Type Glutamate Receptor Subunits Research Articles

GRIA3 p.Met661Thr variant in a female with developmental epileptic encephalopathy

The X-linked human glutamate receptor subunit 3 (GRIA3) gene (MIM *305915, Xq25) encodes ionotropic α amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunit 3, which mediates postsynaptic neurotransmission. Variants in this gene can cause a variety of neurological disorders, primarily reported in male patients. Here, we report a female patient with developmental and epileptic encephalopathy who carries the novel de novo GRIA3 variant NM_007325.5: c.1982T > C: p.Met661Thr.

Error-prone protein synthesis recapitulates early symptoms of Alzheimer disease in aging mice.

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aβ, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.

Aberrant neurogenesis and late onset suppression of synaptic plasticity as well as sustained neuroinflammation in the hippocampal dentate gyrus after developmental exposure to ethanol in rats

Drinking alcohol during pregnancy may cause fetal alcohol spectrum disorder. The present study investigated the effects of maternal oral ethanol (EtOH) exposure (0, 10, or 12.5 % in drinking water) from gestational day 6 until day 21 post-delivery (weaning) on postnatal hippocampal neurogenesis at weaning and in adulthood on postnatal day 77 in rat offspring. At weaning, type-3 neural progenitor cells (NPCs) were decreased in the subgranular zone (SGZ), accompanied by Chrnb2 downregulation and Grin2b upregulation in the dentate gyrus (DG). These results suggested suppression of CHRNB2-mediated cholinergic signaling in γ-aminobutyric acid (GABA)ergic interneurons in the DG hilus and increased glutamatergic signaling through the NR2B subtype of N-methyl-d-aspartate (NMDA) receptors, resulting in NPC reduction. In contrast, upregulation of Chrna7 may increase CHRNA7-mediated cholinergic signaling in immature granule cells, and upregulation of Ntrk2 may cause an increase in somatostatin-immunoreactive (+) GABAergic interneurons, suggesting a compensatory response against NPC reduction. Promotion of SGZ cell proliferation increased type-2a NPCs. Moreover, an increase in calbindin-d-29 K+ interneurons and upregulation of Reln, Drd2, Tgfb2, Il18, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunit genes might participate in this compensatory response. In adulthood, reduction of FOS+ cells and downregulation of Fos and Arc suggested suppression of granule cell synaptic plasticity, reflecting upregulation of Tnf and downregulation of Cntf, Ntrk2, and AMPA-type glutamate receptor genes. In the DG hilus, gliosis and hyper-ramified microglia, accompanying upregulation of C3, appeared at weaning, suggesting contribution to suppressed synaptic plasticity in adulthood. M1 microglia increased throughout adulthood, suggesting sustained neuroinflammation. These results indicate that maternal EtOH exposure temporarily disrupts hippocampal neurogenesis and later suppresses synaptic plasticity. Induction of neuroinflammation might initially ameliorate neurogenesis (as evident by upregulation of Tgfb2 and Il18) but later suppress synaptic plasticity (as evident by upregulation of C3 at weaning and Tnf in adulthood).

An exploratory investigation of antibodies to NMDA-type glutamate receptor subunits in serum and cerebrospinal fluid among psychiatric patients with anti-thyroid antibodies

