Surface Expression Of AMPA Receptors Research Articles

Homeostatic Synaptic Plasticity of Miniature Excitatory Postsynaptic Currents in Mouse Cortical Cultures Requires Neuronal Rab3A.

Following prolonged activity blockade, amplitudes of miniature excitatory postsynaptic currents (mEPSCs) increase, a form of plasticity termed "homeostatic synaptic plasticity." We previously showed that a presynaptic protein, the small GTPase Rab3A, is required for full expression of the increase in miniature endplate current amplitudes following prolonged blockade of action potential activity at the mouse neuromuscular junction in vivo (Wang et al., 2011), but it is unknown whether this form of Rab3A-dependent homeostatic plasticity shares any characteristics with central synapses. We show here that homeostatic synaptic plasticity of mEPSCs is impaired in mouse cortical neuron cultures prepared from Rab3A-/- and mutant mice expressing a single point mutation of Rab3A, Rab3A Earlybird mice. To determine if Rab3A is involved in the well-established homeostatic increase in postsynaptic AMPA-type receptors (AMPARs), we performed a series of experiments in which electrophysiological recordings of mEPSCs and confocal imaging of synaptic AMPAR immunofluorescence were assessed within the same cultures. We found that Rab3A was required for the increase in synaptic AMPARs following prolonged activity blockade, but the increase in mEPSC amplitudes was not always accompanied by an increase in postsynaptic AMPAR levels, suggesting other factors may contribute. Finally, we demonstrate that Rab3A is acting in neurons because only selective loss of Rab3A in neurons, not glia, disrupted the homeostatic increase in mEPSC amplitudes. This is the first demonstration that neuronal Rab3A is required for homeostatic synaptic plasticity and that it does so partially through regulation of the surface expression of AMPA receptors.

ABHD6 drives endocytosis of AMPA receptors to regulate synaptic plasticity and learning flexibility

Trafficking of α‐Amino‐3–hydroxy‐5–methylisoxazole‐4–propionic acid (AMPA) receptors (AMPARs), mediated by AMPAR interacting proteins, enabled neurons to maintain tuning capabilities at rest or active state. α/β-Hydrolase domain-containing 6 (ABHD6), an endocannabinoid hydrolase, was an AMPAR auxiliary subunit found to negatively regulate the surface delivery of AMPARs. While ABHD6 was found to prevent AMPAR tetramerization in endoplasmic reticulum, ABHD6 was also reported to localize at postsynaptic site. Yet, the role of ABHD6 interacting with AMPAR at postsynaptic site, and the physiological significance of ABHD6 regulating AMPAR trafficking remains elusive. Here, we generated the ABHD6 knockout (ABHD6KO) mice and found that deletion of ABHD6 selectively enhanced AMPAR-mediated basal synaptic responses and the surface expression of postsynaptic AMPARs. Furthermore, we found that loss of ABHD6 impaired hippocampal long-term depression (LTD) and synaptic downscaling in hippocampal synapses. AMPAR internalization assays revealed that ABHD6 was essential for neuronal activity-dependent endocytosis of surface AMPARs, which is independent of ABHD6's hydrolase activity. The defects of AMPAR endocytosis and LTD are expressed as deficits in learning flexibility in ABHD6KO mice. Collectively, we demonstrated that ABHD6 is an endocytic accessory protein promoting AMPAR endocytosis, thereby contributes to the formation of LTD, synaptic downscaling and reversal learning.

Experience-Induced Remodeling of the Hippocampal Post-synaptic Proteome and Phosphoproteome

The postsynaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is a key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the hippocampal PSD proteome and phosphoproteome in mice following four different types ofexperience. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated from individual mice to investigate molecular mechanisms underlying experience-dependent remodeling of synapses. We developed a new strategy to identify and quantify the relatively low level of site-specific phosphorylation of PSD proteome from the hippocampus, by using a modified iTRAQ-based TiSH protocol. In the PSD, we identified 3938 proteins and 2761 phosphoproteins in the sequential strategy covering a total of 4968 unique protein groups (at least two peptides including a unique peptide). On the phosphoproteins, we identified a total of 6188 unambiguous phosphosites (75%<site-localization probability). Strikingly, of the significantly IA-regulated phosphoproteins, a large fraction of these displayed an overall decrease in phosphorylation level. Bioinformatic analysis of proteins and phosphoproteins that were regulated by IA were annotated for involvement in the regulation of glutamate receptor functionality, RHO GTPase cycle, and synaptic plasticity. We also identified synaptic kinases, phosphatases, and their respective phosphosites regulated by IA training or immediate shock. Furthermore, we found that AMPA receptor surface expression was regulated by Mg2+/Mn2+ dependent protein phosphatase 1H (Ppm1h). Together, these results unravel the dynamic remodeling ofthe PSD upon IA learning or immediate shock and serve asa resource for elucidating the synaptic proteome dynamics induced by experience-dependent plasticity.

In the fast lane: Receptor trafficking during status epilepticus.

Status epilepticus (SE) remains a significant cause of morbidity and mortality and often is refractory to standard first-line treatments. A rapid loss of synaptic inhibition and development of pharmacoresistance to benzodiazepines (BZDs) occurs early during SE, while NMDA and AMPA receptor antagonists remain effective treatments after BZDs have failed. Multimodal and subunit-selective receptor trafficking within minutes to an hour of SE involves GABA-A, NMDA, and AMPA receptors and contributes to shifts in the number and subunit composition of surface receptors with differential impacts on the physiology, pharmacology, and strength of GABAergic and glutamatergic currents at synaptic and extrasynaptic sites. During the first hour of SE, synaptic GABA-A receptors containing γ2 subunits move to the cell interior while extrasynaptic GABA-A receptors with δ subunits are preserved. Conversely, NMDA receptors containing N2B subunits are increased at synaptic and extrasynaptic sites, and homomeric GluA1 ("GluA2-lacking") calcium permeant AMPA receptor surface expression also is increased. Molecular mechanisms, largely driven by NMDA receptor or calcium permeant AMPA receptor activation early during circuit hyperactivity, regulate subunit-specific interactions with proteins involved with synaptic scaffolding, adaptin-AP2/clathrin-dependent endocytosis, endoplasmic reticulum (ER) retention, and endosomal recycling. Reviewed here is how SE-induced shifts in receptor subunit composition and surface representation increase the excitatory to inhibitory imbalance that sustains seizures and fuels excitotoxicity contributing to chronic sequela such as "spontaneous recurrent seizures" (SRS). A role for early multimodal therapy is suggested both for treatment of SE and for prevention of long-term comorbidities.

Long noncoding RNA Gm2694 drives depressive-like behaviors in male mice by interacting with GRP78 to disrupt endoplasmic reticulum homeostasis.

Long noncoding RNAs (lncRNAs) are involved in various biological processes and implicated in the regulation of neuronal activity, but the potential role of lncRNAs in depression remains largely unknown. Here, we identified that lncRNA Gm2694 was increased in the medial prefrontal cortex (mPFC) of male mice subjected to chronic social defeat stress (CSDS). The down-regulation of Gm2694 in the mPFC alleviated CSDS-induced depressive-like behaviors through enhanced excitatory synaptic transmission. Furthermore, we found that Gm2694 preferentially interacted with the carboxyl-terminal domain of 78-kilodalton glucose-regulated protein (GRP78), which abrogated GRP78 function and disrupted endoplasmic reticulum homeostasis, resulting in a reduction of the surface expression of AMPA receptors (AMPARs). Overexpression of GRP78 in the mPFC promoted the surface expression of AMPARs and attenuated the CSDS-induced depressive-like behaviors of mice. Together, our results unraveled a previously unknown role of Gm2694 in regulating endoplasmic reticulum homeostasis and excitatory synaptic transmission in depression.

