptosomal AMPA receptor subunits GluA1 in WT mice after learning, while no such changes were detected in 7-mo-old 5xFAD mice. Daily training led to an increase in endogenous spine surface GluA1 in Control mice, while this increase was absent in 5xFAD-KI mice which correlates with its learning defects in Go/No-go paradigm. Furthermore, we demonstrated that the onset of abnormal AMPAR dynamics corresponds temporally with microglia and astrocyte overactivation. Our results have shown that impairments in endogenous AMPA receptor dynamics play an important role in learning deficits in 5xFAD mice and AD pathogenesis.

The autism-associated loss of δ-catenin functions disrupts social behavior.

δ-catenin is expressed in excitatory synapses and functions as an anchor for the glutamatergic AMPA receptor (AMPAR) GluA2 subunit in the postsynaptic density. The glycine 34 to serine (G34S) mutation in the δ-catenin gene has been found in autism spectrum disorder (ASD) patients and results in loss of δ-catenin functions at excitatory synapses, which is presumed to underlie ASD pathogenesis in humans. However, how the G34S mutation causes loss of δ-catenin functions to induce ASD remains unclear. Here, using neuroblastoma cells, we identify that the G34S mutation increases glycogen synthase kinase 3ß (GSK3ß)-dependent δ-catenin degradation to reduce δ-catenin levels, which likely contributes to the loss of δ-catenin functions. Synaptic δ-catenin and GluA2 levels in the cortex are significantly decreased in mice harboring the δ-catenin G34S mutation. The G34S mutation increases glutamatergic activity in cortical excitatory neurons while it is decreased in inhibitory interneurons, indicating changes in cellular excitation and inhibition. δ-catenin G34S mutant mice also exhibit social dysfunction, a common feature of ASD. Most importantly, pharmacological inhibition of GSK3ß activity reverses the G34S-induced loss of δ-catenin function effects in cells and mice. Finally, using δ-catenin knockout mice, we confirm that δ-catenin is required for GSK3ß inhibition-induced restoration of normal social behavior in δ-catenin G34S mutant animals. Taken together, we reveal that the loss of δ-catenin functions arising from the ASD-associated G34S mutation induces social dysfunction via alterations in glutamatergic activity and that GSK3ß inhibition can reverse δ-catenin G34S-induced synaptic and behavioral deficits.

G272V and P301L Mutations Induce Isoform Specific Tau Mislocalization to Dendritic Spines and Synaptic Dysfunctions in Cellular Models of 3R and 4R Tau Frontotemporal Dementia.

Tau pathologies are detected in the brains of some of the most common neurodegenerative diseases including Alzheimer's disease (AD), Lewy body dementia (LBD), chronic traumatic encephalopathy (CTE), and frontotemporal dementia (FTD). Tau proteins are expressed in six isoforms with either three or four microtubule-binding repeats (3R tau or 4R tau) due to alternative RNA splicing. AD, LBD, and CTE brains contain pathological deposits of both 3R and 4R tau. FTD patients can exhibit either 4R tau pathologies in most cases or 3R tau pathologies less commonly in Pick's disease, which is a subfamily of FTD. Here, we report the isoform-specific roles of tau in FTD. The P301L mutation, linked to familial 4R tau FTD, induces mislocalization of 4R tau to dendritic spines in primary hippocampal cultures that were prepared from neonatal rat pups of both sexes. Contrastingly, the G272V mutation, linked to familial Pick's disease, induces phosphorylation-dependent mislocalization of 3R tau but not 4R tau proteins to dendritic spines. The overexpression of G272V 3R tau but not 4R tau proteins leads to the reduction of dendritic spine density and suppression of mEPSCs in 5-week-old primary rat hippocampal cultures. The decrease in mEPSC amplitude caused by G272V 3R tau is dynamin-dependent whereas that caused by P301L 4R tau is dynamin-independent, indicating that the two tau isoforms activate different signaling pathways responsible for excitatory synaptic dysfunction. Our 3R and 4R tau studies here will shed new light on diverse mechanisms underlying FTD, AD, LBD, and CTE.

A Necessary Role for PKC-2 and TPA-1 in Olfactory Memory and Synaptic AMPAR Trafficking in Caenorhabditis elegans.

