AMPA-type Glutamate Receptors Research Articles

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.

Inhibiting Hippo pathway kinases releases WWC1 to promote AMPAR-dependent synaptic plasticity and long-term memory in mice.

The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Seizures exacerbate excitatory: inhibitory imbalance in Alzheimer's disease and 5XFAD mice.

Approximately 22% of Alzheimer's disease (AD) patients suffer from seizures, and the co-occurrence of seizures and epileptiform activity exacerbates AD pathology and related cognitive deficits, suggesting that seizures may be a targetable component of AD progression. Given that alterations in neuronal excitatory:inhibitory (E:I) balance occur in epilepsy, we hypothesized that decreased markers of inhibition relative to those of excitation would be present in AD patients. We similarly hypothesized that in five times familial AD (5XFAD) mice, the E:I imbalance would progress from an early stage (prodromal) to later symptomatic stages, and be further exacerbated by pentylenetetrazol (PTZ) kindling. Post-mortem AD temporal cortical tissue from patients with or without seizure history were examined for changes in several markers of E:I balance, including levels of the inhibitory GABAA receptor, the sodium potassium chloride cotransporter 1 (NKCC1) and potassium chloride cotransporter 2 (KCC2), and the excitatory NMDA and AMPA type glutamate receptors. We performed patch clamp electrophysiological recordings from CA1 neurons in hippocampal slices and examined the same markers of E:I balance in prodromal 5XFAD mice. We next examined 5XFAD mice at chronic stages, after PTZ or control protocols, and in response to chronic mTORC1 inhibitor rapamycin, administered following kindled seizures, for markers of E:I balance. We found that AD patients with comorbid seizures had worsened cognitive and functional scores and had decreased GABAA receptor subunit expression, and increased NKCC1/KCC2 ratios, indicative of depolarizing GABA responses. Patch clamp recordings of prodromal 5XFAD CA1 neurons showed increased intrinsic excitability, along with decreased GABAergic inhibitory transmission and altered glutamatergic neurotransmission, indicating that E:I imbalance may occur in early disease stages. Furthermore, seizure induction in prodromal 5XFAD mice led to later dysregulation of NKCC1/KCC2 and a reduction in GluA2 AMPA glutamate receptor subunit expression, indicative of depolarizing GABA receptors and calcium permeable AMPA receptors. Finally, we found that chronic treatment with the mTORC1 inhibitor, rapamycin, at doses we have previously shown to attenuate seizure-induced β-amyloid pathology and cognitive deficits, could also reverse elevations to NKCC1/KCC2 ratio in these mice. Our data demonstrate novel mechanisms of interaction between AD and epilepsy and indicate that targeting E:I balance, potentially with FDA-approved mTOR inhibitors, hold therapeutic promise for AD patients with a seizure history.

Role of GLR-1 in Age-Dependent Short-Term Memory Decline.

As the global elderly population grows, age-related cognitive decline is becoming an increasingly significant healthcare issue, often leading to various neuropsychiatric disorders. Among the many molecular players involved in memory, AMPA-type glutamate receptors are known to regulate learning and memory, but how their dynamics change with age and affect memory decline is not well understood. Here, we examined the in vivo properties of the AMPA-type glutamate receptor GLR-1 in the AVA interneuron of the Caenorhabditis elegans nervous system during physiological aging. We found that both total and membrane-bound GLR-1 receptor levels decrease with age in wild-type worms, regardless of their location along the axon. Using fluorescence recovery after photobleaching, we also demonstrated that a reduction in GLR-1 abundance correlates with decreased local, synaptic GLR-1 receptor dynamics. Importantly, we found that reduced GLR-1 levels strongly correlate with the age-related decline in short-term associative memory. Genetic manipulation of GLR-1 stability, by either deleting msi-1 or expressing a ubiquitination-defective GLR-1 (4KR) variant, prevented this age-related reduction in receptor abundance and improved the short-term memory performance in older animals, which reached performance levels similar to those of young animals. Overall, our data indicate that AMPA-type glutamate receptor abundance and dynamics are key factors in maintaining memory function and that changes in these parameters are linked to age-dependent short-term memory decline.

