Differential Alterations in the Expression of AMPA Receptor and Its Trafficking Proteins in the Hippocampus Are Associated with Recognition Memory Impairment in the Rotenone-Parkinson's Disease Mouse Model: Neuroprotective Role of Bacopa monnieri Extract CDRI 08.

Parkinson's disease (PD), an age-associated neurodegenerative motor disorder, is associated with dementia and cognitive decline. However, the precise molecular insight into PD-induced cognitive decline is not fully understood. Here, we have investigated the possible alterations in the expression of glutamate receptor and its trafficking/scaffolding/regulatory proteins underlying the memory formation and neuroprotective effects of a specialized Bacopa monnieri extract, CDRI-08 (BME) in the hippocampus of the rotenone-induced PD mouse model. Our Western blotting and qRT-PCR data reveal that the PD-induced recognition memory decline is associated with significant upregulation of the AMPA receptor subunit GluR1 and downregulation of GluR2 subunit genes in the hippocampus of rotenone-affected mice as compared to the vehicle control. Further, expressions of the trafficking proteins are significantly upregulated in the hippocampus of rotenone-affected mice compared to the vehicle control. Our results also reveal that the above alterations in the hippocampus are associated with similar expression patterns of total CREB, pCREB, and BDNF. BME (CDRI-08, 200 mg/kg BW) reverses the expression of AMPA receptor subunits, their trafficking proteins differentially, and the transcriptional modulatory proteins depending on whether the BME treatment was given before or after the rotenone treatment. Our data suggest that expression of the above genes is significantly reversed in the BME pre-treated mice subjected to rotenone treatment towards their levels in the control mice compared to its treatment after rotenone administration. Our results provide the possible molecular basis underlying the rotenone-induced recognition memory decline, conditions mimicking the PD symptoms in mouse model and neuroprotective action of bacoside A and bacoside B (58%)-enriched Bacopa monnieri extract (BME) in the hippocampus.

GluR2 can Drive Neuroinflammation and Cognitive Impairments Following Peripherally Repeated Lipopolysaccharide Exposures.

Neuroinflammation is being increasingly recognized as a vital factor in the development of various neurological and neuropsychiatric diseases. Lipopolysaccharides (LPS), an outer membrane component of gram-negative bacteria, can trigger innate immune responses, resulting in neuroinflammation and subsequent cognitive deficits. The expression of glutamate receptors (GluRs) on glial cells can induce glial activation. Therefore, we hypothesized that repeated LPS exposure can increase GluR levels, promoting microglial activation and ultimately affecting synaptic plasticity and cognitive function. In this study, C57/BL6 mice were repeatedly exposed to LPS to construct a neuroinflammation animal model. The levels of GluRs, inflammatory cytokines, ionized calcium-binding adaptor molecule 1, postsynaptic density protein 95, synaptophysin 38, NMDA receptor 2 A, and NMDA receptor 2B (GluN2B) were measured in the hippocampi. Furthermore, dendritic spine density in the CA1 hippocampal region was determined. Repeated LPS exposure induced cognitive impairments and microglial activation and increased GluR1 and GluR2 levels. This was accompanied by a significant decrease in GluN2B expression and dendritic spine density in the hippocampi. However, CFM-2, an α-amino-3- hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist, reversed these anomalies. Furthermore, minocycline, a microglial inhibitor, reversed these anomalies and downregulated GluR2 but not GluR1 expression. In summary, we demonstrated that GluR2 plays an essential role in microglia-induced neuroinflammation, resulting in synaptic plasticity and cognitive impairment induced by repeated exposure to LPS.