IntroductionHashimoto's thyroiditis, which is characterized by anti-thyroid antibodies such as the anti-thyroglobulin (Tg) antibody and anti-thyroid peroxidase (TPO) antibody, is one of the autoimmune diseases associated with psychiatric illnesses. We previously reported a high prevalence of antibodies to N-terminals of N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) subunits (GluN1-NT and GluN2B-NT2) among psychiatric patients with anti-thyroid antibodies. However, it remains unclear whether the presence of anti-thyroid antibodies influences antibodies to GluN1-NT or GluN2B-NT2 among psychiatric patients. The present study aims to examine antibodies to GluN1-NT and GluN2B-NT2 in psychiatric patients with anti-thyroid antibodies (PPATs) and in those without (non-PPATs). Material and methodsWe recruited psychiatric inpatients aged 20–60 years. Patients were excluded if they had a history of neurological diseases, dementia, developmental disorders, tumors, or autoimmune diseases except autoimmune thyroiditis. The rest of the participants were divided into two groups according to the presence of serum anti-Tg and anti-TPO antibodies. We investigated serum and cerebrospinal fluid (CSF) antibodies to GluN1-NT and GluN2B-NT2 using an enzyme-linked immunosorbent assay (ELISA). ResultsWe initially recruited seventy-three psychiatric inpatients. Forty-six patients were excluded because of the exclusion criteria. Eighteen PPATs and nine non-PPATs were ultimately enrolled. We also collected stored sera of eighteen healthy controls (HCs) who were age- and sex-matched with PPATs. The optical densities (ODs) of serum antibodies to GluN1-NT (p = 0.0020) and GluN2B-NT2 (p = 0.039) were significantly higher in PPATs than in HCs. The ODs of CSF antibodies to GluN1-NT (p = 0.030) and GluN2B-NT2 (p = 0.017) as well as the positive ratios of those antibodies were significantly higher in PPATs than in non-PPATs. ConclusionOur finding indicates that detecting anti-thyroid antibodies in psychiatric patients would be a clue to consider psychiatric conditions related to antibodies to GluN1-NT/GluN2B-NT2. Further studies focusing on the relationship between PPATs and antibodies to GluN1-NT/GluN2B-NT2 are needed.

Atlas Copco AB, Sweden

Hashimoto's thyroiditis, which is characterized by anti-thyroid antibodies such as the anti-thyroglobulin (Tg) antibody and anti-thyroid peroxidase (TPO) antibody, is one of the autoimmune diseases associated with psychiatric illnesses. We previously reported a high prevalence of antibodies to N-terminals of N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) subunits (GluN1-NT and GluN2B-NT2) among psychiatric patients with anti-thyroid antibodies. However, it remains unclear whether the presence of anti-thyroid antibodies influences antibodies to GluN1-NT or GluN2B-NT2 among psychiatric patients. The present study aims to examine antibodies to GluN1-NT and GluN2B-NT2 in psychiatric patients with anti-thyroid antibodies (PPATs) and in those without (non-PPATs).We recruited psychiatric inpatients aged 20–60 years. Patients were excluded if they had a history of neurological diseases, dementia, developmental disorders, tumors, or autoimmune diseases except autoimmune thyroiditis. The rest of the participants were divided into two groups according to the presence of serum anti-Tg and anti-TPO antibodies. We investigated serum and cerebrospinal fluid (CSF) antibodies to GluN1-NT and GluN2B-NT2 using an enzyme-linked immunosorbent assay (ELISA).We initially recruited seventy-three psychiatric inpatients. Forty-six patients were excluded because of the exclusion criteria. Eighteen PPATs and nine non-PPATs were ultimately enrolled. We also collected stored sera of eighteen healthy controls (HCs) who were age- and sex-matched with PPATs. The optical densities (ODs) of serum antibodies to GluN1-NT (p = 0.0020) and GluN2B-NT2 (p = 0.039) were significantly higher in PPATs than in HCs. The ODs of CSF antibodies to GluN1-NT (p = 0.030) and GluN2B-NT2 (p = 0.017) as well as the positive ratios of those antibodies were significantly higher in PPATs than in non-PPATs.Our finding indicates that detecting anti-thyroid antibodies in psychiatric patients would be a clue to consider psychiatric conditions related to antibodies to GluN1-NT/GluN2B-NT2. Further studies focusing on the relationship between PPATs and antibodies to GluN1-NT/GluN2B-NT2 are needed.