Tyro3 promotes the maturation of glutamatergic synapses.

The receptor tyrosine kinase Tyro3 is abundantly expressed in neurons of the neocortex, hippocampus, and striatum, but its role in these cells is unknown. We found that neuronal expression of this receptor was markedly up-regulated in the postnatal mouse neocortex immediately prior to the final development of glutamatergic synapses. In the absence of Tyro3, cortical and hippocampal synapses never completed end-stage differentiation and remained electrophysiologically and ultrastructurally immature. Tyro3-/- cortical neurons also exhibited diminished plasma membrane expression of the GluA2 subunits of AMPA-type glutamate receptors, which are essential to mature synaptic function. Correspondingly, GluA2 membrane insertion in wild-type neurons was stimulated by Gas6, a Tyro3 ligand widely expressed in the postnatal brain. Behaviorally, Tyro3-/- mice displayed learning enhancements in spatial recognition and fear-conditioning assays. Together, these results demonstrate that Tyro3 promotes the functional maturation of glutamatergic synapses by driving plasma membrane translocation of GluA2 AMPA receptor subunits.

Amyloid-β-induced dendritic spine elimination requires Ca2+-permeable AMPA receptors, AKAP-Calcineurin-NFAT signaling, and the NFAT target gene Mdm2.

Alzheimer's Disease (AD) is associated with brain accumulation of synaptotoxic amyloid-β (Aβ) peptides produced by the proteolytic processing of amyloid precursor protein (APP). Cognitive impairments associated with AD correlate with dendritic spine and excitatory synapse loss, particularly within the hippocampus. In rodents, soluble Aβ oligomers impair hippocampus-dependent learning and memory, promote dendritic spine loss, inhibit NMDA-type glutamate receptor (NMDAR)-dependent long-term potentiation (LTP), and promote synaptic depression (LTD), at least in part through activation of the Ca2+-CaM-dependent phosphatase calcineurin (CaN). Yet, questions remain regarding Aβ-dependent postsynaptic CaN signaling specifically at the synapse to mediate its synaptotoxicity. Here, we use pharmacologic and genetic approaches to demonstrate a role for postsynaptic signaling via A kinase-anchoring protein 150 (AKAP150)-scaffolded CaN in mediating Aβ-induced dendritic spine loss in hippocampal neurons from rats and mice of both sexes. In particular, we found that Ca2+-permeable AMPA-type glutamate receptors (CP-AMPARs), which were previously shown to signal through AKAP-anchored CaN to promote both LTD and Aβ-dependent inhibition of LTP, are also required upstream of AKAP-CaN signaling to mediate spine loss via overexpression of APP containing multiple mutations linked to familial, early-onset AD and increased Aβ production. In addition, we found that the CaN-dependent nuclear factor of activated T-cells (NFAT) transcription factors are required downstream to promote Aβ-mediated dendritic spine loss. Finally, we identified the E3-ubiquitin ligase Mdm2, which was previously linked to LTD and developmental synapse elimination, as a downstream NFAT target gene upregulated by Aβ whose enzymatic activity is required for Aβ-mediated spine loss.Significance Statement Impaired hippocampal function and synapse loss are hallmarks of AD linked to Aβ oligomers. Aβ exposure acutely blocks hippocampal LTP and enhances LTD and chronically leads to dendritic spine synapse loss. In particular, Aβ hijacks normal plasticity mechanisms, biasing them toward synapse weakening/elimination, with previous studies broadly linking CaN phosphatase signaling to this synaptic dysfunction. However, we do not understand how Aβ engages signaling specifically at synapses. Here we elucidate a synapse-to-nucleus signaling pathway coordinated by the postsynaptic scaffold protein AKAP150 that is activated by Ca2+ influx through CP-AMPARs and transduced to nucleus by CaN-NFAT signaling to transcriptionally upregulate the E3-ubiquitin ligase Mdm2 that is required for Aβ-mediated spine loss. These findings identify Mdm2 as potential therapeutic target for AD.

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.

