Parkinson's-linked LRRK2-G2019S derails AMPAR trafficking, mobility, and composition in striatum with cell-type and subunit specificity.

Parkinson's disease (PD) is a multifactorial disease that affects multiple brain systems and circuits. While defined by motor symptoms caused by degeneration of brainstem dopamine neurons, debilitating non-motor abnormalities in fronto-striatal-based cognitive function are common, appear early, and are initially independent of dopamine. Young adult mice expressing the PD-associated G2019S missense mutation in Lrrk2 also exhibit deficits in fronto-striatal-based cognitive tasks. In mice and humans, cognitive functions require dynamic adjustments in glutamatergic synapse strength through cell-surface trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs), but it is unknown how LRRK2 mutation impacts dynamic features of AMPAR trafficking in striatal projection neurons (SPNs). Here, we used Lrrk2G2019S knockin mice to show that surface AMPAR subunit stoichiometry is altered biochemically and functionally in mutant SPNs in dorsomedial striatum to favor the incorporation of GluA1 over GluA2. GluA1-containing AMPARs were resistant to internalization from the cell surface, leaving an excessive accumulation of GluA1 on the surface within and outside synapses. This negatively impacted trafficking dynamics that normally support synapse strengthening, as GluA1-containing AMPARs failed to increase at synapses in response to a potentiating stimulus and showed significantly reduced surface mobility. Surface GluA2-containing AMPARs were expressed at normal levels in synapses, indicating subunit-selective impairment. Abnormal surface accumulation of GluA1 was independent of PKA activity and was limited to D1R SPNs. Since LRRK2 mutation is thought to be part of a common PD pathogenic pathway, our data suggest that sustained, striatal cell-type specific changes in AMPAR composition and trafficking contribute to cognitive or other impairments associated with PD.

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.

Recovery from desensitization in GluA2 AMPA receptors is affected by a single mutation in the N-terminal domain interface.

AMPA-type ionotropic glutamate receptors (AMPARs) are central to various neurological processes, including memory and learning. They assemble as homo- or heterotetramers of GluA1, GluA2, GluA3, and GluA4 subunits, each consisting of an N-terminal domain (NTD), a ligand-binding domain, a transmembrane domain, and a C-terminal domain. While AMPAR gating is primarily controlled by reconfiguration in the ligand-binding domain layer, our study focuses on the NTDs, which also influence gating, yet the underlying mechanism remains enigmatic. In this investigation, we employ molecular dynamics simulations to evaluate the NTD interface strength in GluA1, GluA2, and NTD mutants GluA2-H229N and GluA1-N222H. Our findings reveal that GluA1 has a significantly weaker NTD interface than GluA2. The NTD interface of GluA2 can be weakened by a single point mutation in the NTD dimer-of-dimer interface, namely H229N, which renders GluA2 more GluA1-like. Electrophysiology recordings demonstrate that this mutation also leads to slower recovery from desensitization. Moreover, we observe that lowering the pH induces more splayed NTD states and enhances desensitization in GluA2. We hypothesized that H229 was responsible for this pH sensitivity; however, GluA2-H229N was also affected by pH, meaning that H229 is not solely responsible and that protons exert their effect across multiple domains of the AMPAR. In summary, our work unveils an allosteric connection between the NTD interface strength and AMPAR desensitization.

GluA1 Degradation by Autophagy Contributes to Circadian Rhythm Effects on Cerebral Ischemia Injury.