Activation of NLRP3-Caspase-1 pathway contributes to age-related impairments in cognitive function and synaptic plasticity

Aging is characterized by a progressive deterioration in physiological functions that is associated with cognitive decline as well as other physical functional impairments. Microglia activation leading to neuroinflammation has been generally recognized as playing a critical role in the development of age-related cognitive decline. NLRP3 inflammasome in microglia is fundamental for IL-1β maturation and subsequent inflammatory events. However, it remains unknown whether NLRP3 activation contributes to aging-induced cognitive decline in vivo. Here, our study demonstrated that aging rats showed declined cognitive function and impaired synaptic plasticity as well as decreased density of dendritic spines. Importantly, our data demonstrated strongly enhanced expression of NLRP3, ASC and Caspase-1 in the hippocampus of aged rats as well as decreased AMPA receptor and phosphorylated levels of CaMKII and CREB in the hippocampus of natural aging rats. Furthermore, NLRP3 inflammasome inhibitor elevated the surface expression of AMPA receptor and the phosphorylated levels of CaMKII, CREB in hippocampus, and finally contributed to the attenuation of hippocampal long-term potentiation (LTP) deficits and the improvement of cognitive decline of natural aging rats. These results revealed an important role for the NLRP3-Caspase-1 pathway in aging-induced cognitive decline and suggested that inhibition of NLRP3 inflammasome represented a novel therapeutic intervention for aging-related cognitive impairment.

Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.

Sustained postsynaptic kainate receptor activation downregulates AMPA receptor surface expression and induces hippocampal LTD

SummaryIt is well established that long-term depression (LTD) can be initiated by either NMDA or mGluR activation. Here we report that sustained activation of GluK2 subunit-containing kainate receptors (KARs) leads to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) endocytosis and induces LTD of AMPARs (KAR-LTDAMPAR) in hippocampal neurons. The KAR-evoked loss of surface AMPARs is blocked by the ionotropic KAR inhibitor UBP 310 indicating that KAR-LTDAMPAR requires KAR channel activity. Interestingly, however, blockade of PKC or PKA also reduces GluA2 surface expression and occludes the effect of KAR activation. In acute hippocampal slices, kainate application caused a significant loss of GluA2-containing AMPARs from synapses and long-lasting depression of AMPAR excitatory postsynaptic currents in CA1. These data, together with our previously reported KAR-LTPAMPAR, demonstrate that KARs can bidirectionally regulate synaptic AMPARs and synaptic plasticity via different signaling pathways.

NGPF2 triggers synaptic scaling up through ALK-LIMK-cofilin-mediated mechanisms.

Synaptic scaling is an extensively studied form of homeostatic plasticity critically involved in various brain functions. Although it is accepted that synaptic scaling is expressed through the postsynaptic accumulation of AMPA receptors (AMPARs), the induction mechanism remains elusive. In this study, we show that TTX treatment induces rapid but transient release of the neurite growth-promoting factor 2 (NGPF2), and this release is necessary and sufficient for TTX-induced scaling up. In addition, we show that inhibition of the anaplastic lymphoma kinase (ALK)-LIMK-cofilin signaling pathway blocks TTX- and NGPF2-induced synaptic scaling up. Furthermore, we show that TTX-induced release of NGPF2 is protein synthesis dependent and requires fragile X mental retardation protein 1 (FMRP1). These results indicate that activity blockade induces NGPF2 synthesis and release to trigger synaptic scaling up through LIMK-cofilin-dependent actin reorganization, spine enlargement, and stabilization of AMPARs at the synapse.

GRASP1 ubiquitination regulates AMPA receptor surface expression and synaptic activity in cultured hippocampal neurons.

The synaptic expression of glutamate receptors of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type is dynamically controlled by interaction with binding partners and auxiliary proteins. These proteins can be regulated by posttranslational modifications, including ubiquitination. In this work, we investigated the regulation of glutamate receptor interacting protein-associated protein 1 (GRASP1) by ubiquitin-dependent mechanisms and its impact on surface expression and activity of synaptic AMPA receptors. Cotransfection of GFP-ubiquitin decreased myc-GRASP1 protein levels in HEK293T cells, and this effect was inhibited upon transfection of an ubiquitin mutant that cannot be ubiquitinated on Lys48. In addition, transfection of cultured hippocampal neurons with GFP-ubiquitin reduced the dendritic levels of endogenous GRASP1 and decreased the surface expression of GluA1 AMPA receptor subunits, an effect that was partly reversed by cotransfection with GRASP1. Similarly, transfection of hippocampal neurons with GFP-ubiquitin decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by Ca2+ -impermeable AMPA receptors, and this effect was abrogated by cotransfection of GRASP1. Together, the results show a role for ubiquitination in the regulation of the postsynaptic protein GRASP1, which has an impact on the surface distribution of AMPA receptors and on their activity at the synapse.