Fused in sarcoma regulates glutamate signaling and oxidative stress response

Mutations in fused in sarcoma (fust-1) are linked to ALS. However, how these ALS causative mutations alter physiological processes and lead to the onset of ALS remains largely unknown. By obtaining humanized fust-1 ALS mutations via CRISPR-CAS9, we generated a C. elegans ALS model. Homozygous fust-1 ALS mutant and fust-1 deletion animals are viable in C. elegans. This allows us to better characterize the molecular mechanisms of fust-1-dependent responses. We found FUST-1 plays a role in regulating superoxide dismutase, glutamate signaling, and oxidative stress. FUST-1 suppresses SOD-1 and VGLUT/EAT-4 in the nervous system. FUST-1 also regulates synaptic AMPA-type glutamate receptor GLR-1. We found that fust-1 ALS mutations act as loss-of-function in SOD-1 and VGLUT/EAT-4 phenotypes, whereas the fust-1 ALS mutations act as gain-of-function in redox homeostasis and the microbe-induced oxidative stress response. We hypothesized that FUST-1 is a link between glutamate signaling and SOD-1. Our results may provide new insights into the human ALS alleles and their roles in pathological mechanisms that lead to ALS.

CNSC-06. REDIRECTING IMMUNOSUPPRESSIVE GLIOMA MICROENVIRONMENT THROUGH PHARMACOLOGICAL INTERVENTION TARGETING NEURONAL ACTIVITY

Abstract Recent single-cell RNA-sequencing analyses have revealed a strong association between glioma-neuronal circuit remodeling and regional immunosuppression. Subsequent preclinical investigations using syngeneic tumor models demonstrated that genetic disruption of circuit assembly genes, including Thrombospondin-1 (TSP-1/Thbs1) in GBM cells suppressed synaptogenesis, excitatory neurotransmitter ligand and receptor density in addition to neuronal activities while simultaneously restoring inflammatory responses, indicating glioma-neuron-immune cross talk. However, the underlying mechanisms and directions of causality remain unclear. Here we test the hypothesis that tumor-derived synaptogenic factors directly reprogram the tumor immune microenvironment (“model 1”). Alternatively, tumor intrinsic neuronal activity may indirectly regulate adaptive immunity (“model 2”). In this study, we performed in-vitro bone-marrow-derived macrophage (BMDM) reprogramming assays evaluating various stimulation conditions and in-vivo experiments evaluating the effect of neuronal activity-oriented therapy on the tumor-associated macrophages (TAMs). Our in-vitro assays demonstrated that, secreted factors from mouse neonatal cortical neurons (not tumor-derived synaptogenic factors) induced an anti-inflammatory, M2-like phenotype of BMDMs, corroborating the proposed model 2. This M2-inducing effect was inhibited when the neuronal activities were suppressed pharmacologically, using perampanel (PER), a noncompetitive inhibitor of neuronal AMPA-type glutamate receptors. Direct treatment of BMDMs with PER did not affect the polarization patterns, further supporting the M2-inducing effect of glutamatergic excitatory neuronal signals. In in-vivo syngeneic orthotopic tumor models, treatment with PER significantly prolonged survival compared to the non-treated group (27.5 [PER] vs. 23 days [control], p = 0.01). Flow cytometric analysis of brain-infiltrating leukocytes showed that TAMs isolated from PER-treated mice exhibited a more proinflammatory M1-like polarization compared to the control group (median M1/M2 ratio, 1.2 [PER] vs. 0.7 [control], p = 0.004), which explained the improved prognosis. Collectively, these findings unveil novel immunosuppression mechanisms induced by glioma-neuronal circuit remodeling and the resulting excitatory neuronal signals, and potential therapeutic vulnerabilities that could be targeted in combination with cancer immunotherapy.