The mechanisms of many diseases, including central nervous system disorders, are regulated by circadian rhythms. The development of brain disorders such as depression, autism, and stroke is strongly associated with circadian cycles. Previous studies have shown that cerebral infarct volume is smaller at night (active phase) than during the day (inactive phase) in ischemic stroke rodent models. However, the underlying mechanisms remain unclear. Increasing evidence suggests that glutamate systems and autophagy play important roles in the pathogenesis of stroke. Here, we report that GluA1 expression was decreased and autophagic activity was increased in active-phase male mouse models of stroke compared with the inactive-phase models. In the active-phase model, induction of autophagy decreased the infarct volume, whereas inhibition of autophagy increased the infarct volume. Meanwhile, GluA1 expression was decreased following activation of autophagy and increased following inhibition of autophagy. We used Tat-GluA1 to uncouple p62, an autophagic adapter, from GluA1 and found that this blocked the degradation of GluA1, an effect similar to that of inhibition of autophagy in the active-phase model. We also demonstrated that knock-out of the circadian rhythm gene Per1 abolished the circadian rhythmicity of the volume of infarction and also abolished GluA1 expression and autophagic activity in wild-type (WT) mice. Our results suggest an underlying mechanism by which the circadian rhythm participates in the autophagy-dependent regulation of GluA1 expression, which influences the volume of infarction in stroke.SIGNIFICANCE STATEMENT Circadian rhythms affect the pathophysiological mechanisms of disease. Previous studies suggested that circadian rhythms affect the infarct volume in stroke, but the underlying mechanisms remain largely unknown. Here, we demonstrate that the smaller infarct volume after middle cerebral artery occlusion/reperfusion (MCAO/R) during the active phase is related to lower GluA1 expression and activation of autophagy. The decrease in GluA1 expression during the active phase is mediated by the p62-GluA1 interaction, followed by direct autophagic degradation. In short, GluA1 is the substrate of autophagic degradation, which mainly occurs after MCAO/R during the active phase but not the inactive phase.

Loss of Depalmitoylation Disrupts Homeostatic Plasticity of AMPARs in a Mouse Model of Infantile Neuronal Ceroid Lipofuscinosis.

Protein palmitoylation is the only reversible post-translational lipid modification. Palmitoylation is held in delicate balance by depalmitoylation to precisely regulate protein turnover. While over 20 palmitoylation enzymes are known, depalmitoylation is conducted by fewer enzymes. Of particular interest is the lack of the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1) that causes the devastating pediatric neurodegenerative condition infantile neuronal ceroid lipofuscinosis (CLN1). While most of the research on Ppt1 function has centered on its role in the lysosome, recent findings demonstrated that many Ppt1 substrates are synaptic proteins, including the AMPA receptor (AMPAR) subunit GluA1. Still, the impact of Ppt1-mediated depalmitoylation on synaptic transmission and plasticity remains elusive. Thus, the goal of the present study was to use the Ppt1 -/- mouse model (both sexes) to determine whether Ppt1 regulates AMPAR-mediated synaptic transmission and plasticity, which are crucial for the maintenance of homeostatic adaptations in cortical circuits. Here, we found that basal excitatory transmission in the Ppt1 -/- visual cortex is developmentally regulated and that chemogenetic silencing of the Ppt1 -/- visual cortex excessively enhanced the synaptic expression of GluA1. Furthermore, triggering homeostatic plasticity in Ppt1 -/- primary neurons caused an exaggerated incorporation of GluA1-containing, calcium-permeable AMPARs, which correlated with increased GluA1 palmitoylation. Finally, Ca2+ imaging in awake Ppt1 -/- mice showed visual cortical neurons favor a state of synchronous firing. Collectively, our results elucidate a crucial role for Ppt1 in AMPAR trafficking and show that impeded proteostasis of palmitoylated synaptic proteins drives maladaptive homeostatic plasticity and abnormal recruitment of cortical activity in CLN1.SIGNIFICANCE STATEMENT Neuronal communication is orchestrated by the movement of receptors to and from the synaptic membrane. Protein palmitoylation is the only reversible post-translational lipid modification, a process that must be balanced precisely by depalmitoylation. The significance of depalmitoylation is evidenced by the discovery that mutation of the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (Ppt1) causes severe pediatric neurodegeneration. In this study, we found that the equilibrium provided by Ppt1-mediated depalmitoylation is critical for AMPA receptor (AMPAR)-mediated plasticity and associated homeostatic adaptations of synaptic transmission in cortical circuits. This finding complements the recent explosion of palmitoylation research by emphasizing the necessity of balanced depalmitoylation.

Spinal nerve transection-induced upregulation of SAP97 via promoting membrane trafficking of GluA1-containing AMPA receptors in the dorsal horn contributes to the pathogenesis of neuropathic pain.