Valmet Oyj, Finland

Hashimoto's thyroiditis, which is characterized by anti-thyroid antibodies such as the anti-thyroglobulin (Tg) antibody and anti-thyroid peroxidase (TPO) antibody, is one of the autoimmune diseases associated with psychiatric illnesses. We previously reported a high prevalence of antibodies to N-terminals of N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) subunits (GluN1-NT and GluN2B-NT2) among psychiatric patients with anti-thyroid antibodies. However, it remains unclear whether the presence of anti-thyroid antibodies influences antibodies to GluN1-NT or GluN2B-NT2 among psychiatric patients. The present study aims to examine antibodies to GluN1-NT and GluN2B-NT2 in psychiatric patients with anti-thyroid antibodies (PPATs) and in those without (non-PPATs).We recruited psychiatric inpatients aged 20–60 years. Patients were excluded if they had a history of neurological diseases, dementia, developmental disorders, tumors, or autoimmune diseases except autoimmune thyroiditis. The rest of the participants were divided into two groups according to the presence of serum anti-Tg and anti-TPO antibodies. We investigated serum and cerebrospinal fluid (CSF) antibodies to GluN1-NT and GluN2B-NT2 using an enzyme-linked immunosorbent assay (ELISA).We initially recruited seventy-three psychiatric inpatients. Forty-six patients were excluded because of the exclusion criteria. Eighteen PPATs and nine non-PPATs were ultimately enrolled. We also collected stored sera of eighteen healthy controls (HCs) who were age- and sex-matched with PPATs. The optical densities (ODs) of serum antibodies to GluN1-NT (p = 0.0020) and GluN2B-NT2 (p = 0.039) were significantly higher in PPATs than in HCs. The ODs of CSF antibodies to GluN1-NT (p = 0.030) and GluN2B-NT2 (p = 0.017) as well as the positive ratios of those antibodies were significantly higher in PPATs than in non-PPATs.Our finding indicates that detecting anti-thyroid antibodies in psychiatric patients would be a clue to consider psychiatric conditions related to antibodies to GluN1-NT/GluN2B-NT2. Further studies focusing on the relationship between PPATs and antibodies to GluN1-NT/GluN2B-NT2 are needed.

Cognitive enhancer ST101 improves schizophrenia-like behaviors in neonatal ventral hippocampus-lesioned rats in association with improved CaMKII/PKC pathway

Atypical antipsychotics improve positive and negative symptoms but are not effective for treating cognitive impairments in patients with schizophrenia. We previously reported that cognitive impairments in neonatal ventral hippocampus (NVH)-lesioned rats show resistance to atypical antipsychotics risperidone and are associated with reduced calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) signaling in memory-related regions. The cognitive enhancer ST101 (spiro[imi-dazo[1,2-a]pyridine-3,2-indan]-2(3H)-one) stimulates CaMKII activity in the hippocampus and medial prefrontal cortex (mPFC). We thus tested ST101 on cognitive impairments in NVH-lesioned rats. Chronic ST101 administration (0.1 and/or 0.5 mg/kg, p.o.) significantly improved deficits in prepulse inhibition (PPI), social interaction, and cognitive function in NVH-lesioned rats. ST101 administration (0.5 mg/kg, p.o.) significantly restored the decreased CaMKII autophosphorylation (Thr-286) in the mPFC and hippocampal CA1 regions of NVH-lesioned rats when assessed by immunohistochemistry. Chronic ST101 administration (0.1 mg/kg, p.o.) improved the decline in phosphorylation levels of CaMKII (Thr-286), PKCα (Ser-657), α-amino-3-hydroxy-5-methyl-4-isoxazol- propionic acid (AMPA)-type glutamate receptor subunit 1 (GluA1: Ser-831), and N-methyl-d-aspartate (NMDA) receptor subunit 1 (GluN1: Ser-896) in the mPFC and hippocampal CA1 regions. Taken together, these results suggest that ST101 improves schizophrenia-like behaviors and cognitive impairment by enhancing CaMKII/PKCα signaling in the mPFC and hippocampus in NVH-lesioned rats.