Outside-in signaling through the major histocompatibility complex class-I cytoplasmic tail modulates glutamate receptor expression in neurons

The interplay between AMPA-type glutamate receptors (AMPARs) and major histocompatibility complex class I (MHC-I) proteins in regulating synaptic signaling is a crucial aspect of central nervous system (CNS) function. In this study, we investigate the significance of the cytoplasmic tail of MHC-I in synaptic signaling within the CNS and its impact on the modulation of synaptic glutamate receptor expression. Specifically, we focus on the Y321 to F substitution (Y321F) within the conserved cytoplasmic tyrosine YXXΦ motif, known for its dual role in endocytosis and cellular signaling of MHC-I. Our findings reveal that the Y321F substitution influences the expression of AMPAR subunits GluA2/3 and leads to alterations in the phosphorylation of key kinases, including Fyn, Lyn, p38, ERK1/2, JNK1/2/3, and p70 S6 kinase. These data illuminate the crucial role of MHC-I in AMPAR function and present a novel mechanism by which MHC-I integrates extracellular cues to modulate synaptic plasticity in neurons, which ultimately underpins learning and memory.

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD have indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3b, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3b activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3b activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3b. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces post-synaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated, key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity and human neuropathology.

Ubiquitination of the GluA1 Subunit of AMPA Receptors Is Required for Synaptic Plasticity, Memory, and Cognitive Flexibility.

Activity-dependent changes in the number of AMPA-type glutamate receptors (AMPARs) at the synapse underpin the expression of LTP and LTD, cellular correlates of learning and memory. Post-translational ubiquitination has emerged as a key regulator of the trafficking and surface expression of AMPARs, with ubiquitination of the GluA1 subunit at Lys-868 controlling the post-endocytic sorting of the receptors into the late endosome for degradation, thereby regulating their stability at synapses. However, the physiological significance of GluA1 ubiquitination remains unknown. In this study, we generated mice with a knock-in mutation in the major GluA1 ubiquitination site (K868R) to investigate the role of GluA1 ubiquitination in synaptic plasticity, learning, and memory. Our results reveal that these male mice have normal basal synaptic transmission but exhibit enhanced LTP and deficits in LTD. They also display deficits in short-term spatial memory and cognitive flexibility. These findings underscore the critical roles of GluA1 ubiquitination in bidirectional synaptic plasticity and cognition in male mice.SIGNIFICANCE STATEMENT Subcellular targeting and membrane trafficking determine the precise number of AMPA-type glutamate receptors at synapses, processes that are essential for synaptic plasticity, learning, and memory. Post-translational ubiquitination of the GluA1 subunit marks AMPARs for degradation, but its functional role in vivo remains unknown. Here we demonstrate that the GluA1 ubiquitin-deficient mice exhibit an altered threshold for synaptic plasticity accompanied by deficits in short-term memory and cognitive flexibility. Our findings suggest that activity-dependent ubiquitination of GluA1 fine-tunes the optimal number of synaptic AMPARs required for bidirectional synaptic plasticity and cognition in male mice. Given that increases in amyloid-β cause excessive ubiquitination of GluA1, inhibiting that GluA1 ubiquitination may have the potential to ameliorate amyloid-β-induced synaptic depression in Alzheimer's disease.

Cocaine-induced sensitization and glutamate plasticity in the nucleus accumbens core: effects of sex

BackgroundThe development and persistence of addiction is mediated in part by drug-induced alterations in nucleus accumbens (NAc) function. AMPA-type glutamate receptors (AMPARs) provide the main source of excitatory drive to the NAc and enhancements in transmission of calcium-permeable AMPARs (CP-AMPARs) mediate increased cue-triggered drug-seeking following prolonged withdrawal. Cocaine treatment regimens that result in psychomotor sensitization enhance subsequent drug-seeking and drug-taking behaviors. Furthermore, cocaine-induced locomotor sensitization followed by 14 days of withdrawal results in an increase in glutamatergic synaptic transmission. However, very few studies have examined cocaine-induced alterations in synaptic transmission of females or potential effects of experimenter-administered cocaine on NAc CP-AMPAR-mediated transmission in either sex.MethodsMale and female rats were given repeated systemic cocaine injections to induce psychomotor sensitization (15 mg/kg, i.p. 1 injection/day, 8 days). Controls received repeated saline (1 mL/kg, i.p). After 14–16 days of withdrawal brain slices were prepared and whole-cell patch-clamp approaches in the NAc core were used to measure spontaneous excitatory post-synaptic currents (sEPSC), paired pulse ratio, and CP-AMPAR transmission. Additional female rats from this same cohort were also given a challenge injection of cocaine at withdrawal day 14 to assess the expression of sensitization.ResultsRepeated cocaine produced psychomotor sensitization in both sexes. In males this was accompanied by an increase in sEPSC frequency, but not amplitude, and there was no effect on the paired pulse ratio. Males treated with cocaine and saline had similar sensitivity to Naspm. In contrast, in females there were no significant differences between cocaine and saline groups on any measure, despite females showing robust psychomotor sensitization both during the induction and expression phase.ConclusionsOverall, these data reveal striking sex differences in cocaine-induced NAc glutamate plasticity that accompany the induction of psychomotor sensitization. This suggests that the neural adaptations that contribute to sensitization vary by sex.

