Comparative transcriptome analysis reveals growth and molecular pathway of body color regulation in turbot (Scophthalmus maximus) exposed to different light spectrum.

Fish body color changes play vital roles in adapting to ecological light environment and influencing market value. However, the initial mechanisms governing the changes remain unknown. Here, we scrutinized the impact of light spectrum on turbot (Scophthalmus maximus) body coloration, exposing them to red, blue, and full light spectra from embryo to 90 days post hatch. Transcriptome and quantitative real-time PCR (qRT-PCR) analyses were employed to elucidate underlying biological processes. The results showed that red light induced dimorphism in turbot juvenile skin pigmentation: some exhibited black coloration (Red_Black_Surface, R_B_S), while others displayed lighter skin (Red_White_Bottom, R_W_B), with red light leading to reduced skin lightness (L*) and body weight, particularly in R_B_S group. Transcriptomic and qRT-PCR analyses showcased upregulated gene expressions related to melanin synthesis in R_B_S individuals, notably tyrosinase (tyr), tyrosinase-related protein 1 (tyrp1), and dopachrome tautomerase (dct), alongside solute carrier family 24 member 5 (slc24a5) and oculocutaneous albinism type II (oca2) as pivotal regulators. Nervous system emerged as a critical mediator in spectral environment-driven color regulation. N-methyl d-aspartate (NMDA) glutamate receptor, and calcium signaling pathway emerged as pivotal links intertwining spectral conditions, neural signal transduction, and color regulation. The individual differences in NMDA glutamate receptor expression and subsequent neural excitability seemed responsible for dichromatic body coloration in red light-expose juveniles. This study provides new insights into the comprehending of fish adaptation to environment and methods for fish body color regulation and could potentially help enhance the economic benefit of fish farming industry.

AGAP3: A novel BRAF fusion partner in pediatric pancreatic-type acinar cell carcinoma.

Most available molecular data on pancreatic acinar cell carcinoma (PACC) are provided by studies of adult cases. BRAF, RAF1, or RET rearrangements have been described in approximately 30% of cases. To the best of our knowledge, only seven cases with molecular data have been reported in pediatric PACC. We report here the comprehensive study of a pancreatic-type ACC from a 6-year-old patient. We detected an AGAP3::BRAF fusion. This result showing a BRAF rearrangement demonstrates a molecular link between adult and pediatric PACC. Moreover, it identifies AGAP3, a gene located at 7q36.1 that encodes a major component of the N-methyl-d-aspartate (NMDA) receptor signaling complex, as a partner gene of BRAF. The variability of BRAF partners is consistent with a driver role of BRAF alterations in PACC. The identification of such alterations is noteworthy for considering the use of MEK inhibitors in metastatic cases. We did not detect associated genomic instability. The better outcome of pediatric cases might be related to their stable genomic background.

An Epilepsy-Associated Mutation of Salt-Inducible Kinase 1 Increases the Susceptibility to Epileptic Seizures and Interferes with Adrenocorticotropic Hormone Therapy for Infantile Spasms in Mice.

Six mutations in the salt-inducible kinase 1 (SIK1) have been identified in developmental and epileptic encephalopathy (DEE-30) patients, and two of the mutations are nonsense mutations that truncate the C-terminal region of SIK1. In a previous study, we generated SIK1 mutant (SIK1-MT) mice recapitulating the C-terminal truncated mutations using CRISPR/Cas9-mediated genome editing and found an increase in excitatory synaptic transmission and enhancement of neural excitability in neocortical neurons in SIK1-MT mice. NMDA was injected into SIK1-MT males to induce epileptic seizures in the mice. The severity of the NMDA-induced seizures was estimated by the latency and the number of tail flickering and hyperflexion. Activated brain regions were evaluated by immunohistochemistry against c-fos, Iba1, and GFAP. As another epilepsy model, pentylenetetrazol was injected into the adult SIK1 mutant mice. Seizure susceptibility induced by both NMDA and PTZ was enhanced in SIK1-MT mice. Brain regions including the thalamus and hypothalamus were strongly activated in NMDA-induced seizures. The epilepsy-associated mutation of SIK1 canceled the pharmacological effects of the ACTH treatment on NMDA-induced seizures. These results suggest that SIK1 may be involved in the neuropathological mechanisms of NMDA-induced spasms and the pharmacological mechanism of ACTH treatment.