Protein kinase C (PKC) functions are essential for synaptic plasticity, learning, and memory. However, the roles of specific members of the PKC family in synaptic function, learning, and memory are poorly understood. Here, we investigated the role of individual PKC homologs for synaptic plasticity in Caenorhabditis elegans and found a differential role for pkc-2 and tpa-1, but not pkc-1 and pkc-3 in associative olfactory learning and memory. More specifically we show that PKC-2 is essential for associative learning and TPA-1 for short-term associative memory (STAM). Using endogenous labeling and cell-specific rescues, we show that TPA-1 and PKC-2 are required in AVA for their functions. Previous studies demonstrated that olfactory learning and memory in C. elegans are tied to proper synaptic content and trafficking of AMPA-type ionotropic glutamate receptor homolog GLR-1 in the AVA command interneurons. Therefore, we quantified synaptic content, transport, and delivery of GLR-1 in AVA and showed that loss of pkc-2 and tpa-1 leads to decreased transport and delivery but only a subtle decrease in GLR-1 levels at synapses. AVA-specific expression of both PKC-2 and TPA-1 rescued these defects. Finally, genetic epistasis showed that PKC-2 and TPA-1 likely act in the same pathway to control GLR-1 transport and delivery, while regulating different aspects of olfactory learning and STAM. Thus, our data tie together cell-specific functions of 2 PKCs to neuronal and behavioral outcomes in C. elegans, enabling comparative approaches to understand the evolutionarily conserved role of PKC in synaptic plasticity, learning, and memory.

Tau regulates Arc stability in neuronal dendrites via a proteasome-sensitive but ubiquitin-independent pathway.

Tauopathies are neurodegenerative disorders characterized by the deposition of aggregates of the microtubule-associated protein tau, a main component of neurofibrillary tangles. Alzheimer's disease (AD) is the most common type of tauopathy and dementia, with amyloid-beta pathology as an additional hallmark feature of the disease. Besides its role in stabilizing microtubules, tau is localized at postsynaptic sites and can regulate synaptic plasticity. The activity-regulated cytoskeleton-associated protein (Arc) is an immediate early gene that plays a key role in synaptic plasticity, learning, and memory. Arc has been implicated in AD pathogenesis and regulates the release of amyloid-beta. We found that decreased Arc levels correlate with AD status and disease severity. Importantly, Arc protein was upregulated in the hippocampus of Tau KO mice and dendrites of Tau KO primary hippocampal neurons. Overexpression of tau decreased Arc stability in an activity-dependent manner, exclusively in neuronal dendrites, which was coupled to an increase in the expression of dendritic and somatic surface GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. The tau-dependent decrease in Arc was found to be proteasome-sensitive, yet independent of Arc ubiquitination and required the endophilin-binding domain of Arc. Importantly, these effects on Arc stability and GluA1 localization were not observed in the commonly studied tau mutant, P301L. These observations provide a potential molecular basis for synaptic dysfunction mediated through the accumulation of tau in dendrites. Our findings confirm that Arc is misregulated in AD and further show a physiological role for tau in regulating Arc stability and AMPA receptor targeting.

Activation of innate immune receptor TLR9 by mitochondrial DNA plays essential roles in the chemical long-term depression of hippocampal neurons.

Synaptic plasticity is believed to be the cellular basis for experience-dependent learning and memory. Although long-term depression (LTD), a form of synaptic plasticity, is caused by the activity-dependent reduction of cell surface α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPA receptors) at postsynaptic sites, its regulation by neuronal activity is not completely understood. In this study, we showed that the inhibition of toll-like receptor-9 (TLR9), an innate immune receptor, suppresses N-methyl-d-aspartate (NMDA)-induced reduction of cell surface AMPA receptors in cultured hippocampal neurons. We found that inhibition of TLR9 also blocked NMDA-induced activation of caspase-3, which plays an essential role in the induction of LTD. siRNA-based knockdown of TLR9 also suppressed the NMDA-induced reduction of cell surface AMPA receptors, although the scrambled RNA had no effect on the NMDA-induced trafficking of AMPA receptors. Overexpression of the siRNA-resistant form of TLR9 rescued the AMPA receptor trafficking abolished by siRNA. Furthermore, NMDA stimulation induced rapid mitochondrial morphological changes, mitophagy, and the binding of mitochondrial DNA (mtDNA) to TLR9. Treatment with dideoxycytidine and mitochondrial division inhibitor-1, which block mtDNA replication and mitophagy, respectively, inhibited NMDA-dependent AMPA receptor internalization. These results suggest that mitophagy induced by NMDA receptor activation releases mtDNA and activates TLR9, which plays an essential role in the trafficking of AMPA receptors during the induction of LTD.