Revisiting Glutamate Excitotoxicity in Amyotrophic Lateral Sclerosis and Age-Related Neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest benefits. To design new and more effective therapies for ALS, particularly for sporadic ALS of unknown and diverse etiologies, we must identify key, convergent mechanisms of disease pathogenesis. This review focuses on the origin and effects of glutamate-mediated excitotoxicity in ALS (the cortical hyperexcitability hypothesis), in which increased glutamatergic signaling causes motor neurons to become hyperexcitable and eventually die. We characterize both primary and secondary contributions to excitotoxicity, referring to processes taking place at the synapse and within the cell, respectively. 'Primary pathways' include upregulation of calcium-permeable AMPA receptors, dysfunction of the EAAT2 astrocytic glutamate transporter, increased release of glutamate from the presynaptic terminal, and reduced inhibition by cortical interneurons-all of which have been observed in ALS patients and model systems. 'Secondary pathways' include changes to mitochondrial morphology and function, increased production of reactive oxygen species, and endoplasmic reticulum (ER) stress. By identifying key targets in the excitotoxicity cascade, we emphasize the importance of this pathway in the pathogenesis of ALS and suggest that intervening in this pathway could be effective for developing therapies for this disease.

Neuroprotective effects of quinpirole on lithium chloride pilocarpine-induced epilepsy in rats and its underlying mechanisms.

INTRODUCTION: Epilepsy is a common neurological disorder that presents with challenging mechanisms and treatment strategies. This study investigated the neuroprotective effects of quinpirole on lithium chloride pilocarpine-induced epileptic rats and explored its potential mechanisms. METHODS: Lithium chloride pilocarpine was used to induce an epileptic model in rats, and the effects of quinpirole on seizure symptoms and cognitive function were evaluated. The Racine scoring method, electroencephalography, and Morris water maze test were used to assess seizure severity and learning and memory functions in rats in the epileptic group. Additionally, immunohistochemistry and Western blot techniques were used to analyze the protein expression levels and morphological changes in glutamate receptor 2 (GluR2; GRIA2), BAX, and BCL2 in the hippocampi of rats in the epileptic group. RESULTS: First, it was confirmed that the symptoms in rats in the epileptic group were consistent with features of epilepsy. Furthermore, these rats demonstrated decreased learning and memory function in the Morris water maze test. Additionally, gene and protein levels of GluR2 in the hippocampi of rats in the epileptic group were significantly reduced. Quinpirole treatment significantly delayed seizure onset and decreased the mortality rate after the induction of a seizure. Furthermore, electroencephalography showed a significant decrease in the frequency of the spike waves. In the Morris water maze test, rats from the quinpirole treatment group demonstrated a shorter latency period to reach the platform and an increased number of crossings through the target quadrant. Network pharmacology analysis revealed a close association between quinpirole and GluR2 as well as its involvement in the cAMP signaling pathway, cocaine addiction, and dopaminergic synapses. Furthermore, immunohistochemistry and Western blot analysis showed that quinpirole treatment resulted in a denser arrangement and a more regular morphology of the granule cells in the hippocampi of rats in the epileptic group. Additionally, quinpirole treatment decreased the protein expression of BAX and increased the protein expression of BCL2. CONCLUSION: The current study demonstrated that quinpirole exerted neuroprotective effects in the epileptic rat model induced by lithium chloride pilocarpine. Additionally, it was found that the treatment not only alleviated the rats' seizure symptoms, but also improved their learning and memory abilities. This improvement was linked to the modulation of protein expression levels of GLUR2, BAX, and BCL2. These findings provided clues that would be important for further investigation of the therapeutic potential of quinpirole and its underlying mechanisms for epilepsy treatment.

Intravenous Injection of GluR2-3Y Inhibits Repeated Morphine-Primed Reinstatement of Drug Seeking in Rats.

Studies have demonstrated that the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is essential to drug addiction. In this study, we explored the influence of GluR2-3Y, an interfering peptide to prevent the endocytosis of AMPA receptors containing the GluR2 subunit, on morphine-seeking behavior in the rat self-administration model. After self-administration was established, the rats received intravenous injections of GluR2-3Y during the extinction sessions. There were no significant differences in both active and inactive pokes compared to the control group of rats that received GluR2-3S, indicating that GluR2-3Y has no significant influences on the extinction of morphine self-administration. The other two groups of rats were trained, extinguished, and reinstated by repeated morphine priming (respectively, called Prime 1, Prime 2, and Prime 3). Only one intravenous injection of GluR2-3Y was performed before Prime 1. Compared to the control group, GluR2-3Y did not affect Prime 1, but significantly attenuated the morphine-seeking behavior during repeated morphine-primed reinstatement, indicating an inhibitory after effect of GluR2-3Y on morphine-seeking behavior in rats. The long-term depression (LTD) in the nucleus accumbens (NAc) shell was also assessed. Pretreatment with GluR2-3Y altered the ability of LTD induction to the level of that in the naive group, while pretreatment with GluR2-3S had no effects on LTD. Our results demonstrated that the intravenous injection of GluR2-3Y, to block the endocytosis of AMPA receptors, inhibited the reinstatement of morphine-seeking behavior, which may be induced by modulating the neuronal plasticity in the NAc shell of rats.