ABCC1 regulates cocaine-associated memory, spine plasticity and GluA1 and GluA2 surface expression.

ATP-binding cassettes C1 (ABCC1s) are expressed in the neurons of the brain, but their function in neurological diseases is far from clear. In this study, we investigated the role of ABCC1 in the hippocampus in cocaine-associated memory and spine plasticity. We also investigated the role of ABCC1 in AMPA receptors (AMPARs) surface expression in primary prefrontal cortex (PFC) neurons following dopamine treatment, which was used to mimic exposure to cocaine. We found that cocaine increased ABCC1 expression in the hippocampus, and ABCC1-siRNA blocked cocaine-induced place preference. Furthermore, a morphological study showed that ABCC1-siRNA reduced the total spine density, including thin, stubby and mushroom spines in both cocaine and basal treatments compared with controls. Meanwhile, in vitro tests showed that ABCC1-siRNA decreased GluA1 and GluA2 surface expression induced by dopamine, while a decreased number of synapses in primary PFC neurons was observed following dopamine treatment. The data show that ABCC1 in the hippocampus is critically involved in cocaine-associated memory and spine plasticity and that dopamine induces AMPARs surface expression in primary PFC neurons. ABCC1 is thus presented as a new signaling molecule involved in cocaine addiction, which may provide a new target for the treatment of cocaine addiction.

Missense mutation of Fmr1 results in impaired AMPAR-mediated plasticity and socio-cognitive deficits in mice

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.

Selective coactivation of α7- and α4β2-nicotinic acetylcholine receptors reverses beta-amyloid–induced synaptic dysfunction

Beta-amyloid (Aβ) has been recognized as an early trigger in the pathogenesis of Alzheimer's disease (AD) leading to synaptic and cognitive impairments. Aβ can alter neuronal signaling through interactions with nicotinic acetylcholine receptors (nAChRs), contributing to synaptic dysfunction in AD. The three major nAChR subtypes in the hippocampus are composed of α7-, α4β2-, and α3β4-nAChRs. Aβ selectively affects α7- and α4β2-nAChRs, but not α3β4-nAChRs in hippocampal neurons, resulting in neuronal hyperexcitation. However, how nAChR subtype selectivity for Aβ affects synaptic function in AD is not completely understood. Here, we showed that Aβ associated with α7- and α4β2-nAChRs but not α3β4-nAChRs. Computational modeling suggested that two amino acids in α7-nAChRs, arginine 208 and glutamate 211, were important for the interaction between Aβ and α7-containing nAChRs. These residues are conserved only in the α7 and α4 subunits. We therefore mutated these amino acids in α7-containing nAChRs to mimic the α3 subunit and found that mutant α7-containing receptors were unable to interact with Aβ. In addition, mutant α3-containing nAChRs mimicking the α7 subunit interact with Aβ. This provides direct molecular evidence for how Aβ selectively interacted with α7- and α4β2-nAChRs, but not α3β4-nAChRs. Selective coactivation of α7- and α4β2-nAChRs also sufficiently reversed Aβ-induced AMPA receptor dysfunction, including Aβ-induced reduction of AMPA receptor phosphorylation and surface expression in hippocampal neurons. Moreover, costimulation of α7- and α4β2-nAChRs reversed the Aβ-induced disruption of long-term potentiation. These findings support a novel mechanism for Aβ's impact on synaptic function in AD, namely, the differential regulation of nAChR subtypes.

Hippo-Released WWC1 Primes Ampa Receptor Regulatory Complexes for Hippocampal Learning

Learning and memory rely on changes in postsynaptic AMPA-type glutamate receptor (AMPA receptor) number, spatial organization and function. The Hippo pathway component WWC1 regulates AMPA receptor surface expression and impacts on memory performance. However, synaptic binding partners of WWC1 and its hierarchical position in AMPA receptor complexes are largely unknown. Using cell-surface proteomics in hippocampal tissue of Wwc1-deficient mice, and by generating a hippocampus-specific interactome we show that WWC1 is a major regulatory platform in AMPA receptor signalling networks. Interaction of the Hippo pathway members WWC1 and LATS limited the impact of WWC1 on synaptic proteins. Truncation of WWC1/LATS-binding through a point mutation at WWC1 elevated the abundance of WWC1 in AMPA receptor complexes and improved hippocampal-dependent learning and memory. Thus, relocation of WWC1 from the Hippo-pathway to AMPA receptor regulatory complexes provides an innovative strategy for the treatment of cognitive impairments.