GSK3α, not GSK3ß, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring.

Glycogen synthase kinase-3 (GSK3) is an important signalling protein in the brain and modulates different forms of synaptic plasticity. Neuronal functions of GSK3 are typically attributed to one of its two isoforms, GSK3ß, simply because of its prevalent expression in the brain. Consequently, the importance of isoform-specific functions of GSK3 in synaptic plasticity has not been fully explored. We now directly address this question for NMDA receptor-dependent long-term depression (LTD) in the hippocampus. Here, we specifically target the GSK3 isoforms with shRNA knock-down in mouse hippocampus and with novel isoform-selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3α, but not GSK3ß, is required for LTD. The specific engagement of GSK3α occurs via its transient anchoring in dendritic spines during LTD induction. We find that the major GSK3 substrate, the microtubule-binding protein tau, is required for this spine anchoring of GSK3α and mediates GSK3α-induced LTD. These results link GSK3α and tau in a common mechanism for synaptic depression and rule out a major role for GSK3ß in this process.

Identification and functional evaluation of GRIA1 missense and truncation variants in individuals with ID: An emerging neurodevelopmental syndrome.

GRIA1 encodes the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, which are ligand-gated ion channels that act as excitatory receptors for the neurotransmitter L-glutamate (Glu). AMPA receptors (AMPARs) are homo- or heteromeric protein complexes with four subunits, each encoded by different genes, GRIA1 to GRIA4. Although GluA1-containing AMPARs have a crucial role in brain function, the human phenotype associated with deleterious GRIA1 sequence variants has not been established. Subjects with de novo missense and nonsense GRIA1 variants were identified through international collaboration. Detailed phenotypic and genetic assessments of the subjects were carried out and the pathogenicity of the variants was evaluated in vitro to characterize changes in AMPAR function and expression. In addition, two Xenopus gria1 CRISPR-Cas9 F0 models were established to characterize the in vivo consequences. Seven unrelated individuals with rare GRIA1 variants were identified. One individual carried a homozygous nonsense variant (p.Arg377Ter), and six had heterozygous missense variations (p.Arg345Gln, p.Ala636Thr, p.Ile627Thr, and p.Gly745Asp), of which the p.Ala636Thr variant was recurrent in three individuals. The cohort revealed subjects to have a recurrent neurodevelopmental disorder mostly affecting cognition and speech. Functional evaluation of major GluA1-containing AMPAR subtypes carrying the GRIA1 variant mutations showed that three of the four missense variants profoundly perturb receptor function. The homozygous stop-gain variant completely destroys the expression of GluA1-containing AMPARs. The Xenopus gria1 models show transient motor deficits, an intermittent seizure phenotype, and a significant impairment to working memory in mutants. These data support a developmental disorder caused by both heterozygous and homozygous variants in GRIA1 affecting AMPAR function.

Mechanisms That Underlie Expression of Estradiol-Induced Excitatory Synaptic Potentiation in the Hippocampus Differ between Males and Females.

17ß-estradiol (E2) is synthesized in the hippocampus of both sexes and acutely potentiates excitatory synapses in each sex. Previously, we found that the mechanisms for initiation of E2-induced synaptic potentiation differ between males and females, including in the molecular signaling involved. Here, we used electrical stimulation and two-photon glutamate uncaging in hippocampal slices from adult male and female rats to investigate whether the downstream consequences of distinct molecular signaling remain different between the sexes or converge to the same mechanism(s) of expression of potentiation. This showed that synaptic activity is necessary for expression of E2-induced potentiation in females but not males, which paralleled a sex-specific requirement in females for calcium-permeable AMPARs (cpAMPARs) to stabilize potentiation. Nonstationary fluctuation analysis of two-photon evoked unitary synaptic currents showed that the postsynaptic component of E2-induced potentiation occurs either through an increase in AMPAR conductance or in nonconductive properties of AMPARs (number of channels × open probability) and never both at the same synapse. In females, most synapses (76%) were potentiated via increased AMPAR conductance, whereas in males, more synapses (60%) were potentiated via an increase in nonconductive AMPAR properties. Inhibition of cpAMPARs eliminated E2-induced synaptic potentiation in females, whereas some synapses in males were unaffected by cpAMPAR inhibition; these synapses in males potentiated exclusively via increased AMPAR nonconductive properties. This sex bias in expression mechanisms of E2-induced synaptic potentiation underscores the concept of latent sex differences in mechanisms of synaptic plasticity in which the same outcome in each sex is achieved through distinct underlying mechanisms.SIGNIFICANCE STATEMENT Estrogens are synthesized in the brains of both sexes and potentiate excitatory synapses to the same degree in each sex. Despite this apparent similarity, the molecular signaling that initiates estrogen-induced synaptic potentiation differs between the sexes. Here we show that these differences extend to the mechanisms of expression of synaptic potentiation and result in distinct patterns of postsynaptic neurotransmitter receptor modulation in each sex. Such latent sex differences, in which the same outcome is achieved through distinct underlying mechanisms in males versus females, indicate that molecular mechanisms targeted for drug development may differ between the sexes even in the absence of an overt sex difference in behavior or disease.