Emerging evidence has implicated an important role of synapse-associated protein-97 (SAP97)-regulated GluA1-containing AMPARs membrane trafficking in cocaine restate and in contextual episodic memory of schizophrenia. Herein, we investigated the role of SAP97 in neuropathic pain following lumbar 5 spinal nerve transection (SNT) in rats. Our results showed that SNT led to upregulation of SAP97, enhanced the interaction between SAP97 and GluA1, and increased GluA1-containing AMPARs membrane trafficking in the dorsal horn. Microinjection of AAV-EGFP-SAP97 shRNA in lumbar 5 spinal dorsal horn inhibited SAP97 production, decreased SAP97-GluA1 interaction, reduced the membrane trafficking of GluA1-containing AMPARs, and partially attenuated neuropathic pain following SNT. Intrathecal injections of SAP97 siRNA or NASPM, an antagonist of GluA1-containing AMPARs, also partially reversed neuropathic pain on day 7, but not on day 14, after SNT. Spinal overexpression of SAP97 by AAV-EGFP-SAP97 enhanced SAP97-GluA1 interaction, increased the membrane insertion of GluA1-containing AMPARs, and induced abnormal pain in naïve rats. In addition, treatment with SAP97 siRNA or NASPM i.t. injection alleviated SNT-induced allodynia and hyperalgesia and exhibited a longer effect in female rats. Together, our results indicate that the SNT-induced upregulation of SAP97 via promoting GluA1-containing AMPARs membrane trafficking in the dorsal horn contributes to the pathogenesis of neuropathic pain. Targeting spinal SAP97 might be a promising therapeutic strategy to treatment of chronic pain.

Insufficient support for retinoic acid receptor control of synaptic plasticity through a non-genomic mechanism.

It is well established that retinoic acid receptors (RARs) function as nuclear receptors that control gene expression in response to binding of the ligand retinoic acid (RA). However, some studies have proposed that RAR-alpha (RARa) controls synaptic plasticity via non-genomic effects outside the nucleus, i.e. effects on mRNA translation of GluA1, a sub-unit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. In order to support this non-genomic mechanism, studies have reported RARa knockout mice or treatment with pharmacological levels of RA and RAR antagonists to propose that RARa is required to control normal synaptic plasticity. A major shortcoming of the non-genomic hypothesis is that there have been no mutational studies showing that RARa can bind the GluA1 mRNA to control GLUA1 protein levels in a non-genomic manner. Also, without a genetic study that removes the endogenous ligand RA, it is impossible to conclude that RARa and its ligand RA control synaptic plasticity through a non-genomic signaling mechanism.

Gender-related variation expressions of neuroplastin TRAF6, GluA1, GABA(A) receptor, and PMCA in cortex, hippocampus, and brainstem in an experimental epilepsy model.

Epileptic seizures are seen as a result of changing excitability balance depending on the deterioration in synaptic plasticity in the brain. Neuroplastin, and its related molecules which are known to play a role in synaptic plasticity, neurotransmitter activities that provide balance of excitability and, different neurological diseases, have not been studied before in epilepsy. In this study, a total of 34 Sprague-Dawley male and female rats, 2 months old, weighing 250-300 g were used. The epilepsy model in rats was made via pentylenetetrazole (PTZ). After the completion of the experimental procedure, the brain tissue of the rats were taken and the histopathological changes in the hippocampus and cortex parts and the brain stem were investigated, as well as the immunoreactivity of the proteins related to the immunohistochemical methods. As a result of the histopathological evaluation, it was determined that neuron degeneration and the number of dilated blood vessels in the hippocampus, frontal cortex, and brain stem were higher in the PTZ status epilepticus (SE) groups than in the control groups. It was observed that neuroplastin and related proteins TNF receptor-associated factor 6 (TRAF6), Gamma amino butyric acid type A receptors [(GABA(A)], and plasma membrane Ca2+ ATPase (PMCA) protein immunoreactivity levels increased especially in the male hippocampus, and only AMPA receptor subunit type 1 (GluA1) immunoreactivity decreased, unlike other proteins. We believe this may be caused by a problem in the mechanisms regulating the interaction of neuroplastin and GluA1 and may cause problems in synaptic plasticity in the experimental epilepsy model. It may be useful to elucidate this mechanism and target GluA1 when determining treatment strategies.

Inhibition of neuronal pentraxin 2 relieved epileptic seizure via reducing GluA1 phosphorylation.