ANTI-NMDA receptor encephalitis after vaccination: A case report

Hashimoto's thyroiditis, which is characterized by anti-thyroid antibodies such as the anti-thyroglobulin (Tg) antibody and anti-thyroid peroxidase (TPO) antibody, is one of the autoimmune diseases associated with psychiatric illnesses. We previously reported a high prevalence of antibodies to N-terminals of N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) subunits (GluN1-NT and GluN2B-NT2) among psychiatric patients with anti-thyroid antibodies. However, it remains unclear whether the presence of anti-thyroid antibodies influences antibodies to GluN1-NT or GluN2B-NT2 among psychiatric patients. The present study aims to examine antibodies to GluN1-NT and GluN2B-NT2 in psychiatric patients with anti-thyroid antibodies (PPATs) and in those without (non-PPATs).We recruited psychiatric inpatients aged 20–60 years. Patients were excluded if they had a history of neurological diseases, dementia, developmental disorders, tumors, or autoimmune diseases except autoimmune thyroiditis. The rest of the participants were divided into two groups according to the presence of serum anti-Tg and anti-TPO antibodies. We investigated serum and cerebrospinal fluid (CSF) antibodies to GluN1-NT and GluN2B-NT2 using an enzyme-linked immunosorbent assay (ELISA).We initially recruited seventy-three psychiatric inpatients. Forty-six patients were excluded because of the exclusion criteria. Eighteen PPATs and nine non-PPATs were ultimately enrolled. We also collected stored sera of eighteen healthy controls (HCs) who were age- and sex-matched with PPATs. The optical densities (ODs) of serum antibodies to GluN1-NT (p = 0.0020) and GluN2B-NT2 (p = 0.039) were significantly higher in PPATs than in HCs. The ODs of CSF antibodies to GluN1-NT (p = 0.030) and GluN2B-NT2 (p = 0.017) as well as the positive ratios of those antibodies were significantly higher in PPATs than in non-PPATs.Our finding indicates that detecting anti-thyroid antibodies in psychiatric patients would be a clue to consider psychiatric conditions related to antibodies to GluN1-NT/GluN2B-NT2. Further studies focusing on the relationship between PPATs and antibodies to GluN1-NT/GluN2B-NT2 are needed.

Neurotoxicity Mechanism of Environmental Chemicals and Its Evaluation System

It is pivotal to assess the toxicity and safety of chemicals, including medicines, in the research field of environmental health science. Here we introduce neurotoxic mechanisms in mammals of environmental organotin and Parkinson's disease-related chemicals. We clarified that low concentrations of tributyltin decrease α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunit GluA2 (GluR2) expression, leading to the vulnerability of cultured neurons. That is, tributyltin reduces GluA2 prior to neuronal death. This GluA2 decrease can be used as a sensitive evaluation index of neurotoxicity, since low levels of certain chemicals, for example some agrochemicals, decrease GluA2 expression. We also elucidated the mechanisms of abnormal protein metabolism induced by low levels of two Parkinson's disease-related chemicals: 1-methyl-4-phenylpyridinium ion (MPP+) and 1,2,3,4-tetrahydroisoquinoline derivatives. It is expected that these findings will become clues in accurately evaluating the toxicity of chemicals and/or in investigating the causes of disease.

Adult–onset Rasmussen encephalitis with high anti N–methyl–D aspartate type glutamate receptor subunit antibody titer in serum and cerebrospinal fluid : Effects of high–dose intravenous methylprednisolone pulse therapy implemented eleven years after onset

Adult–onset Rasmussen encephalitis with high anti N–methyl–D aspartate type glutamate receptor subunit antibody titer in serum and cerebrospinal fluid : Effects of high–dose intravenous methylprednisolone pulse therapy implemented eleven years after onset

Low-Concentration Tributyltin Decreases GluR2 Expression via Nuclear Respiratory Factor-1 Inhibition.

Tributyltin (TBT), which has been widely used as an antifouling agent in paints, is a common environmental pollutant. Although the toxicity of high-dose TBT has been extensively reported, the effects of low concentrations of TBT are relatively less well studied. We have previously reported that low-concentration TBT decreases α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor subunit 2 (GluR2) expression in cortical neurons and enhances neuronal vulnerability to glutamate. However, the mechanism of this TBT-induced GluR2 decrease remains unknown. Therefore, we examined the effects of TBT on the activity of transcription factors that control GluR2 expression. Exposure of primary cortical neurons to 20 nM TBT for 3 h to 9 days resulted in a decrease in GluR2 mRNA expression. Moreover, TBT inhibited the DNA binding activity of nuclear respiratory factor-1 (NRF-1), a transcription factor that positively regulates the GluR2. This result indicates that TBT inhibits the activity of NRF-1 and subsequently decreases GluR2 expression. In addition, 20 nM TBT decreased the expression of genes such as cytochrome c, cytochrome c oxidase (COX) 4, and COX 6c, which are downstream of NRF-1. Our results suggest that NRF-1 inhibition is an important molecular action of the neurotoxicity induced by low-concentration TBT.