NMDA Receptor Activation-Dependent Antidepressant-Relevant Behavioral and Synaptic Actions of Ketamine.

Ketamine is a well-characterized NMDA receptor (NMDAR) antagonist, although the relevance of this pharmacology to its rapid (within hours of administration) antidepressant actions, which depend on mechanisms convergent with strengthening of excitatory synapses, is unclear. Activation of synaptic NMDARs is necessary for the induction of canonical long-term potentiation (LTP) leading to a sustained expression of increased synaptic strength. We tested the hypothesis that induction of rapid antidepressant effects requires NMDAR activation, by using behavioral pharmacology, western blot quantification of hippocampal synaptoneurosomal protein levels, and ex vivo hippocampal slice electrophysiology in male mice. We found that ketamine exerts an inverted U-shaped dose-response in antidepressant-sensitive behavioral tests, suggesting that an excessive NMDAR inhibition can prevent ketamine's antidepressant effects. Ketamine's actions to induce antidepressant-like behavioral effects, up-regulation of hippocampal AMPAR subunits GluA1 and GluA2, as well as metaplasticity measured ex vivo using electrically-stimulated LTP, were abolished by pretreatment with other non-antidepressant NMDAR antagonists, including MK-801 and CPP. Similarly, the antidepressant-like actions of other putative rapid-acting antidepressant drugs (2R,6R)-hydroxynorketamine (ketamine metabolite), MRK-016 (GABAAα5 negative allosteric modulator), and LY341495 (mGlu2/3 receptor antagonist) were blocked by NMDAR inhibition. Ketamine acted synergistically with an NMDAR positive allosteric modulator to exert antidepressant-like behavioral effects and activation of the NMDAR subunit GluN2A was necessary and sufficient for such relevant effects. We conclude rapid-acting antidepressant compounds share a common downstream NMDAR-activation dependent effector mechanism, despite variation in initial pharmacological targets. Promoting NMDAR signaling or other approaches that enhance NMDAR-dependent LTP-like synaptic potentiation may be an effective antidepressant strategy.SIGNIFICANCE STATEMENT The anesthetic and antidepressant drug ketamine is well-characterized as an NMDA receptor (NMDAR) antagonist; though, the relevance and full impact of this pharmacology to its antidepressant actions is unclear. We found that NMDAR activation, which occurs downstream of their initial actions, is necessary for the beneficial effects of ketamine and several other putative antidepressant compounds. As such, promoting NMDAR signaling, or other approaches that enhance NMDAR-dependent long-term potentiation (LTP)-like synaptic potentiation in vivo may be an effective antidepressant strategy directly, or acting synergistically with other drug or interventional treatments.

Alternative Splicing of the Flip/Flop Cassette and TARP Auxiliary Subunits Engage in a Privileged Relationship That Fine-Tunes AMPA Receptor Gating.