Synaptic mechanisms underlying onset and progression of memory deficits caused by hippocampal and midbrain synucleinopathy

Cognitive deficits, including working memory, and visuospatial deficits are common and debilitating in Parkinson’s disease. α-synucleinopathy in the hippocampus and cortex is considered as the major risk factor. However, little is known about the progression and specific synaptic mechanisms underlying the memory deficits induced by α-synucleinopathy. Here, we tested the hypothesis that pathologic α-Synuclein (α-Syn), initiated in different brain regions, leads to distinct onset and progression of the pathology. We report that overexpression of human α-Syn in the murine mesencephalon leads to late onset memory impairment and sensorimotor deficits accompanied by reduced dopamine D1 expression in the hippocampus. In contrast, human α-Syn overexpression in the hippocampus leads to early memory impairment, altered synaptic transmission and plasticity, and decreased expression of GluA1 AMPA-type glutamate receptors. These findings identify the synaptic mechanisms leading to memory impairment induced by hippocampal α-synucleinopathy and provide functional evidence of the major neuronal networks involved in disease progression.

Enhanced functional detection of synaptic calcium-permeable AMPA receptors using intracellular NASPM.

Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent channel block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits - including transmembrane AMPAR regulatory proteins, cornichons, and GSG1L - which are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here, we show that the inclusion of the spider toxin analog 1-naphthylacetyl spermine (NASPM) in the intracellular solution results in a complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, completely blocks outward synaptic currents. Thus, our results identify a functional measure of CP-AMPARs, that is unaffected by their auxiliary subunit content.

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 through 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, 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.

Sex differences in glutamate AMPA receptor subunits mRNA with fast gating kinetics in the mouse cochlea.

Evidence shows that females have increased supra-threshold peripheral auditory processing compared to males. This is indicated by larger auditory brainstem responses (ABR) wave I amplitude, which measures afferent spiral ganglion neuron (SGN)-auditory nerve synchrony. However, the underlying molecular mechanisms of this sex difference are mostly unknown. We sought to elucidate sex differences in ABR wave I amplitude by examining molecular markers known to affect synaptic transmission kinetics. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate fast excitatory transmission in mature SGN afferent synapses. Each AMPAR channel is a tetramer composed of GluA2, 3, and 4 subunits (Gria2, 3, and 4 genes), and those lacking GluA2 subunits have larger currents, are calcium-permeable, and have faster gating kinetics. Moreover, alternatively spliced flip and flop isoforms of each AMPAR subunit affect channel kinetics, having faster kinetics those AMPARs containing Gria3 and Gria4 flop isoforms. We hypothesized that SGNs of females have more fast-gating AMPAR subunit mRNA than males, which could contribute to more temporally precise synaptic transmission and increased SGN synchrony. Our data show that the index of Gria3 relative to Gria2 transcripts on SGN was higher in females than males (females: 48%; males: 43%), suggesting that females have more SGNs with higher Gria3 mRNA relative to Gria2. Analysis of the relative abundance of the flip and flop alternatively spliced isoforms showed that females have a 2-fold increase in fast-gating Gria3 flop mRNA, while males have more slow-gating (2.5-fold) of the flip. We propose that Gria3 may in part mediate greater SGN synchrony in females. Significance Statement: Females of multiple vertebrate species, including fish and mammals, have been reported to have enhanced sound-evoked synchrony of afferents in the auditory nerve. However, the underlying molecular mediators of this physiologic sex difference are unknown. Elucidating potential molecular mechanisms related to sex differences in auditory processing is important for maintaining healthy ears and developing potential treatments for hearing loss in both sexes. This study found that females have a 2-fold increase in Gria3 flop mRNA, a fast-gating AMPA-type glutamate receptor subunit. This difference may contribute to greater neural synchrony in the auditory nerve of female mice compared to males, and this sex difference may be conserved in all vertebrates.

