Expression Of GluD1 Research Articles

Energy metabolism-related GLUD1 contributes to favorable clinical outcomes of IDH-mutant glioma

BackgroundGlioma is the most common brain tumor. IDH mutations occur frequently in glioma, indicating a more favorable prognosis. We aimed to explore energy metabolism-related genes in glioma to promote the research and treatment.MethodsDatasets were obtained from TCGA and GEO databases. Candidate genes were screened by differential gene expression analysis, then functional enrichment analysis was conducted on the candidate genes. PPI was also carried out to help determine the target gene. GSEA and DO analysis were conducted in the different expression level groups of the target gene. Survival analysis and immune cell infiltrating analysis were performed as well.ResultsWe screened 34 candidate genes and selected GLUD1 as the target gene. All candidate genes were significantly enriched in 10 KEGG pathways and 330 GO terms. GLUD1 expression was higher in IDH-mutant samples than IDH-wildtype samples, and higher in normal samples than tumor samples. Low GLUD1 expression was related to poor prognosis according to survival analysis. Most types of immune cells were negatively related to GLUD1 expression, but monocytes and activated mast cells exhibited significantly positive correlation with GLUD1 expression. GLUD1 expression was significantly related to 119 drugs and 6 immune checkpoint genes. GLUD1 was able to serve as an independent prognostic indicator of IDH-mutant glioma.ConclusionIn this study, we identified an energy metabolism-related gene GLUD1 potentially contributing to favorable clinical outcomes of IDH-mutant glioma. In glioma, GLUD1 related clinical outcomes and immune landscape were clearer, and more valuable information was provided for immunotherapy.

PoRVA G9P[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism.

PoRVA and PEDV coinfections are extremely common in clinical practice. Although coinfections of PoRVA and PEDV are known to result in increased mortality, the underlying mechanism remains unknown. Here, we found that PoRVA infection promoted PEDV infection in vivo and in vitro and that PoRVA G9P[23] (RVA-HNNY strain) enhanced PEDV replication more significantly than did PoRVA G5P[7] (RVA-SXXA strain). Metabolomic analysis revealed that RVA-HNNY more efficiently induced an increase in the intracellular glutamine content in porcine small intestinal epithelial cells than did RVA-SXXA, which more markedly promoted ATP production to facilitate PEDV replication, whereas glutamine deprivation abrogated the effect of PoRVA infection on promoting PEDV replication. Further studies showed that PoRVA infection promoted glutamine uptake by upregulating the expression of the glutamine transporter protein SLC1A5. In SLC1A5 knockout cells, PoRVA infection neither elevated intracellular glutamine nor promoted PEDV replication. During PoRVA infection, the activity and protein expression levels of glutamine catabolism-related enzymes (GLS1 and GLUD1) were also significantly increased promoting ATP production through glutamine anaplerosis into the TCA cycle. Consistent with that, siRNAs or inhibitors of GLS1 and GLUD1 significantly inhibited the promotion of PEDV replication by PoRVA. Notably, RVA-HNNY infection more markedly promoted SLC1A5, GLS1 and GLUD1 expression to more significantly increase the uptake and catabolism of glutamine than RVA-SXXA infection. Collectively, our findings illuminate a novel mechanism by which PoRVA infection promotes PEDV infection and reveal that the modulation of glutamine uptake is key for the different efficiencies of PoRVA G9P[23] and PoRVA G5P[7] in promoting PEDV replication.

Intravenous recombinant cerebellin 1 treatment restores signalling by spinal glutamate delta 1 receptors and mitigates chronic pain.

Chronic pain remains a major clinical problem that needs effective therapeutic agents. Glutamate delta 1 (GluD1) receptors and the protein cerebellin 1 (Cbln1) are down-regulated in the central amygdala (CeA) in models of inflammatory and neuropathic pain. One treatment with Cbln1, intracerebroventricularly (ICV) or in CeA, normalized GluD1 and reduced AMPA receptor expression, resulting in lasting (7-10days) pain relief. Unlike many CNS-targeting biological agents, the structure of Cbln1 suggests potential blood-brain barrier penetration. Here, we have tested whether systemic administration of Cbln1 provides analgesic effects via action in the CNS. Analgesic effects of intravenous recombinant Cbln1 was assessed in complete Freund's adjuvant inflammatory pain model in mice. GluD1 knockout and a mutant form of Cbln1 were used. A single intravenous injection of Cbln1 mitigated nocifensive and averse behaviour in both inflammatory and neuropathic pain models. This effect of Cbln1 was dependent on GluD1 receptors and required binding to the amino terminal domain of GluD1. Time course of analgesic effect was similar to previously reported ICV and intra-CeA injection. GluD1 in both spinal cord and CeA was down -regulated in the inflammatory pain model, whereas GluD1 expression in spinal cord but not in CeA, was partly normalized by intravenous Cbln1. Importantly, recombinant Cbln1 was detected in the synaptoneurosomes in spinal cord but not in the CeA. Our results describe a novel mechanism by which systemic Cbln1 induces analgesia potentially by central actions involving normalization of signalling by spinal cord GluD1 receptors.

Rett and Rett-related disorders: Common mechanisms for shared symptoms?