Neuronal pentraxin 2 (Nptx2), a member of the synaptic protein family linked to excitatory synaptic formation, is found to be upregulated in epileptic mice, yet its role in epilepsy has been unclear. In vivo, we constructed a mouse model of epilepsy by using kainic acid induction. In vitro experiments, a Mg2+-free medium was used to induce epileptiform discharges in neurons. The results showed that the Nptx2 was upregulated in epileptic mice. Moreover, Nptx2 knockdown reduced the number of seizures and seizure duration. Knocking down Nptx2 not only reduced the number and duration of seizures but also showed a decrease in electroencephalogram amplitude. Behavioral tests indicated improvements in learning and memory abilities after Nptx2 knockdown. The Nissl staining and Timms staining revealed that Nptx2 silencing mitigated epilepsy-induced brain damage. The immunofluorescence staining revealed that Nptx2 absence resulted in a reduction of apoptosis. Nptx2 knockdown reduced Bax, cleaved caspase3, and cleaved caspase9 expression, while increased Bcl-2 expression. Notably, Nptx2 knockdown inhibited GluA1 phosphorylation at the S831 site and reduced the GluA1 membrane expression. The PSD95 expression declined in the epilepsy model, while the Nptx2 knockdown reversed it. Collectively, our study indicated that Nptx2 silencing not only alleviated brain damage and neuron apoptosis but also improved learning and memory ability in epileptic mice, suggesting Nptx2 as a promising target for epilepsy treatment.

The amino-terminal domain of GluA1 mediates LTP maintenance via interaction with neuroplastin-65.

Long-term potentiation (LTP) has long been considered as an important cellular mechanism for learning and memory. LTP expression involves NMDA receptor-dependent synaptic insertion of AMPA receptors (AMPARs). However, how AMPARs are recruited and anchored at the postsynaptic membrane during LTP remains largely unknown. In this study, using CRISPR/Cas9 to delete the endogenous AMPARs and replace them with the mutant forms in single neurons, we have found that the amino-terminal domain (ATD) of GluA1 is required for LTP maintenance. Moreover, we show that GluA1 ATD directly interacts with the cell adhesion molecule neuroplastin-65 (Np65). Neurons lacking Np65 exhibit severely impaired LTP maintenance, and Np65 deletion prevents GluA1 from rescuing LTP in AMPARs-deleted neurons. Thus, our study reveals an essential role for GluA1/Np65 binding in anchoring AMPARs at the postsynaptic membrane during LTP.

[Tacrolimus Induces Pain by Upregulating the Synaptic Expression of AMPA Receptors in the Spinal Cord Dorsal Horn]. / ä»–å ‹èŽ«å¸é€šè¿‡ä¸Šè°ƒè„Šé«“èƒŒè§’AMPA受体的突触表达诱发疼痛.

Objective: To investigate the effects of long-term administration of tacrolimus (also known as FK506) on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the spinal cord dorsal horn of mice. Methods: 1) A total of 24 mice were evenly and randomly assigned to two groups, a FK506 group and a Saline group. The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline. Both groups received injection once a day for 7 days in a row. Some of the mice ( n=6 in each group) were monitored for the changes in the paw withdrawal threshold (PWT), the paw withdrawal latency (PWL), and the spontaneous pain behaviors to establish the pain model. The other mice ( n=6 in each group) of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection. Then, immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors. 2) The mice were randomly divided into two groups, FK506+calcineurin (CaN) group and FK506+Saline group ( n=6 in each group). After the pain model was constructed, the mice were given intrathecal injection of recombinant CaN (also know as 33 U) or normal saline. Then, 60 minutes later, the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain. 3) Another18 mice were selected. The mice were randomly and evenly assigned to three groups, a control group (receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline), FK506+Saline group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline) and FK506+CaN group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN). Then, 60 minutes later, the spinal cords were isolated and subjected to immunoblotting assay to determine the role of CaN in FK506-induced AMPA receptor modification. Results: 1) After 7 consecutive days of intraperitoneal injection of FK506, the PWT and PWL of mice dropped significantly, reaching on day 7 as low as 22.3%±0.05% and 66.6%±0.05% of the control group, respectively ( P<0.01). The FK506-treated mice displayed evident spontaneous pain behavior, presenting significantly increased licking activities ( P<0.01). These results indicated that FK506-induced pain model was successfully established. Immunoblotting assay showed that the total expressions of GluA1 and GluA2 subunits in the spinal dorsal horn of the FK506 group remained unchanged in comparison with those of the Saline group. However, FK506 specifically induced an increase in the synaptic expression of GluA1. In addition, the phosphorylation levels of GluA1 at Ser845 and Ser831 in FK506-treated mice were significantly increased in comparison with those of the control group ( P<0.05). 2) Compared with those of the mice in the FK506+Saline group, the PWT and the PWL of mice in the FK506+CaN group were significantly increased ( P<0.05). 3) Compared with those of the FK506+Saline group, the synaptic expression of GluA1 were decreased in FK506+CaN group ( P<0.01) and the phosphorylation levels of GluA1 at Ser845 and Ser831 were significantly downregulated ( P<0.001). Conclusion: The hyper-expression and hyperphosphorylation of GluA1 subunit in the spinal cord dorsal horn resulting from CaN inhibition contributes to the FK506-induced pain syndrome. FK506 induces the synaptic hyper-expression and hyperphosphorylation of GluA1 in the dorsal horn of the spinal cord through CaN inhibition, thereby inducing pain.