Alternative splicing of AMPA-type glutamate receptors (AMPARs) and allosteric modulation by auxiliary subunits, such as transmembrane AMPAR regulatory proteins (TARPs), are two important mechanisms that regulate the time course of glutamatergic neurotransmission. Prior work has shown that alternative splicing of the flip/flop cassette profoundly regulates TARP γ2 modulation, where flip receptor gating exhibits robust sensitivity to TARPs while flop isoforms are relatively insensitive to TARP modulation. Whether this splice variant-specific regulation extends to other auxiliary subunit families, such as cornichons (CNIHs), GSG1L, or CKAMPs, remains unknown. Here, we demonstrate that CNIH-3 modulation is unaffected by AMPAR alternative splicing due to inherent differences in how CNIH-3 and TARP γ2 modify channel gating. CNIH-3 slows receptor deactivation from the outset of current decay, consistent with structural evidence showing its point of contact at the level of the pore. In contrast, TARP γ2 acts via the KGK site of the ligand-binding domain (LBD) to slow the onset of desensitization. Although GSG1L and CKAMP44 primarily slow recovery from desensitization, their effects on channel gating are unaffected by alternative splicing, further underlining that structural events leading to the onset and recovery from desensitization are separable. Together, this work establishes that alternative splicing and TARP auxiliary subunits form a unique partnership that governs fast glutamatergic signaling at central synapses. Since proteomic studies suggest that all native AMPARs co-assemble with at least two TARPs, allosteric coupling between the flip/flop cassette and TARPs may represent a common design element in all AMPAR complexes of the mammalian brain.SIGNIFICANCE STATEMENT All fast excitatory neurotransmission in the mammalian brain is mediated by AMPA-type glutamate receptors (AMPARs). The time course of AMPAR gating can be regulated by two distinct mechanisms: alternative splicing of the flip/flop cassette and association with auxiliary subunits. Although these regulatory mechanisms have been well studied individually, it is not clear whether alternative splicing impacts auxiliary protein modulation of AMPARs. Here, we compare the four main families of AMPAR auxiliary subunits, transmembrane AMPAR regulatory proteins (TARPs; γ2), cornichons (CNIH-3), GSG1L and CKAMPs (CKAMP44), and find a privileged relationship between TARPs and the flip/flop cassette that is not shared by others. The flop cassette acts as a master switch to override TARP action, and this coupling represents a way to fine-tune AMPAR signaling.

Protein 4.1N Plays a Cell Type-Specific Role in Hippocampal Glutamatergic Synapse Regulation.

Many glutamatergic synapse proteins contain a 4.1N protein binding domain. However, a role for 4.1N in the regulation of glutamatergic neurotransmission has been controversial. Here, we observe significantly higher expression of protein 4.1N in granule neurons of the dentate gyrus (DG granule neurons) compared with other hippocampal regions. We discover that reducing 4.1N expression in rat DG granule neurons of either sex results in a significant reduction in glutamatergic synapse function that is caused by a decrease in the number of glutamatergic synapses. By contrast, we find reduction of 4.1N expression in hippocampal CA1 pyramidal neurons has no impact on basal glutamatergic neurotransmission. We also find 4.1N's C-terminal domain (CTD) to be nonessential to its role in the regulation of glutamatergic synapses of DG granule neurons. Instead, we show that 4.1N's four-point-one, ezrin, radixin, and moesin (FERM) domain is essential for supporting synaptic AMPA receptor (AMPAR) function in these neurons. Altogether, this work demonstrates a novel, cell type-specific role for protein 4.1N in governing glutamatergic synapse function.SIGNIFICANCE STATEMENT Glutamatergic synapses exhibit immense molecular diversity. In comparison to heavily studied Schaffer collateral, CA1 glutamatergic synapses, significantly less is known about perforant path-dentate gyrus (DG) synapses. Our data demonstrate that compromising 4.1N function in CA1 pyramidal neurons produces no alteration in basal glutamatergic synaptic transmission. However, in DG granule neurons, compromising 4.1N function leads to a significant decrease in the strength of glutamatergic neurotransmission at perforant pathway synapses. Together, our data identifies 4.1N as a cell type-specific regulator of synaptic transmission within the hippocampus and reveals a unique molecular program that governs perforant pathway synapse function.

s-Afadin binds to MAGUIN/Cnksr2 and regulates the localization of the AMPA receptor and glutamatergic synaptic response in hippocampal neurons.

A hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. Afadin has two splice variants: l-afadin and s-afadin. l-Afadin, but not s-afadin, regulates the formation of the PAJs but the roles of s-afadin in synaptogenesis remain unknown. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin in vivo and in vitro. MAGUIN/CNKSR2 is one of the causative genes for nonsyndromic X-linked intellectual disability accompanied by epilepsy and aphasia. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABAA receptor antagonist. These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model.