AMPA Receptor Surface Expression Is Regulated by S-Nitrosylation of Thorase and Transnitrosylation of NSF

SUMMARYThe regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking affects multiple brain functions, such as learning and memory. We have previously shown that Thorase plays an important role in the internalization of AMPARs from the synaptic membrane. Here, we show that N-methyl-d-aspartate receptor (NMDAR) activation leads to increased S-nitrosylation of Thorase and N-ethylmaleimide-sensitive factor (NSF). S-nitrosylation of Thorase stabilizes Thorase-AMPAR complexes and enhances the internalization of AMPAR and interaction with protein-interacting C kinase 1 (PICK1). S-nitrosylated NSF is dependent on the S-nitrosylation of Thorase via trans-nitrosylation, which modulates the surface insertion of AMPARs. In the presence of the S-nitrosylation-deficient C137L Thorase mutant, AMPAR trafficking, long-term potentiation, and long-term depression are impaired. Overall, our data suggest that both S-nitrosylation and interactions of Thorase and NSF/PICK1 are required to modulate AMPAR-mediated synaptic plasticity. This study provides critical information that elucidates the mechanism underlying Thorase and NSF-mediated trafficking of AMPAR complexes.

Getting a GRIP on AMPARs

Neuroscience Regulation of synaptic AMPA receptors (AMPARs) is a key process implicated in synaptic plasticity. Glutamate receptor interacting protein 1 (GRIP1) is a scaffolding protein that interacts directly with several members of the AMPAR family. GRIP1 regulates the surface expression and synaptic stabilization of AMPARs, but its role in the expression of long-term potentiation (LTP) is unknown. Tan et al. investigated the function of GRIP1 in LTP and its impact on learning and memory. GRIP1 was recruited into synapses with AMPARs. The GRIP1-AMPAR interaction was enhanced during LTP. Loss of GRIP1 blocked the activity-induced accumulation of synaptic AMPARs and mice deficient in Grip1 exhibited learning and memory deficits. These findings indicate an essential role for GRIP1 in synaptic plasticity and cognitive functions. Proc. Natl. Acad. Sci. U.S.A. 10.1073/pnas.2014827117 (2020).

GSK-3β regulates the synaptic expression of NMDA receptors via phosphorylation of phosphatidylinositol 4 kinase type IIα.

Deregulation of GSK‐3β is strongly implicated in a variety of serious brain conditions, such as Alzheimer disease, bipolar disorder and schizophrenia. To understand how GSK‐3β becomes dysregulated in these conditions, it is important to understand its physiological functions in the central nervous system. In this context, GSK‐3β plays a role in the induction of NMDA receptor‐dependent long‐term depression (LTD) and several substrates for GSK‐3β have been identified in this form of synaptic plasticity, including KLC‐2, PSD‐95 and tau. Stabilization of NMDA receptors at synapses has also been shown to involve GSK‐3β, but the substrates involved are currently unknown. Recent work has identified phosphatidylinositol 4 kinase type IIα (PI4KIIα) as a neuronal GSK‐3β substrate that can potentially regulate the surface expression of AMPA receptors. In the present study, we investigated the synaptic role of PI4KIIα in organotypic rat hippocampal slices. We found that knockdown of PI4KIIα has no effect on synaptic AMPA receptor‐mediated synaptic transmission but substantially reduces NMDA receptor‐mediated synaptic transmission. Furthermore, the ability of the selective GSK‐3 inhibitor, CT99021, to reduce the amplitude of NMDA receptor‐mediated currents was occluded in shRNA‐PI4KIIα transfected neurons. The effects of knocking down PI4KIIα were fully rescued by a shRNA‐resistant wild‐type construct, but not by a mutant construct that cannot be phosphorylated by GSK‐3β. These data suggest that GSK‐3β phosphorylates PI4KIIα to stabilize NMDA receptors at the synapse.

A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain.