LRFN2 binding to NMDAR inhibits the progress of ESCC via regulating the Wnt/ß-Catenin and NF-κB signaling pathway.

As a neuronal transmembrane protein, leucine-rich repeat and fibronectin type-III domain-containing protein 2 (LRFN2) can recruit and combine with N-methyl-d-aspartate receptors (NMDARs) to promote nerve growth. Genetic studies suggest that mutations in LRFN2 are associated with various cancers. However, the role and mechanism of LRFN2 in the progression of ESCC have not been elucidated. In this study, we demonstrated that LRFN2 was significantly downregulated in ESCC tissues by qRT-PCR and immunohistochemistry. Low LRFN2 expression was an adverse prognostic factor in patients with ESCC. Overexpression of LRFN2 effectively suppressed the proliferation, migration, invasion, and epithelial-to-mesenchymal transition in vitro and tumor growth in vivo. Bioinformatics analysis indicated that Wnt/ß-catenin signaling regulation was one of the most potential mechanisms and studies confirmed that overexpression of LFRN2 obviously downregulated the expression of ß-catenin, c-Myc, and cyclin D1 in ESCC cells and tumor tissues. Further studies revealed that LRFN2 plays an anti-ESCC role by binding with NMDAR-GRIN2B and this effect can be weakened by NR2B-selective NMDA antagonist-NMDA-IN-1. Moreover, the bioinformatics analysis showed that the interaction of GRIN2B and GSK3ß affects the NF-κB pathway, which was demonstrated by western blot experiments. Collectively, our results indicate that LRFN2 binding to NMDARs inhibits the progression of ESCC by regulating the Wnt/ß-catenin and NF-κB pathway, which provides a new therapeutic target for improving the prognosis of patients with ESCC.

Modification by genetic polymorphism of lead-induced IQ alteration: a systematic review.

As well as a lead-related environmental factor, genetic factors could also corroborate important changes in intelligence quotient (IQ) through single-nucleotide polymorphisms. Thus, a systematic review was carried out to evaluate the possible influence of polymorphism on blood Pb levels and IQ points in pediatric patients (0-19 years old). Following the PRISMA guideline, the studies were systematically collected on PubMed, Scopus, and Embase databases. Six genes (transferrin (TF); glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A); glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B); dopamine receptor D2/ankyrin repeat and kinase domain containing 1 ankyrin repeat and kinase domain containing 1 (DRD2/ANKK1); aminolevulinate dehydratase (ALAD); vitamin D receptor (VDR)) were found in six selected articles. In these genes, 11 single-nucleotide polymorphisms were searched and six different types of variations (missense variant, intron variant, synonymous variant, stop, stop gained) were observed. Due to the few studies in the literature, there is no conclusive data to point out that there is a direct relationship between polymorphisms, Pb levels, and reduction of IQ points.

Association of the D-amino acid oxidase gene with methadone dose in heroin dependent patients under methadone maintenance treatment.

Methadone is a synthetic opioid used for the maintenance treatment (MMT) of heroin dependence. It primarily binds to the µ-opioid receptor (MOR; with its gene, namely OPRM1). Methadone is also an N-methyl-D-aspartate (NMDA) receptor antagonist. The role of NMDA receptor in the regulatory mechanisms of methadone dosage in heroin dependent patients is so far not clear. D-amino acid oxidase (DAO) is an important enzyme that indirectly activates the NMDA receptor through its effect on the D-serine level. To test the hypothesis that genetic polymorphisms in the DAO gene are associated with methadone treatment dose and responses, we selected four single nucleotide polymorphisms (SNPs) in DAO from the literature reports of the Taiwanese population. SNPs were genotyped in 344 MMT patients. In this study, we identified a functional SNP rs55944529 in the DAO gene that reveals a modest but significant association with the methadone dosage in the recessive model of analysis (P = 0.003) and plasma concentrations (P = 0.003) in MMT patients. However, it did not show association with plasma methadone concentration in multiple linear regression analysis. It is also associated with the methadone adverse reactions of dry mouth (P = 0.002), difficulty with urination (P = 0.0003) in the dominant model, and the withdrawal symptoms of yawning (P = 0.005) and gooseflesh skin (P = 0.004) in the recessive model. Our results suggest a role of the indirect regulatory mechanisms of the NMDA reporter, possibly via the DAO genetic variants, in the methadone dose and some adverse reactions in MMT patients.

A homozygous GRIN1 null variant causes a more severe phenotype of early infantile epileptic encephalopathy.