SNS alleviates depression-like behaviors in CUMS mice by regluating dendritic spines via NCOA4-mediated ferritinophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Depression is one of the most common mood disturbances worldwide. The Si-ni-san formula (SNS) is a famous classic Traditional Chinese Medicine (TCM) widely used to treat depression for thousands of years in clinics. However, the mechanism underlying the therapeutic effect of SNS in improving depression-like behaviors following chronic unpredictable mild stress (CUMS) remains unknown. AIM OF THE STUDY: This study aimed to investigate whether SNS alleviates depression-like behaviors in CUMS mice by regulating dendritic spines via NCOA4-mediated ferritinophagy in vitro and in vivo. STUDY DESIGN AND METHODS: In vivo, mice were exposed to CUMS for 42 days, and SNS (4.9, 9.8, 19.6 g/kg/d), fluoxetine (10 mg/kg/d), 3-methyladenine (3-MA) (30 mg/kg/d), rapamycin(1 mg/kg/d), and deferoxamine (DFO) (200 mg/kg/d) were conducted once daily during the last 3 weeks of the CUMS procedure. In vitro, a depressive model was established by culture of SH-SY5Y cells with corticosterone, followed by treatment with different concentrations of freeze-dried SNS (0.001, 0.01, 0.1 mg/mL) and rapamycin (10 nM), NCOA4-overexpression, Si-NCOA4. After the behavioral test (open-field test (OFT), sucrose preference test (SPT), forced swimming test (FST) and tail suspension test (TST), dendritic spines, GluR2 protein expression, iron concentration, and ferritinophagy-related protein levels (P62, FTH, NCOA4, LC3-II/LC3-I) were tested in vitro and in vivo using immunohistochemistry, golgi staining, immunofluorescence, and Western blot assays. Finally, HEK-293T cells were transfected by si-NCOA4 or GluR2-and NCOA4-overexpression plasmid and treated with corticosterone(100 µM), freeze-dried SNS(0.01 mg/mL), rapamycin(25 nM), and 3-MA(5 mM). The binding amount of GluR2, NCOA4, and LC3 was assessed by the co-immunoprecipitation (CO-IP) assay. RESULTS: 3-MA, SNS, and DFO promoted depressive-like behaviors in CUMS mice during OFT, SPT, FST and TST, improved the amount of the total, thin, mushroom spine density and enhanced GluR2 protein expression in the hippocampus. Meanwhile, treatment with SNS decreased iron concentrations and inhibited NCOA4-mediated ferritinophagy activation in vitro and in vivo. Importantly, 3-MA and SNS could prevent the binding of GluR2, NCOA4 and LC3 in corticosterone-treated HEK-293T, and rapamycin reversed this phenomenon after treatment with SNS. CONCLUSION: SNS alleviates depression-like behaviors in CUMS mice by regulating dendritic spines via NCOA4-mediated ferritinophagy.

The changing levels of GluR2 phosphorylation in oligodendrocyte precursor cells in mice model of ischaemic hypoxia / 神经解剖学杂志