Maladaptive plasticity involving increased expression of AMPA‐type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell‐permeable, high‐affinity (~2 nM) peptide inhibitor, Tat‐P4‐(C5)2, of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat‐P4‐(C5)2 disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA‐receptor surface expression in vivo. Moreover, Tat‐P4‐(C5)2 administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat‐P4‐(C5)2 as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non‐tandem protein–protein interaction domains.

Tomosyn regulates the small RhoA GTPase to control the dendritic stability of neurons and the surface expression of AMPA receptors.

Tomosyn, a protein encoded by syntaxin‐1‐binding protein 5 (STXBP5) gene, has a well‐established presynaptic role in the inhibition of neurotransmitter release and the reduction of synaptic transmission by its canonical interaction with the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor machinery. However, the postsynaptic role of tomosyn in dendritic arborization, spine stability, and trafficking of ionotropic glutamate receptors remains to be elucidated. We used short hairpin RNA to knock down tomosyn in mouse primary neurons to evaluate the postsynaptic cellular function and molecular signaling regulated by tomosyn. Knockdown of tomosyn led to an increase of RhoA GTPase activity accompanied by compromised dendritic arborization, loss of dendritic spines, decreased surface expression of AMPA receptors, and reduced miniature excitatory postsynaptic current frequency. Inhibiting RhoA signaling was sufficient to rescue the abnormal dendritic morphology and the surface expression of AMPA receptors. The function of tomosyn regulating RhoA is mediated through the N‐terminal WD40 motif, where two variants each carrying a single nucleotide mutation in this region were found in individuals with autism spectrum disorder (ASD). We demonstrated that these variants displayed loss‐of‐function phenotypes. Unlike the wild‐type tomosyn, these two variants failed to restore the reduced dendritic complexity, spine density, as well as decreased surface expression of AMPA receptors in tomosyn knockdown neurons. This study uncovers a novel role of tomosyn in maintaining neuronal function by inhibiting RhoA activity. Further analysis of tomosyn variants also provides a potential mechanism for explaining cellular pathology in ASD.

Neuropathic Pain Causes Memory Deficits and Dendrite Tree Morphology Changes in Mouse Hippocampus.

IntroductionNeuropathic pain manifests in a diverse combination of sensory symptoms and disorders of higher nervous activity, such as memory deficiency, anxiety, depression, anhedonia, etc. This suggests the participation of brain structures, including the hippocampus, in the pathogenesis of neuropathic pain. The elucidation of central sensitization mechanisms underlying neuropathic pain cognitive and affective symptoms may be useful in the development of new and effective treatments for these common disorders. The study aims to elucidate the effect of chronic neuropathic pain on cognitive function and underlying neuronal plasticity in the hippocampus.MethodsChronic constriction injury of mouse right hind limb sciatic nerve was used as a model of neuropathic pain. The presence of neuropathic pain was confirmed by the thermal and mechanical allodynia. The morphology of the CA1 pyramidal neurons and the dentate gyrus (DG) granule neurons were studied using Golgi-Cox staining. The hippocampal proteins concentration was determined by immunohistochemistry and ELISA.ResultsBehavioral testing revealed reduced locomotor activity as well as impaired working and long-term memory in mice with a ligated nerve. We revealed changes in the dendritic tree morphology in CA1 and the dentate gyrus hippocampal subregions. We found the atrophy of the CA1 pyramidal neurons and an increase in the dendritic tree complexity in DG. Moreover, changes in the density of dendritic spines were observed in these regions. In addition, we revealed increased expression of the Arc protein in DG granule neurons and decreased surface expression of AMPA receptors within the hippocampus. Decreased AMPA receptors expression underlies observed altered dendrite arborization and dendritic spines morphology.DiscussionWe found that pain information entering the hippocampus causes neuronal plasticity changes. The changes in neurite arborization, dendritic length and dendritic spines morphology as well as protein expression are observed within the hippocampal regions involved in the processing of pain information. Moreover, changes in the dendrite morphology in hippocampal subregions are different due to the anatomical and functional heterogeneity of the hippocampus. Apparently, the detected morphological and biochemical changes can underlie the observed hippocampus-dependent behavioral and cognitive impairment in animals with neuropathic pain.