Pathogenic variants in glutamate receptor, ionotropic, NMDA-1 (GRIN1) cause an autosomal dominant or recessive neurodevelopmental disorder with global developmental delay, with or without seizures (AD or AR GRIN1-NDD). Here, we describe a novel homozygous canonical splice site variant in GRIN1 in a 12-month-old boy with early infantile epileptic encephalopathy and severe global developmental delay. This represents only the second family with a homozygous predicted-null variant in GRIN1 reported to date. We review the published literature on AR GRIN1-NDD and find that the phenotype in our patient is much more severe than those seen with homozygous missense variants. A similarly severe phenotype of intractable epilepsy and infantile death has only been reported in one other family with a homozygous nonsense variant in GRIN1. We, therefore, propose that biallelic predicted-null variants in GRIN1 can cause a markedly more severe clinical phenotype than AR GRIN1-NDD caused by missense variants.

CYFIP1 Dosages Exhibit Divergent Behavioral Impact via Diametric Regulation of NMDA Receptor Complex Translation in Mouse Models of Psychiatric Disorders.

BACKGROUND: Gene dosage imbalance caused by copy number variations (CNVs) is a prominent contributor to brain disorders. In particular, 15q11.2 CNV duplications and deletions have been associated with autism spectrum disorder and schizophrenia, respectively. The mechanism underlying these diametric contributions remains unclear. METHODS: We established both loss-of-function and gain-of-function mouse models of Cyfip1, one of four genes within 15q11.2 CNVs. To assess the functional consequences of altered CYFIP1 levels, we performed systematic investigations on behavioral, electrophysiological, and biochemical phenotypes in both mouse models. In addition, we utilized RNA immunoprecipitation sequencing (RIP-seq) analysis to reveal molecular targets of CYFIP1 in vivo. RESULTS: Cyfip1 loss-of-function and gain-of function mouse models exhibited distinct and shared behavioral abnormalities related to autism spectrum disorder and schizophrenia. RIP-seq analysis identified messenger RNA targets of CYFIP1 in vivo, including postsynaptic NMDA receptor (NMDAR) complex components. In addition, these mouse models showed diametric changes in levels of postsynaptic NMDAR complex components at synapses because of dysregulated protein translation, resulting in bidirectional alteration of NMDAR-mediated signaling. Importantly, pharmacological balancing of NMDAR signaling in these mouse models with diametric Cyfip1 dosages rescues behavioral abnormalities. CONCLUSIONS: CYFIP1 regulates protein translation of NMDAR and associated complex components at synapses to maintain normal synaptic functions and behaviors. Our integrated analyses provide insight into how gene dosage imbalance caused by CNVs may contribute to divergent neuropsychiatric disorders.

N-methyl-d-aspartate (NMDA) receptor genetics: The power of paralog homology and protein dynamics in defining dominant genetic variants.

Predicting genotype-to-phenotype correlations from genomic variants has been challenging, particularly for genes that have a complex balance of dominant and recessive inheritance for phenotypes. Variants in NMDA receptor components GRIN1, GRIN2A, and GRIN2B cause a myriad of dominant disease phenotypes, with the most common being epilepsy and autism spectrum disorder. Starting from the analysis of a variant of uncertain significance (VUS, GRIN2A G760S), we realized the need for tools to map dominant variants for the components of the NMDA receptor. Some variants within GRIN1, GRIN2A, and GRIN2B exert dominant epilepsy and developmental delay, yet other amino acid variants are conserved and predicted to alter protein function but do not have dominant phenotypes. Common variant annotation tools are not powered to determine pathogenic dominant outcomes. To address this gap, we integrated sequence and structural analyses for GRIN1, GRIN2A, and GRIN2B. Using this approach, we determined that paralog homology mapping and topology can segregate dominant variants, with an elevation of intermolecular contacts between the subunits. Furthermore, demonstrating the general utility of our methodology, we show that 25 VUS within ClinVar also reach a dominant variant annotation, including the GRIN2A G760S variant. Our work suggests paralog homology and protein topology as a powerful strategy within the receptor complex to resolve dominant genetic variants relative to variants that would fit a recessive inheritance, requiring two damaging variants. These strategies should be tested in additional dominant genetic disorders to determine the broader utility.

Pemafibrate prevents retinal neuronal cell death in NMDA-induced excitotoxicity via inhibition of p-c-Jun expression.