Protein kinase D promotes activity-dependent AMPA receptor endocytosis in hippocampal neurons.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the protein kinase D (PKD) family promotes basal and activity-induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1-containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their colocalization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.

Microglia modulate TNFα-mediated synaptic plasticity.

The pro-inflammatory cytokine tumor necrosis factor α (TNFα) tunes the capacity of neurons to express synaptic plasticity. It remains, however, unclear how TNFα mediates synaptic positive (=change) and negative (=stability) feedback mechanisms. We assessed effects of TNFα on microglia activation and synaptic transmission onto CA1 pyramidal neurons of mouse organotypic entorhino-hippocampal tissue cultures. TNFα mediated changes in excitatory and inhibitory neurotransmission in a concentration-dependent manner, where low concentration strengthened glutamatergic neurotransmission via synaptic accumulation of GluA1-only-containing AMPA receptors and higher concentration increased inhibition. The latter induced the synaptic accumulation of GluA1-only-containing AMPA receptors as well. However, activated, pro-inflammatory microglia mediated a homeostatic adjustment of excitatory synapses, that is, an initial increase in excitatory synaptic strength at 3 h returned to baseline within 24 h, while inhibitory neurotransmission increased. In microglia-depleted tissue cultures, synaptic strengthening triggered by high levels of TNFα persisted and the impact of TNFα on inhibitory neurotransmission was still observed and dependent on its concentration. These findings underscore the essential role of microglia in TNFα-mediated synaptic plasticity. They suggest that pro-inflammatory microglia mediate synaptic homeostasis, that is, negative feedback mechanisms, which may affect the ability of neurons to express further plasticity, thereby emphasizing the importance of microglia as gatekeepers of synaptic change and stability.

Phosphorylation of the AMPA receptor subunit GluA1 regulates clathrin-mediated receptor internalization.

Synaptic strength is altered during synaptic plasticity by controlling the number of AMPA receptors (AMPARs) at excitatory synapses. During long-term potentiation and synaptic upscaling, AMPARs are accumulated at synapses to increase synaptic strength. Neuronal activity leads to phosphorylation of AMPAR subunit GluA1 (also known as GRIA1) and subsequent elevation of GluA1 surface expression, either by an increase in receptor forward trafficking to the synaptic membrane or a decrease in receptor internalization. However, the molecular pathways underlying GluA1 phosphorylation-induced elevation of surface AMPAR expression are not completely understood. Here, we employ fluorescence recovery after photobleaching (FRAP) to reveal that phosphorylation of GluA1 serine 845 (S845) predominantly plays a role in receptor internalization, rather than forward trafficking, during synaptic plasticity. Notably, internalization of AMPARs depends upon the clathrin adaptor AP2, which recruits cargo proteins into endocytic clathrin-coated pits. In fact, we further reveal that an increase in GluA1 S845 phosphorylation upon two distinct forms of synaptic plasticity diminishes the binding of the AP2 adaptor, reducing internalization and resulting in elevation of GluA1 surface expression. We thus demonstrate a mechanism of GluA1 phosphorylation-regulated clathrin-mediated internalization of AMPARs.