Semi-quantitative analyses of antibodies to N-methyl-d-aspartate type glutamate receptor subunits (GluN2B & GluN1) in the clinical course of Rasmussen syndrome

In Rasmussen syndrome (RS), in addition to the predominant involvement of cytotoxic T cells, heterogeneous autoantibodies against neural molecules are also found, but their function has not been elucidated. We examined antibodies to N-methyl-d-aspartate (NMDA) type glutamate receptor (GluR) subunits (GluN2B & GluN1) semi-quantitatively in cerebrospinal fluid (CSF) samples from RS patients, and evaluated their changes over time and their roles in immunopathogenesis. Autoantibodies against N-terminal and C-terminal of GluN2B and GluN1 were examined in 40 CSF samples collected from 18 RS patients 5 to 180 months after the onset of RS. Epileptic patients without infectious etiology or progressive clinical course served as disease controls (n=23). Synthesized peptides encoding the extracellular and intracellular domains of human GluN2B and GluN1 subunits were used as antigens in ELISA. We defined the cut-off for these antibodies as mean +2 standard deviations (optimal density) of the disease controls. MRI were evaluated according to the MRI staging proposed by Bien et al. (2002b, Neurology 58, 250). CSF levels of antibodies against N-terminal and C-terminal of GluN2B were higher in RS patients than in disease controls (p<0.01). Likewise, CSF levels of antibodies against N-terminal and C-terminal of GluN1 were also higher in RS patients than in disease controls (p<0.01). All four antibodies tested were below cut-off levels in almost all CSF samples collected within one year from epilepsy onset. The proportions of CSF samples with these antibodies above cut-off levels were highest from 12 to 23 months after epilepsy onset, and declined after 24 months. CSF levels of these antibodies were higher when seizure occurred daily than when seizure occurred less frequently (p<0.01), and were higher at MRI stage 3 than at MRI stages 0, 2 and 4 (p<0.05), except for anti-GluN1-CT antibody at stage 2. Broad epitope recognition spectrum and delayed production of autoantibodies to NMDA type GluR in CSF of RS patients suggest that the autoantibodies are produced against NMDA type GluR antigens derived from cytotoxic T cell-mediated neuronal damages. These antibodies may impact the pathophysiology of RS in the most active stage, and could be a marker for active inflammation in the clinical course of RS. Further studies including passive transfer of the antibodies to mice may reveal the pivotal roles of the antibodies in RS.

Genetic dissection of memory for associative and non‐associative learning in Caenorhabditis elegans

The distinction between non-associative and associative forms of learning has historically been based on the behavioral training paradigm. Through discovering the molecular mechanisms that mediate learning, we can develop a deeper understanding of the relationships between different forms of learning. Here, we genetically dissect short- and long-term memory for a non-associative form of learning, habituation and an associative form of learning, context conditioning for habituation, in the nematode Caenorhabditis elegans. In short-term chemosensory context conditioning for habituation, worms trained and tested in the presence of either a taste (sodium acetate) or smell (diacetyl) context cue show greater retention of habituation to tap stimuli when compared with animals trained and tested without a salient cue. Long-term memory for olfactory context conditioning was observed 24 h after a training procedure that does not normally induce 24 h memory. Like long-term habituation, this long-term memory was dependent on the transcription factor cyclic AMP-response element-binding protein. Worms with mutations in glr-1 [a non-N-methyl-d-aspartate (NMDA)-type glutamate receptor subunit] showed short-term but not long-term habituation or short- or long-term context conditioning. Worms with mutations in nmr-1 (an NMDA-receptor subunit) showed normal short- and long-term memory for habituation but did not show either short- or long-term context conditioning. Rescue of nmr-1 in the RIM interneurons rescued short- and long-term olfactory context conditioning leading to the hypothesis that these interneurons function to integrate information from chemosensory and mechanosensory systems for associative learning.

Overexpression of Calcium-Permeable Glutamate Receptors in Glioblastoma Derived Brain Tumor Initiating Cells