Excitotoxicity is involved in the retinal neuronal cell death in diabetic retinopathy. Although fenofibrate has been shown to ameliorate the progression of diabetic retinopathy, the effect of pemafibrate, which is highly selective for peroxisome proliferator-activated receptor α on retinal neuronal cell death has not been documented. Here, we investigated whether pemafibrate exerts a beneficial effect against retinal ganglion cell (RGC) death induced by N-methyl-D-aspartate (NMDA) in rats. Experiments were performed on adult male Wistar rats that received an intravitreal injection of 20 nmol NMDA. Fluoro-Gold labeled RGC morphometry showed that oral intake of pemafibrate once a day for 7 days resulted in significant protection on RGC death induced by NMDA. Phosphorylated c-Jun protein, which is involved in apoptosis, was upregulated after NMDA exposure, and this increase was significantly lessened by the systemic pemafibrate treatment. Phosphorylated c-Jun immunopositive cells were colocalized with Thy-1 immunopositive cells, and the increased these cells were ameliorated by the pemafibrate treatment. An increase in TUNEL-positive cells was significantly suppressed by the pemafibrate treatment. Phosphorylated c-Jun immunopositive cells were colocalized with TUNEL-positive cells, and they were decreased by pemafibrate treatment. These results suggest that the RGC protection achieved with pemafibrate appears to be associated with inhibition of phosphorylated c-Jun and its anti-apoptotic effect.

Advances in D-Amino Acids in Neurological Research.

D-amino acids have been known to exist in the human brain for nearly 40 years, and they continue to be a field of active study to today. This review article aims to give a concise overview of the recent advances in D-amino acid research as they relate to the brain and neurological disorders. This work has largely been focused on modulation of the N-methyl-D-aspartate (NMDA) receptor and its relationship to Alzheimer's disease and Schizophrenia, but there has been a wealth of novel research which has elucidated a novel role for several D-amino acids in altering brain chemistry in a neuroprotective manner. D-amino acids which have no currently known activity in the brain but which have active derivatives will also be reviewed.

Over-activation of NMDA receptors promotes ABCA1 degradation and foam cell formation.

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1ß and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.

Treatment of diabetic retinopathy through neuropeptide Y-mediated enhancement of neurovascular microenvironment.

Diabetic retinopathy (DR) is one of the most severe clinical manifestations of diabetes mellitus and a major cause of blindness. DR is principally a microvascular disease, although the pathogenesis also involves metabolic reactive intermediates which induce neuronal and glial activation resulting in disruption of the neurovascular unit and regulation of the microvasculature. However, the impact of neural/glial activation in DR remains controversial, notwithstanding our understanding as to when neural/glial activation occurs in the course of disease. The objective of this study was to determine a potential protective role of neuropeptide Y (NPY) using an established model of DR permissive to N-methyl-D-aspartate (NMDA)-induced excitotoxic apoptosis of retinal ganglion cells (RGC) and vascular endothelial growth factor (VEGF)-induced vascular leakage. In vitro evaluation using primary retinal endothelial cells demonstrates that NPY promotes vascular integrity, demonstrated by maintained tight junction protein expression and reduced permeability in response to VEGF treatment. Furthermore, ex vivo assessment of retinal tissue explants shows that NPY can protect RGC from excitotoxic-induced apoptosis. In vivo clinical imaging and ex vivo tissue analysis in the diabetic model permitted assessment of NPY treatment in relation to neural and endothelial changes. The neuroprotective effects of NPY were confirmed by attenuating NMDA-induced retinal neural apoptosis and able to maintain inner retinal vascular integrity. These findings could have important clinical implications and offer novel therapeutic approaches for the treatment in the early stages of DR.

Caffeine as a potential arousal enhancer: altered NMDA subunit gene expression without improving cognitive performance in REM sleep deprived rats.