Objective:To investigate the expression of phosphorylated glutamate receptor 2[p-GluR2(S880)]in oligodendrocyte precursor cells(OPCs)of mice model of hypoxic ischemic brain injury(HIBI).Methods:The HIBI model of C57BL/6 neonatal mice was established by right common carotid artery ligation and hypoxia for 90 min.The anxiety-like behavior of the mice was evaluated by elevated plus maze(EPM)and open field test(OFT).The expres-sion of p-GluR2(S880),oligodendrocyte marker 4(O4)and myelin basic protein(MBP)in brain tissue of HIBI model mice was detected by immunofluorescence staining.What's more,the expression levels of p-GluR2(S880)and MBP were detected by Western Blot.Results:Compared with sham operation group,there were significant anxiety-like behaviors 90 days after HIBI operation(P＜0.05).The expression of MBP protein decreased significantly in 14 and 28 days after HIBI operation.The expression of p-GluR2(S880)protein was up-regulated at all time points after HIBI op-eration(P＜0.05),and the number of O4 and p-GluR2(S880)double positive cells in brain tissue of HIBI group was significantly increased(P＜0.05).Conclusion:The up-regulation of p-GluR2(S880)expression in OPCs may lead to myelination disorder in HIBI model mice.

The recycling of AMPA receptors/GABAa receptors is related to neuronal excitation/inhibition imbalance and may be regulated by KIF5A.

Background: Excitation/inhibition imbalance (E/I imbalance), which involves an increase of alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptors (AMPARs) and decrease of gamma-aminobutyric acid type A (GABA) type A receptors (GABAaRs) on the neuron surface, has been documented in the pathogenesis of seizures. Notably, it has been established that both the glutamate receptor subunit 2 (GluR2) of AMPARs and beta 2/3 subunits of GABAaRs (Gabrb2+3) participate in the recycling mechanism mediated by the kinesin heavy chain isoform 5A (KIF5A), which determines the number of neuron surface receptors. However, it remains unclear whether receptor recycling is involved in the pathogenesis of seizures. Methods: Twelve adult male Sprague-Dawley rats were randomly allocated to the normal control (Ctl) group (n=6) and the pentylenetetrazol (PTZ)-induced seizure (Sez) group (n=6). The rats in the Ctl group were treated with saline. The rats in the Sez group received an intraperitoneal injection of PTZ at an initial dose of 40 mg/kg. Primary cultured neurons were obtained from newborn rats (24-hour-old). The neurons were exposed to magnesium-free (Mg2+-free) extracellular fluid for 3 hours to establish the seizure model in vitro. We detected the electrophysiology of the seizure model, the expression levels of KIF5A, GluR2, and Gabrb2+3, the recycling ratio of GluR2 and Gabrb2+3, the interaction between KIF5A and GluR2, and the interaction between KIF5A and Gabrb2+3. Results: In the Sez group, the expression of GluR2 on the cell surface was increased and the expression of Gabrb2+3 on the cell surface was decreased. The amplitude and frequency of action potentials were significantly increased in the Mg2+-free group. The amplitude and decay time of AMPAR-mediated miniature excitatory postsynaptic currents were increased in the Mg2+-free group. The amplitude and decay time of miniature inhibitory postsynaptic currents were decreased in the Mg2+-free group. The recycling ratio of GluR2 was increased and the recycling ratio of Gabrb2+3 was decreased in the Mg2+-free group. The interaction between KIF5A and GluR2 was increased, and the interaction between KIF5A and Gabrb2+3 was decreased in the seizure model in vivo and in vitro. Conclusions: The recycling of AMPA receptors/GABAa receptors is related to E/I imbalance and may be regulated by KIF5A.

RNA gene editing in the eye and beyond: The neglected tool of the gene editing armatorium?

RNA editing allows correction of pathological point mutations without permanently altering genomic DNA. Theoretically targetable to any RNA type and site, its flexibility and reversibility makes it a potentially powerful gene editing tool. RNA editing offers a host of potential advantages in specific niches when compared to currently available alternative gene manipulation techniques. Unlike DNA editors, which are currently too large to be delivered in vivo using a viral vector, smaller RNA editors fit easily within the capabilities of an adeno-associated virus (AAV). Unlike gene augmentation, which is limited by gene size and viral packaging constraints, RNA editing may correct transcripts too long to fit within a viral vector. In this article we examine the development of RNA editing and discuss potential applications and pitfalls. We argue that, although in its infancy, an RNA editing approach can offer unique advantages for selected retinal diseases.

The dorsal hippocampal CA3 regulates spatial reference memory through the CtBP2/GluR2 pathway.