Glioblastoma multiforme is the most malignant type of primary brain tumor with a poor prognosis. These tumors consist of a heterogeneous population of malignant cells, including well-differentiated tumor cells and less differentiated cells with stem cell properties. These cancer stem cells, known as brain tumor initiating cells, likely contribute to glioma recurrence, as they are highly invasive, mobile, resistant to radiation and chemotherapy, and have the capacity to self-renew. Glioblastoma tumor cells release excitotoxic levels of glutamate, which may be a key process in the death of peritumoral neurons, formation of necrosis, local inflammation, and glioma-related seizures. Moreover, elevated glutamate levels in the tumor may act in paracrine and autocrine manner to activate glutamate receptors on glioblastoma tumor cells, resulting in proliferation and invasion. Using a previously described culturing condition that selectively promotes the growth of brain tumor initiating cells, which express the stem cell markers nestin and SOX-2, we characterize the expression of α-amino-3-hydroxy-5-methyl-4-isozolepropionic acid (AMPA)-type glutamate receptor subunits in brain tumor initiating cells derived from glioblastomas. Here we show for the first time that glioblastoma brain tumor initiating cells express high concentrations of functional calcium-permeable AMPA receptors, compared to the differentiated tumor cultures consisting of non-stem cells. Up-regulated calcium-permeable AMPA receptor expression was confirmed by immunoblotting, immunocytochemistry, and intracellular calcium imaging in response to specific agonists. Our findings raise the possibility that glutamate secretion in the GBM tumor microenvironment may stimulate brain tumor derived cancer stem cells.

HNK-1 Glyco-epitope Regulates the Stability of the Glutamate Receptor Subunit GluR2 on the Neuronal Cell Surface

HNK-1 (human natural killer-1) glyco-epitope, a sulfated glucuronic acid attached to N-acetyllactosamine on the nonreducing termini of glycans, is highly expressed in the nervous system. Our previous report showed that mice lacking a glucuronyltransferase (GlcAT-P), a key enzyme for biosynthesis of the HNK-1 epitope, showed reduced long term potentiation at hippocampal CA1 synapses. In this study, we identified an alpha-amino-3-hydroxy-5-methylisoxazole propionate (AMPA)-type glutamate receptor subunit, GluR2, which directly contributes to excitatory synaptic transmission and synaptic plasticity, as a novel HNK-1 carrier molecule. We demonstrated that the HNK-1 epitope is specifically expressed on the N-linked glycan(s) on GluR2 among the glutamate receptors tested, and the glycan structure, including HNK-1 on GluR2, was determined using liquid chromatography-tandem mass spectrometry. As for the function of HNK-1 on GluR2, we found that the GluR2 not carrying HNK-1 was dramatically endocytosed and expressed less on the cell surface compared with GluR2 carrying HNK-1 in both cultured hippocampal neurons and heterologous cells. These results suggest that HNK-1 stabilizes GluR2 on neuronal surface membranes and regulates the number of surface AMPA receptors. Moreover, we showed that the expression of the HNK-1 epitope enhanced the interaction between GluR2 and N-cadherin, which has important roles in AMPA receptor trafficking. Our findings suggest that the HNK-1 epitope on GluR2 regulates cell surface stability of GluR2 by modulating the interaction with N-cadherin.

HNK-1 (human natural killer-1) glyco-epitope is essential for normal spine morphogenesis in developing hippocampal neurons

The human natural killer-1 (HNK-1) glyco-epitope possesses a unique structural feature, a sulfated glucuronic acid attached to lactosamine on the non-reducing termini of glycans. The expression of HNK-1 is temporally and spatially regulated by glucuronyltransferase (GlcAT-P) in the brain. Our previous report showed that mice lacking GlcAT-P almost completely lost HNK-1 expression in the brain and exhibited reduced long-term potentiation (LTP) at hippocampal CA1 synapses. GlcAT-P-deficient mice also showed impaired hippocampus-dependent spatial learning. Although HNK-1 plays an essential role in synaptic plasticity and memory formation, it remains unclear how HNK-1 regulates these functions. In this study, we showed that loss of the HNK-1 epitope resulted in an increase of filopodium-like immature spines and a decrease of mushroom-like mature spines in both the early postnatal mouse hippocampus and cultured hippocampal neurons. However, HNK-1 had no influence on spine density or filopodium formation. Immunofluorescence staining revealed that loss of HNK-1 altered the distribution of postsynaptic proteins such as α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-type glutamate receptor subunit GluR2 and PSD-95 from spine heads onto dendritic shafts without affecting synapse formation, resulting in an increase of shaft synapses in cultured GlcAT-P-deficient neurons. GluR2, a major HNK-1 carrier glycoprotein in postsynaptic density, has the ability to promote spine morphogenesis. Overexpression of GluR2 promoted spine growth in both wild-type and GlcAT-P-deficient neurons, but the increase in GlcAT-P-deficient neurons was lower than that in wild-type neurons. This is the first evidence that HNK-1 is a key factor for normal dendritic spine maturation and is involved in the distribution of postsynaptic proteins.