Caffeine is one of the most extensively consumed stimulants in the world and has been suggested to induce wakefulness by antagonizing the function of the adenosine A2A receptor. Therefore, we investigated the effects of chronic caffeine consumption on learning and memory in the REM sleep-deprived rats.Male Wistar rats (n = 50), were randomly assigned into 5 groups: Control (C), Caffeine (Cf), Pedestal Control (PC), Sleep Deprivation (SD), Sleep Deprivation and Caffeine (SD + Cf). Sleep deprivation procedure was applied as the flower-pot technique. SD and SD + Cf groups were deprived for 18 hours in a day for 21 days. Caffeine was administered daily in drinking water (0.3 g/L) for 5 weeks. For evaluated learning and memory function, Morris Water Maze Test (MWM) was used. Fluidigm Access Array was used for Grin2a, Grin2b, BDNF, cdk5/cdk5r1, CaMKIIa genes expression in the hippocampus. Distance moved and escape latency were decreased through trial days (p<0.05). However, there is no significant difference between groups for time spent in targeted quadrant during probe test for memory performance. Grin2a up-regulation was found in Cf and SD+Cf (p<0.05), and cdk5r1 increased in Cf and PC control (p<0.05). Also, BDNF up-regulation was found in PC group. Grin2b, Cdk5, CaMKIIa expression levels were not changed significantly. We showed chronic caffeine altered some of the hippocampal genes without changing learning and memory in REM sleep deprived rats. Chronic consumption of caffeine caused up-regulation in Grin2a that subunit of NMDA receptor. We supposed that chronic caffeine consumption maintained arousal without affecting learning and memory performance.

NMDA Receptors in Astrocytes: In Search for Roles in Neurotransmission and Astrocytic Homeostasis.

Studies of the last two decades have demonstrated the presence in astrocytic cell membranes of N-methyl-d-aspartate (NMDA) receptors (NMDARs), albeit their apparently low abundance makes demonstration of their presence and function more difficult than of other glutamate (Glu) receptor classes residing in astrocytes. Activation of astrocytic NMDARs directly in brain slices and in acutely isolated or cultured astrocytes evokes intracellular calcium increase, by mutually unexclusive ionotropic and metabotropic mechanisms. However, other than one report on the contribution of astrocyte-located NMDARs to astrocyte-dependent modulation of presynaptic strength in the hippocampus, there is no sound evidence for the significant role of astrocytic NMDARs in astrocytic-neuronal interaction in neurotransmission, as yet. Durable exposure of astrocytic and neuronal co-cultures to NMDA has been reported to upregulate astrocytic synthesis of glutathione, and in this way to increase the antioxidative capacity of neurons. On the other hand, overexposure to NMDA decreases, by an as yet unknown mechanism, the ability of cultured astrocytes to express glutamine synthetase (GS), aquaporin-4 (AQP4), and the inward rectifying potassium channel Kir4.1, the three astroglia-specific proteins critical for homeostatic function of astrocytes. The beneficial or detrimental effects of astrocytic NMDAR stimulation revealed in the in vitro studies remain to be proven in the in vivo setting.

Excessive activation of ionotropic glutamate receptors induces apoptotic hair-cell death independent of afferent and efferent innervation.

Accumulation of excess glutamate plays a central role in eliciting the pathological events that follow intensely loud noise exposures and ischemia-reperfusion injury. Glutamate excitotoxicity has been characterized in cochlear nerve terminals, but much less is known about whether excess glutamate signaling also contributes to pathological changes in sensory hair cells. I therefore examined whether glutamate excitotoxicity damages hair cells in zebrafish larvae exposed to drugs that mimic excitotoxic trauma. Exposure to ionotropic glutamate receptor (iGluR) agonists, kainic acid (KA) or N-methyl-D-aspartate (NMDA), contributed to significant, progressive hair cell loss in zebrafish lateral-line organs. To examine whether hair-cell loss was a secondary effect of excitotoxic damage to innervating neurons, I exposed neurog1a morphants-fish whose hair-cell organs are devoid of afferent and efferent innervation-to KA or NMDA. Significant, dose-dependent hair-cell loss occurred in neurog1a morphants exposed to either agonist, and the loss was comparable to wild-type siblings. A survey of iGluR gene expression revealed AMPA-, Kainate-, and NMDA-type subunits are expressed in zebrafish hair cells. Finally, hair cells exposed to KA or NMDA appear to undergo apoptotic cell death. Cumulatively, these data reveal that excess glutamate signaling through iGluRs induces hair-cell death independent of damage to postsynaptic terminals.

Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration.