Inhibiting NLRP3 inflammasome signaling pathway promotes neurological recovery following hypoxic-ischemic brain damage by increasing p97-mediated surface GluA1-containing AMPA receptors.

BACKGROUND: The nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is believed to be a key mediator of neuroinflammation and subsequent secondary brain injury induced by ischemic stroke. However, the role and underlying mechanism of the NLRP3 inflammasome in neonates with hypoxic-ischemic encephalopathy (HIE) are still unclear. METHODS: The protein expressions of the NLRP3 inflammasome including NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1) and interleukin-1ß (IL-1ß), the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor (AMPAR) subunit, and the ATPase valosin-containing protein (VCP/p97), were determined by Western blotting. The interaction between p97 and AMPA glutamate receptor 1 (GluA1) was determined by co-immunoprecipitation. The histopathological level of hypoxic-ischemic brain damage (HIBD) was determined by triphenyltetrazolium chloride (TTC) staining. Polymerase chain reaction (PCR) and Western blotting were used to confirm the genotype of the knockout mice. Motor functions, including myodynamia and coordination, were evaluated by using grasping and rotarod tests. Hippocampus-dependent spatial cognitive function was measured by using the Morris-water maze (MWM). RESULTS: We reported that the NLRP3 inflammasome signaling pathway, such as NLRP3, caspase-1 and IL-1ß, was activated in rats with HIBD and oxygen-glucose deprivation (OGD)-treated cultured primary neurons. Further studies showed that the protein level of the AMPAR GluA1 subunit on the hippocampal postsynaptic membrane was significantly decreased in rats with HIBD, and it could be restored to control levels after treatment with the specific caspase-1 inhibitor AC-YVAD-CMK. Similarly, in vitro studies showed that OGD reduced GluA1 protein levels on the plasma membrane in cultured primary neurons, whereas AC-YVAD-CMK treatment restored this reduction. Importantly, we showed that OGD treatment obviously enhanced the interaction between p97 and GluA1, while AC-YVAD-CMK treatment promoted the dissociation of p97 from the GluA1 complex and consequently facilitated the localization of GluA1 on the plasma membrane of cultured primary neurons. Finally, we reported that the deficits in motor function, learning and memory in animals with HIBD, were ameliorated by pharmacological intervention or genetic ablation of caspase-1. CONCLUSION: Inhibiting the NLRP3 inflammasome signaling pathway promotes neurological recovery in animals with HIBD by increasing p97-mediated surface GluA1 expression, thereby providing new insight into HIE therapy.

A strategy can be used to analyze intracellular interaction proteomics of cell-surface receptors.

Comprehensive knowledge of the intracellular protein interactions of cell-surface receptors will greatly advance our comprehension of the underlying trafficking mechanisms. Hence, development of effective and high-throughput approaches is highly desired. In this work, we presented a strategy aiming to tailor toward the analysis of intracellular protein interactome of cell-surface receptors. We used α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors subunit GluA1 as an example to illustrate the methodological application. To capture intracellular proteins that interact with GluA1, after surface biotinylation of the prepared hippocampal neurons and slices, the non-biotinylated protein components as intracellular protein-enriched fraction were unconventionally applied for the following co-immunoprecipitation. The co-immuno-precipitated proteins were then analyzed through mass spectrometry-based proteomics and bioinformatics platforms. The detailed localizations indicated that intracellular proteins accounted for up to 93.7 and 90.3% of the analyzed proteins in the neurons and slices, respectively, suggesting that our protein preparation was highly effective to characterize intracellular interactome of GluA1. Further, we systematically revealed the protein functional profile of GluA1 intracellular interactome, thereby providing complete overview and better comprehension of diverse intracellular biological processes correlated with the complex GluA1 trafficking. All experimental results demonstrated that our methodology would be applicable and useful for intracellular interaction proteomics of general cell-surface receptors.

SYNPLA, a method to identify synapses displaying plasticity after learning.

Which neural circuits undergo synaptic changes when an animal learns? Although it is widely accepted that changes in synaptic strength underlie many forms of learning and memory, it remains challenging to connect changes in synaptic strength at specific neural pathways to specific behaviors and memories. Here we introduce SYNPLA (synaptic proximity ligation assay), a synapse-specific, high-throughput, and potentially brain-wide method capable of detecting circuit-specific learning-induced synaptic plasticity.