The dorsal hippocampus plays a pivotal role in spatial memory. However, the role of subregion-specific molecular pathways in spatial cognition remains unclear. We observed that the transcriptional coregulator C-terminal binding protein 2 (CtBP2) presented CA3-specific enrichment in expression. RNAi interference of CtBP2 in the dorsal CA3 (dCA3) neurons, but not the ventral CA3 (vCA3), specifically impaired spatial reference memory and reduced the expression of GluR2, the calcium permeability determinant subunit of AMPA receptors. Application of an antagonist for GluR2-absent calcium permeable AMPA receptors rescued spatial memory deficits in dCA3 CtBP2 knockdown animals. Transcriptomic analysis suggest that CtBP2 may regulate GluR2 protein level through post-translational mechanisms, especially by the endocytosis pathway which regulates AMPA receptor sorting. Consistently, CtBP2 deficiency altered the mRNA expression of multiple endocytosis-regulatory genes, and CtBP2 knockdown in primary hippocampal neurons enhanced GluR2-containing AMPA receptor endocytosis. Together, our results provide evidence that the dCA3 regulates spatial reference memory by the CtBP2/GluR2 pathway through the modulation of calcium permeable AMPA receptors.

Coumestrol pre-treatment improves spatial learning and memory deficits following transient cerebral ischemia recruiting hippocampal GluR2 AMPA receptors.

Transient global ischemia is a leading cause of learning and memory dysfunction and induces a pattern of delayed neuronal death in the CA1 subfield of the hippocampus by down-regulating GluR2 mRNA AMPA receptors in this cerebral area. This study sought to investigate the neuroprotective effect of coumestrol against spatial memory impairment induced by global ischemia that leads to neural death by reducing the GluR2 receptors content in the hippocampal CA1 area. Our studies demonstrated that coumestrol administration prevented spatial memory deficits in mice. These findings suggest a cognitive enhancement role of coumestrol against cognitive impairment in ischemic events.

α2δ-1 protein promotes synaptic expression of Ca2+ permeable-AMPA receptors by inhibiting GluA1/GluA2 heteromeric assembly in the hypothalamus in hypertension.

Glutamate AMPA receptors (AMPARs) lacking GluA2 subunit are calcium permeable (CP-AMPARs), which are increased in the hypothalamic paraventricular nucleus (PVN) and maintain sympathetic outflow in hypertension. Here, we determined the role of α2δ-1, an NMDA receptor-interacting protein, in regulating synaptic CP-AMPARs in the hypothalamus in spontaneously hypertensive rats (SHR). Co-immunoprecipitation showed that levels of GluA1/GluA2, but not GluA2/GluA3, protein complexes in hypothalamic synaptosomes were reduced in SHR compared with Wistar-Kyoto rats (WKY). The level of GluA1/GluA2 heteromers in endoplasmic reticulum-enriched fractions of the hypothalamus was significantly lower in SHR than in WKY, which was restored by inhibiting α2δ-1 with gabapentin. Gabapentin also switched AMPAR-mediated excitatory postsynaptic currents (AMPAR-EPSCs) from inward rectifying to linear and attenuated the inhibitory effect of IEM-1460, a selective CP-AMPAR blocker, on AMPAR-EPSCs in spinally projecting PVN neurons in SHR. Furthermore, co-immunoprecipitation revealed that α2δ-1 directly interacted with GluA1 and GluA2 in the hypothalamus of rats and humans. Levels of α2δ-1/GluA1 and α2δ-1/GluA2 protein complexes in the hypothalamus were significantly greater in SHR than in WKY. Disrupting the α2δ-1-AMPAR interaction with an α2δ-1 C terminus peptide normalized GluA1/GluA2 heteromers in the endoplasmic reticulum of the hypothalamus diminished in SHR. In addition, α2δ-1 C terminus peptide diminished inward rectification of AMPAR-EPSCs and the inhibitory effect of IEM-1460 on AMPAR-EPSCs of PVN neurons in SHR. Thus, α2δ-1 augments synaptic CP-AMPARs by inhibiting GluA1/GluA2 heteromeric assembly in the hypothalamus in hypertension. These findings extend our understanding of the molecular basis of sustained sympathetic outflow in neurogenic hypertension.