Despite the recognized role of tumor necrosis factor (TNF) in inflammation and neuronal degeneration, anti-TNF therapeutics failed to treat neurodegenerative diseases. Animal disease models had revealed the antithetic effects of the two TNF receptors (TNFR) in the central nervous system, whereby TNFR1 has been associated with inflammatory degeneration and TNFR2 with neuroprotection. We here show the therapeutic potential of selective inhibition of TNFR1 and activation of TNFR2 by ATROSAB, a TNFR1-selective antagonistic antibody, and EHD2-scTNFR2, an agonistic TNFR2-selective TNF, respectively, in a mouse model of NMDA-induced acute neurodegeneration. Coadministration of either ATROSAB or EHD2-scTNFR2 into the magnocellular nucleus basalis significantly protected cholinergic neurons and their cortical projections against cell death, and reverted the neurodegeneration-associated memory impairment in a passive avoidance paradigm. Simultaneous blocking of TNFR1 and TNFR2 signaling, however, abrogated the therapeutic effect. Our results uncover an essential role of TNFR2 in neuroprotection. Accordingly, the therapeutic activity of ATROSAB is mediated by shifting the balance of the antithetic activity of endogenous TNF toward TNFR2, which appears essential for neuroprotection. Our data also explain earlier results showing that complete blocking of TNF activity by anti-TNF drugs was detrimental rather than protective and argue for the use of next-generation TNFR-selective TNF therapeutics as an effective approach in treating neurodegenerative diseases.

Gene expression of NMDA and AMPA receptors in different facial motor neurons.

OBJECTIVES/HYPOTHESIS: Facial motor neurons (FMNs) are involved in the remodeling of the facial nucleus in response to peripheral injury. This study aimed to examine the gene expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) in reinnervating dormant FMNs after facial nerve axotomy. STUDY DESIGN: Animal study. METHODS: Rat models of facial-facial anastomosis were set up and raised until the 90th day. By laser capture microdissection (LCM), the reinnervating neurons labeled by Fluoro-Ruby (FR) were first captured, and the remaining (dormant) neurons identified by Nissl staining were captured in the facial nucleus of the operated side. The total RNA of two types of neurons were extracted, and the gene expressions of AMPAR and NMDAR were studied by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Messenger RNA (mRNA) of AMPAR subunits (GluR1, GluR2, GluR3, and GluR4) and NMDAR subunits (NR1, NR2a, NR2b, NR2c, and NR2d) was detected in reinnervating and dormant neurons. The relative ratios exhibited that the expressions of GluR1, GluR4, NR2a, NR2b, NR2c, and NR2d mRNA were lower, whereas the expressions of GluR2, GluR3, and NR1 mRNA were higher in dormant FMNs than in reinnervating counterparts. CONCLUSIONS: LCM in combination with real-time qRT-PCR can be employed for the examination of gene expression of different FMNs in a heterogeneous nucleus. The adaptive changes in AMPAR and NMDAR subunit mRNA might dictate the regenerative fate of FMNs in response to the peripheral axotomy and thereby play a unique role in the pathogenesis of facial nerve injury and regeneration.

Proteomic and genomic evidence implicates the postsynaptic density in schizophrenia.

The postsynaptic density (PSD) contains a complex set of proteins of known relevance to neuropsychiatric disorders, and schizophrenia specifically. We enriched for this anatomical structure, in the anterior cingulate cortex, of 20 schizophrenia samples and 20 controls from the Stanley Medical Research Institute, and used unbiased shotgun proteomics incorporating label-free quantitation to identify differentially expressed proteins. Quantitative investigation of the PSD revealed more than 700 protein identifications and 143 differentially expressed proteins. Prominent among these were altered expression of proteins involved in clathrin-mediated endocytosis (CME) (Dynamin-1, adaptor protein 2) and N-methyl-D-aspartate (NMDA)-interacting proteins such as CYFIP2, SYNPO, SHANK3, ESYT and MAPK3 (all P<0.0015). Pathway analysis of the differentially expressed proteins implicated the cellular processes of endocytosis, long-term potentiation and calcium signaling. Both single-gene and gene-set enrichment analyses in genome-wide association data from the largest schizophrenia sample to date of 13,689 cases and 18,226 controls show significant association of HIST1H1E and MAPK3, and enrichment of our PSD proteome. Taken together, our data provide robust evidence implicating PSD-associated proteins and genes in schizophrenia, and suggest that within the PSD, NMDA-interacting and endocytosis-related proteins contribute to disease pathophysiology.

Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression.

Long-term depression (LTD) underlies learning and memory in various brain regions. Although postsynaptic AMPA receptor trafficking mediates LTD, its underlying molecular mechanisms remain largely unclear. Here we show that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, stargazin's interaction with AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Thus, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors--transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes--through its sequential binding to AP-2 and AP-3A.