Fragile X Mental Retardation Protein Regulates the Levels of Scaffold Proteins and Glutamate Receptors in Postsynaptic Densities

Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1-3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3'-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients.

Estrogen dissociates Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit in postischemic hippocampus

During cerebral ischemia, part of the damage associated with the hyperactivation of glutamate receptors results from the hyperphosphorylation of the microtubule-associated protein Tau. Previous studies have shown that estradiol treatment reduces neural damage after cerebral ischemia. Here, we show that transient occlusion of the middle cerebral artery results in the hyperphosphorylation of Tau and in a significant increase in the association of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type glutamate receptor subunits 2/3 in the hippocampus. Estradiol treatment decreased hippocampal injury, inhibited glycogen synthase kinase-3beta and decreased the hyperphosphorylation of Tau and the interaction of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. These findings suggest that ischemia produces a strong association between Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, and estradiol can exert at least part of its neuroprotective activity through inhibition of glycogen synthase kinase-3beta.

Interaction of LDL receptor‐related protein 4 (LRP4) with postsynaptic scaffold proteins via its C‐terminal PDZ domain‐binding motif, and its regulation by Ca2+/calmodulin‐dependent protein kinase II

We cloned here a full-length cDNA of Dem26[Tian et al. (1999)Mol. Brain Res., 72, 147-157], a member of the low-density lipoprotein (LDL) receptor gene family from the rat brain. We originally named the corresponding protein synaptic LDL receptor-related protein (synLRP) [Tian et al. (2002) Soc. Neurosci. Abstr., 28, 405] and have renamed it LRP4 to accord it systematic nomenclature (GenBank(TM) accession no. AB073317). LRP4 protein interacted with postsynaptic scaffold proteins such as postsynaptic density (PSD)-95 via its C-terminal tail sequence, and associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor subunit. The mRNA of LRP4 was localized to dendrites, as well as somas, of neuronal cells, and the full-length protein of 250 kDa was highly concentrated in the brain and localized to various subcellular compartments in the brain, including synaptic fractions. Immunocytochemical study using cultured cortical neurons suggested surface localization in the neuronal cells both in somas and dendrites. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylated the C-terminal cytoplasmic region of LRP4 at Ser1887 and Ser1900, and the phosphorylation at the latter site suppressed the interaction of the protein with PSD-95 and synapse-associated protein 97 (SAP97). These findings suggest a postsynaptic role for LRP4, a putative endocytic multiligand receptor, and a mechanism in which CaMKII regulates PDZ-dependent protein-protein interactions and receptor dynamics.

Kainate induces rapid redistribution of the actin cytoskeleton in ameboid microglia

Microglia are key mediators of the immune response in the central nervous system (CNS). They are closely related to macrophages and undergo dramatic morphological and functional changes after CNS trauma or excitotoxic lesions. Microglia can be directly stimulated by excitatory neurotransmitters and are known to express many neurotransmitter receptors. The role of these receptors, however, is not clear. This study describes the microglial response to the glutamate receptor agonist kainate (KA) and shows via immunochemistry that the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunit GluR1 is present on cultured microglia. In the presence of 100 microM or 1 mM KA, cultured microglia underwent dramatic morphological and cytoskeletal changes as observed by time-lapse photography and quantitative confocal analysis of phalloidin labeling. KA-stimulated microglia showed condensation of cytoplasmic actin filaments, rapid de- and repolymerization, and cytoplasmic redistribution of condensed actin bundles. Rearrangement of actin filaments-thought to be involved in locomotion and phagocytosis and to indicate an increased level of activation (for reviews see Greenberg [ 1995] Trends Cell Biol. 5:93-99; Imai and Kohsaka [ 2002] Glia 40:164-174)-was significantly increased in treated vs. control cultures. Morphological plasticity and membrane ruffling were also seen. These findings suggest direct microglial excitation via glutamate receptor pathways. Thus, neurotransmitter release after brain or spinal cord injury might directly modulate the inflammatory response.