Exosomes Derived Neuronal Markers: Immunoaffinity Isolation and Characterization.

Neuronal exosomes play a crucial role in intercellular communication in the brain and represent a promising biomarker for neurological diseases, including stroke. However, limited techniques are available for isolating neuronal exosomes due to their small number in the serum exosomes. Thus, the development of efficient tools with brain-specific markers is needed. Here, we show the optimization of an immunoaffinity assay-based isolation protocol for specific exosomes or neuronally derived exosomes (NDE). Our results demonstrated that one-micron functionalized magnetic beads successfully separated CD63+ and L1CAM+ exosomes from serum. The size and shape of exosomes or exosomes pulled by beads were confirmed by Dynamic light scattering and Transmission electron microscopy; also, beads were well resolved in conventional flow cytometry analysis, which revealed that CD63-pulled serum exosomes had 5% expression of L1CAM. Furthermore, transmission electron microscopy showed that exosomes eluted from magnetic beads retained their original size, shape, and form without any damage. Furthermore, we showed isolation of NDE using GluR2/3-capturing antibody (α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor) using an optimized immunoaffinity bead assay utilizing 100 µl serum of stroke patients or age-matched healthy group. GluR2/3-captured exosomes were confirmed by western blot analysis. The western blot analysis showed a significant increase in the 35KDa subunit of GluR2/3 receptor protein in the exosomes of stroke patients compared to the healthy group. In addition, the multimeric GluR2/3 receptor protein in exosomes was further validated by the presence of the GluR2 subunit. Thus, our study shows GluR3/2 may be an effective candidate to isolate neuronal exosomes.

The molecular mechanism underlying mitophagy-mediated hippocampal neuron apoptosis in diabetes-related depression.

Diabetes-related depression (DD) is a major complication of diabetes mellitus. Our previous studies indicated that glutamate (Glu) and hippocampal neuron apoptosis are key signal and direct factor leading to diabetes-related depression, respectively. However, the accurate pathogenesis remains to be unclear. We hypothesized that diabetes-related depression might be associated with the mitophagy-mediated hippocampal neuron apoptosis, triggered by aberrant Glu-glutamate receptor2 (GluR2)-Parkin pathway. To testify this hypothesis, here the rat model of DD in vivo and in vitro were both established so as to uncover the potential mechanism of DD based on mitophagy and apoptosis. We found that DD rats exhibit an elevated glutamate levels followed by monoamine neurotransmitter deficiency and depressive-like behaviour, and DD modelling promoted autophagosome formation and caused mitochondrial impairment, eventually leading to hippocampal neuron apoptosis via aberrant Glu-GluR2-Parkin pathway. Further, in vitro study demonstrated that the simulated DD conditions resulted in an abnormal glutamate and monoamine neurotransmitter levels followed by autophagic flux increment, mitochondrial membrane potential reduction and mitochondrial reactive oxygen species and lactic dehydrogenase elevation. Interestingly, both GluR2 and mammalian target of rapamycin (mTOR) receptor blocker aggravated mitophagy-induced hippocampal neuron apoptosis and abnormal expression of apoptotic protein. In contrast, both GluR2 and mTOR receptor agonist ameliorated those apoptosis in simulated DD conditions. Our findings revealed that mitophagy-mediated hippocampal neuron apoptosis, triggered by aberrant Glu-GluR2-Parkin pathway, is responsible for depressive-like behaviour and monoamine neurotransmitter deficiency in DD rats. This work provides promising molecular targets and strategy for the treatment of DD.

α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly.