Anti-glutamatergic Effects of Three Lignan Compounds: Arctigenin, Matairesinol and Trachelogenin - An ex vivo Study on Rat Brain Slices.

Arctigenin is a bioactive dibenzylbutyrolactone-type lignan exhibiting various pharmacological activities. The neuroprotective effects of arctigenin were demonstrated to be mediated via inhibition of AMPA and KA type glutamate receptors in the somatosensory cortex of the rat brain. The aim of this study was to compare the effects of arctigenin with matairesinol and trachelogenin on synaptic activity in ex vivo rat brain slices. Arctigenin, matairesinol and trachelogenin were isolated from Arctium lappa, Centaurea scabiosa and Cirsium arvense, respectively, and applied on brain slices via perfusion medium at the concentration range of 0.5 - 40 µM. The effects of the lignans were examined in the CA1 hippocampus and the somatosensory cortex by recording electrically evoked field potentials. Arctigenin and trachelogenin caused a significant dose-dependent decrease in the amplitude of hippocampal population spikes (POPS) and the slope of excitatory postsynaptic potentials (EPSPs), whereas matairesinol (1 µM and 10 µM) decreased EPSP slope but had no effect on POPS amplitude. Trachelogenin effect (0.5 µM, 10 µM, 20 µM) was comparable to arctigenin (1 µM, 20 µM, 40 µM) (p > 0.05). In the neocortex, arctigenin (10 µM, 20 µM) and trachelogenin (10 µM) significantly decreased the amplitude of evoked potential early component, while matairesinol (1 µM and 10 µM) had no significant effect (p > 0.05). The results suggest that trachelogenin and arctigenin act via inhibition of AMPA and KA receptors in the brain and trachelogenin has a higher potency than arctigenin. Thus, trachelogenin and arctigenin could serve as lead compounds in the development of neuroprotective drugs.

Inhibition mechanism of AMPA receptors by the pore blocker memantine.

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by AMPA-type ionotropic glutamate receptors. AMPA receptors co-assemble with auxiliary proteins, such as transmembrane AMPA receptor regulatory proteins (TARPs), cornichons, cysteine knot proteins and GSG1; which regulate AMPAR assembly, trafficking, gating, and pharmacology. TARPs are the most studied AMPAR auxiliary proteins for their role in the synaptic plasticity. The coupling between AMPA and TARPs change their kinetic and pharmacology properties. Here we show that in the presence of TARPs GluA2 receptors are inhibited by memantine. Memantine inhibition is dependent on charge at site 607, glutamine to arginine mutation showing a larger inhibition by memantine relative to the that GluA2(R) and the GluA2(Q). The inhibition by memantine is activity- and voltage-dependent, suggesting a pore block mechanism.

Low-Density Lipoprotein Receptor LRP-2 regulates GLR-1 glutamate receptors and glutamatergic behavior in C. elegans.

We identified the Low-Density Lipoprotein (LDL) Receptor Related Protein-2 (LRP-2) in a RNAi screen for genes that regulate glutamatergic behavior in C. elegans . lrp-2 loss-of-function mutants have defects in glutamatergic mechanosensory nose-touch behavior and suppress increased spontaneous reversals induced by GLR-1(A/T), a constitutively-active form of the AMPA-type glutamate receptor GLR-1. Total and surface levels of GLR-1 are increased throughout the ventral nerve cord of lrp-2 mutants suggesting that LRP-2 promotes glutamatergic signaling by regulating some aspect of GLR-1 trafficking, localization or function.