Loss of Arc attenuates the behavioral and molecular responses for sleep homeostasis in mice.

The activity-regulated cytoskeleton-associated protein (Arc) gene is a neural immediate early gene that is involved in synaptic downscaling and is robustly induced by prolonged wakefulness in rodent brains. Converging evidence has led to the hypothesis that wakefulness potentiates, and sleep reduces, synaptic strengthening. This suggests a potential role for Arc in these and other sleep-related processes. However, the role of Arc in sleep remains unknown. Here, we demonstrated that Arc is important for the induction of multiple behavioral and molecular responses associated with sleep homeostasis. Arc knockout (KO) mice displayed increased time spent in rapid eye movement (REM) sleep under baseline conditions and marked attenuation of sleep rebound to both 4 h of total sleep deprivation (SD) and selective REM deprivation. At the molecular level, the following homeostatic sleep responses to 4-h SD were all blunted in Arc KO mice: increase of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 and its phosphorylation in synaptoneurosomes; induction of a subset of SD-response genes; and suppression of the GluA1 messenger RNA in the cortex. In wild-type brains, SD increased Arc protein expression in multiple subcellular locations, including the nucleus, cytoplasm, and synapse, which is reversed in part by recovery sleep. Arc is critical for these behavioral and multiple molecular responses to SD, thus providing a multifunctional role for Arc in the maintenance of sleep homeostasis, which may be attributed by the sleep/wake-associated changes in subcellular location of Arc.

Ca2+-Permeable AMPA Receptors Contribute to Changed Dorsal Horn Neuronal Firing and Inflammatory Pain.

The dorsal horn (DH) neurons of the spinal cord play a critical role in nociceptive input integration and processing in the central nervous system. Engaged neuronal classes and cell-specific excitability shape nociceptive computation within the DH. The DH hyperexcitability (central sensitisation) has been considered a fundamental mechanism in mediating nociceptive hypersensitivity, with the proven role of Ca2+-permeable AMPA receptors (AMPARs). However, whether and how the DH hyperexcitability relates to changes in action potential (AP) parameters in DH neurons and if Ca2+-permeable AMPARs contribute to these changes remain unknown. We examined the cell-class heterogeneity of APs generated by DH neurons in inflammatory pain conditions to address these. Inflammatory-induced peripheral hypersensitivity increased DH neuronal excitability. We found changes in the AP threshold and amplitude but not kinetics (spike waveform) in DH neurons generating sustained or initial bursts of firing patterns. In contrast, there were no changes in AP parameters in the DH neurons displaying a single spike firing pattern. Genetic knockdown of the molecular mechanism responsible for the upregulation of Ca2+-permeable AMPARs allowed the recovery of cell-specific AP changes in peripheral inflammation. Selective inhibition of Ca2+-permeable AMPARs in the spinal cord alleviated nociceptive hypersensitivity, both thermal and mechanical modalities, in animals with peripheral inflammation. Thus, Ca2+-permeable AMPARs contribute to shaping APs in DH neurons and nociceptive hypersensitivity. This may represent a neuropathological mechanism in the DH circuits, leading to aberrant signal transfer to other nociceptive pathways.

Long-term exposure to high glucose induces changes in the expression of AMPA receptor subunits and glutamate transmission in primary cultured cortical neurons.

Hyperglycemia, which occurs under the diabetic conditions, induces serious diabetic complications. Diabetic encephalopathy has been defined as one of the major complications of diabetes, and is characterized by neurochemical and neurodegenerative changes. However, little is known about the effect of long-term exposure to high glucose on neuronal cells. In the present study, we showed that exposure to glutamate (100 mM) for 7 days induced toxicity in primary cortical neurons using the MTT assay. Additionally, high glucose increased the sensitivity of AMPA- or NMDA-induced neurotoxicity, and decreased extracellular glutamate levels in primary cortical neurons. In Western blot analyses, the protein levels of the GluA1 and GluA2 subunits of the AMPA receptor as well as synaptophysin in neurons treated with high glucose were significantly increased compared with the control (25 mM glucose). Therefore, long-term exposure to high glucose induced neuronal death through the disruption of glutamate homeostasis.