Many neurological disorders show an increased prevalence of GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs), which dramatically alters synaptic function. However, the molecular mechanism underlying this distinct synaptic plasticity remains enigmatic. Here, we show that nerve injury potentiates postsynaptic, but not presynaptic, CP-AMPARs in the spinal dorsal horn via α2δ-1. Overexpressing α2δ-1, previously regarded as a Ca2+ channel subunit, augments CP-AMPAR levels at the cell surface and synapse. Mechanistically, α2δ-1 physically interacts with both GluA1 and GluA2 via its C terminus, inhibits the GluA1/GluA2 heteromeric assembly, and increases GluA2 retention in the endoplasmic reticulum. Consequently, α2δ-1 diminishes the availability and synaptic expression of GluA1/GluA2 heterotetramers in the spinal cord in neuropathic pain. Inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-AMPAR complex fully restores the intracellular assembly and synaptic dominance of heteromeric GluA1/GluA2 receptors. Thus, α2δ-1 is a pivotal AMPAR-interacting protein that controls the subunit composition and Ca2+ permeability of postsynaptic AMPARs.

Maternal separation increases cocaine intake through a mechanism involving plasticity in glutamate signalling.

Early-life stress (ELS) is associated with negative consequences, including maladaptive long-lasting brain effects. These alterations seem to increase the likelihood of developing substance use disorders. However, the molecular consequences of ELS are poorly understood. In the present study, we tested the impact of ELS induced by maternal separation with early weaning (MSEW) in CD1 male mice at different phases of cocaine self-administration (SA). We also investigated the subsequent alterations on GluR2, GluR1, cAMP response element-binding (CREB), and CREB-phosphorylation (pCREB) in ventral tegmental area (VTA) and nucleus accumbens (NAc) induced by both MSEW and cocaine SA. Our results show that MSEW animals expressed a higher cocaine intake, an increased vulnerability to the acquisition of cocaine SA, and incapacity to extinguish cocaine SA behaviour. MSEW mice showed decreased GluR2 and increased GluR1 and pCREB in NAc. Also, results displayed reduction of basal levels of GluR1 and CREB and an elevation of GluR1/GluR2 ratio in the VTA. Such results hint at an enhanced glutamatergic function in NAc and increased excitability of VTA DA neurons in maternally separated mice. Altogether, our results suggest that MSEW induces molecular alterations in the brain areas related to reward processing, increasing the vulnerability to depression and cocaine-seeking behaviour.

PKC epsilon as a neonatal target to correct FXS-linked AMPA receptor translocation in the hippocampus, boost PVN oxytocin expression, and normalize adult behavior in Fmr1 knockout mice.

Fragile X Syndrome (FXS) is an inherited developmental disorder caused by the non-expression of the Fmr1 gene. FXS is associated with abnormal social and anxiety behavior that is more prominent among males. Given that oxytocin (OXT) regulates both social and anxiety behavior, we studied the effect of FXS in the hypothalamic paraventricular nucleus (PVN), the major central source of OXT. We observed a significant suppression of protein kinase C epsilon (PKCε) (34%) in the ventral hippocampal CA1 region of postnatal day-18 (P18) male Fmr1 knockout (KO) mice, which displayed social behavior deficits and hyper-anxiety in adulthood. These mice also displayed a 39% increase in cell surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) at P18 (measured by the surface level of the AMPAR subunit GluR2), thereby indicating excitation of the CA1 neurons. It is known that neuronal activation at CA1 is linked to an inhibition of the PVN neurons. As expected, these mice also displayed a 25% suppression of oxytocin+ (OXT+) cells in the PVN at P20. Stimulating PKCε during postnatal days 6-,14 (P6-14) mice using a selective activator, dicyclopropyl-linoleic acid (DCP-LA), corrected AMPAR externalization in CA1 and suppression of OXT+ cell number in PVN in a PKCε dependent manner. Most notably, neonatal DCP-LA treatment rescued social behavior deficits and hyper-anxiety, displayed by adult (≥P60) male but not female KO mice. Thus, neonatal stimulation of PKCε could be a strategy to correct endophenotypic anomalies during brain development and aberrant adult behavior of the FXS males to the wild-type levels.

Internalization of AMPA-type Glutamate Receptor in the MIN6 Pancreatic ß-cell Line.

The activity of AMPA-type glutamate receptor is involved in insulin release from pancreatic ß-cells. However, the mechanism and dynamics that underlie AMPA receptor-mediated insulin release in ß-cells is largely unknown. Here, we show that AMPA induces internalization of glutamate receptor 2/3 (GluR2/3), AMPA receptor subtype, in the mouse ß-cell line MIN6. Immunofluorescence experiments showed that GluR2/3 appeared as fine dots that were distributed throughout MIN6 cells. Intracellular GluR2/3 co-localized with AP2 and clathrin, markers for clathrin-coated pits and vesicles. Immunoelectron microscopy revealed that GluR2/3 was also localized at plasma membrane. Surface biotinylation and immunofluorescence measurements showed that addition of AMPA caused an approximate 1.8-fold increase in GluR2/3 internalization under low-glucose conditions. Furthermore, internalized GluR2 largely co-localized with EEA1, an early endosome marker. In addition, GluR2/3 co-immunoprecipitated with cortactin, a F-actin binding protein. Depletion of cortactin by RNAi in MIN6 cells altered the intracellular distribution of GluR2/3, suggesting that cortactin is involved in internalization of GluR2/3 in MIN6 cells. Taken together, our results suggest that pancreatic ß-cells adjust the amount of AMPA-type GluR2/3 on the cell surface to regulate the receptive capability of the cell for glutamate.Key words: endocytosis, GluR2, AMPA, cortactin, MIN6.

DRG Neurons Promote Perineural Invasion of Endometrial Cancer via GluR2.

Background: Perineural invasion (PNI) is correlated with negative prognosis in multiple cancers, but its role in endometrial cancer (EC) is still largely unknown; thus, targeted treatment for nerve infiltration is lacking as well. Methods: The interaction between nerve and EC cells were investigated by in vitro neural invasion assay and transwell coculture system. Then the nerve-related receptor gene glutamate ionotropic receptor AMPA type subunit 2 (GRIA2) was detected in EC tissues and cells using PCR array, western blotting, and immunohistochemistry. The role of GluR2 (gene name GRIA2) on EC proliferation, migration and invasion was evaluated by a GluR2 antagonist and shRNA. At the same time, the neurotransmitter effect on GluR2 (glutamate) from the cocultured conditional medium was measured using high-performance liquid chromatography (HPLC). Results: EC cell line Ishikawa (ISK) showed the ability to migrate along neurites in vitro and the numbers of migrated/invaded EC cells in the DRG neuron coculture group were significantly increased. The expression of GluR2 in EC tissue was found to be higher than that in para-carcinoma tissue. After GluR2 antagonist and GluR2 shRNA treatment, the proliferation, migration and invasion of ISK cells was markedly inhibited. Moreover, the ability of DRG neurons to promote the migration and invasion of ISK cells could also be attenuated by downregulation of GluR2, and the concentration of the neurotransmitter glutamate was notably increased in the coculture conditional medium compared to that in the DRG neuron or ISK cells alone. Conclusions: DRG neurons promote metastasis of EC cells via GluR2, which might be a risk factor for PNI in EC. Moreover, the perineural system may promote tumor invasion and metastasis under certain circumstances.

Stimulation of mGluR1/5 Improves Defective Internalization of AMPA Receptors in NPC1 Mutant Mouse.

Niemann-Pick type C1 (NPC1) disease is characterized by neurodegeneration caused by cholesterol accumulation in the late endosome/lysosome. In this study, a defective basal and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-stimulated internalization of GluR2-containing AMPA receptors in NPC1-/- cortical neurons was detected. Our results show that the amount of cholesterol and group I metabotropic glutamate receptors (mGluR1/5) in lipid rafts of NPC1-/- cortical tissue and neurons are decreased and their downstream signals of p-ERK are defective, which are restored by a rebalance of cholesterol homeostasis through ß-cyclodextrin (ß-CD) treatment. Application of 3,5-dihydroxyphenylglycine (DHPG)-a mGluR1/5 agonist-and ß-CD markedly increases the internalization of AMPA receptors and decreases over-influx of calcium in NPC1-/- neurons, respectively. Furthermore, the defective phosphorylated GluR2 and protein kinase C signals are ameliorated by the treatment with DHPG and ß-CD, respectively, suggesting an involvement of them in internalization dysfunction. Taken together, our data imply that abnormal internalization of AMPA receptors is a critical mechanism for neuronal dysfunction and the correction of dysfunctional mGluR1/5 is a potential therapeutic strategy for NPC1 disease.