Rett syndrome is a neurodevelopmental disorder caused by loss-of-function mutations in the methyl-CpG binding protein-2 (MeCP2) gene that is characterized by epilepsy, intellectual disability, autistic features, speech deficits, and sleep and breathing abnormalities. Neurologically, patients with all three disorders display microcephaly, aberrant dendritic morphology, reduced spine density, and an imbalance of excitatory/inhibitory signaling. Loss-of-function mutations in the cyclin-dependent kinase-like 5 (CDKL5) and FOXG1 genes also cause similar behavioral and neurobiological defects and were referred to as congenital or variant Rett syndrome. The relatively recent realization that CDKL5 deficiency disorder (CDD), FOXG1 syndrome, and Rett syndrome are distinct neurodevelopmental disorders with some distinctive features have resulted in separate focus being placed on each disorder with the assumption that distinct molecular mechanisms underlie their pathogenesis. However, given that many of the core symptoms and neurological features are shared, it is likely that the disorders share some critical molecular underpinnings. This review discusses the possibility that deregulation of common molecules in neurons and astrocytes plays a central role in key behavioral and neurological abnormalities in all three disorders. These include KCC2, a chloride transporter, vGlut1, a vesicular glutamate transporter, GluD1, an orphan-glutamate receptor subunit, and PSD-95, a postsynaptic scaffolding protein. We propose that reduced expression or activity of KCC2, vGlut1, PSD-95, and AKT, along with increased expression of GluD1, is involved in the excitatory/inhibitory that represents a key aspect in all three disorders. In addition, astrocyte-derived brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), and inflammatory cytokines likely affect the expression and functioning of these molecules resulting in disease-associated abnormalities.

Glutamate regulates gliosis of BMSCs to promote ENS regeneration through α-KG and H3K9/H3K27 demethylation

BackgroundThere is a lack of effective therapies for enteric nervous system (ENS) injury. Our previous study showed that transplanted bone marrow-derived mesenchymal stem cells (BMSCs) play a “glia-like cells” role in initiating ENS regeneration in denervated mice. Cellular energy metabolism is an important factor in maintaining the biological characteristics of stem cells. However, how cellular energy metabolism regulates the fate of BMSCs in the ENS-injured microenvironment is unclear.MethodsThe biological characteristics, energy metabolism, and histone methylation levels of BMSCs following ENS injury were determined. Then, glutamate dehydrogenase 1 (Glud1) which catalyzes the oxidative deamination of glutamate to α-KG was overexpressed (OE) in BMSCs. Further, OE-Glud1 BMSCs were targeted–transplanted into the ENS injury site of denervated mice to determine their effects on ENS regeneration.ResultsIn vitro, in the ENS-injured high-glutamate microenvironment, the ratio of α-ketoglutarate (α-KG) to succinate (P < 0.05), the histone demethylation level (P < 0.05), the protein expression of glial cell markers (P < 0.05), and the gene expression of Glud1 (P < 0.05) were significantly increased. And the binding of H3K9me3 to the GFAP, S100B, and GDNF promoter was enhanced (P < 0.05). Moreover, α-KG treatment increased the monomethylation and decreased the trimethylation on H3K9 (P < 0.01) and H3K27 (P < 0.05) in BMSCs and significantly upregulated the protein expression of glial cell markers (P < 0.01), which was reversed by the α-KG competitive inhibitor D-2-hydroxyglutarate (P < 0.05). Besides, overexpression of Glud1 in BMSCs exhibited increases in monomethylation and decreases in trimethylation on H3K9 (P < 0.05) and H3K27 (P < 0.05), and upregulated protein expression of glial cell markers (P < 0.01). In vivo, BMSCs overexpressing Glud1 had a strong promotion effect on ENS regeneration in denervated mice through H3K9/H3K27 demethylation (P < 0.05), and upregulating the expression of glial cell protein (P < 0.05).ConclusionsBMSCs overexpressing Glud1 promote the expression of glial cell markers and ENS remodeling in denervated mice through regulating intracellular α-KG and H3K9/H3K27 demethylation.

Loss of SDHB Induces a Metabolic Switch in the hPheo1 Cell Line toward Enhanced OXPHOS.

Background: Enzymes of tricarboxylic acid (TCA) have recently been recognized as tumor suppressors. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) cause pheochromocytomas and paragangliomas (PCCs/PGLs) and predispose patients to malignant disease with poor prognosis. Methods: Using the human pheochromocytoma cell line (hPheo1), we knocked down SDHB gene expression using CRISPR-cas9 technology. Results: Microarray gene expression analysis showed that >500 differentially expressed gene targets, about 54%, were upregulated in response to SDHB knock down. Notably, genes involved in glycolysis, hypoxia, cell proliferation, and cell differentiation were up regulated, whereas genes involved in oxidative phosphorylation (OXPHOS) were downregulated. In vitro studies show that hPheo1 proliferation is not affected negatively and the cells that survive by shifting their metabolism to the use of glutamine as an alternative energy source and promote OXPHOS activity. Knock down of SDHB expression results in a significant increase in GLUD1 expression in hPheo1 cells cultured as monolayer or as 3D culture. Analysis of TCGA data confirms the enhancement of GLUD1 in SDHB mutated/low expressed PCCs/PGLs. Conclusions: Our data suggest that the downregulation of SDHB in PCCs/PGLs results in increased GLUD1 expression and may represent a potential biomarker and therapeutic target in SDHB mutated tumors and SDHB loss of activity-dependent diseases.

グルタミン酸受容体GluD1は高次脳機能領域に強く発現し、小脳では平行線維－プルキンエ細胞シナプスの形成に関与する

Of the two members of the δ subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum, and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knockout mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons, and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knockout mice, the density of PF synapses on interneuron somata was significantly reduced, and the size and number of interneurons were significantly diminished. Thus, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex, i,e., GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses, and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.

GLUD1 Promotes Cetuximab Resistance in Metastasis Colorectal Cancer

Background: Colorectal cancer (CRC) is one of the most common causes of cancer mortality worldwide, among which metastatic colorectal cancer (mCRC) is the deadliest one. Cetuximab as a monoclonal antibody targeting epidermal growth factor receptor (EGFR) has improved the survival of mCRC patients. However, primary and acquired resistance to cetuximab are often found during therapy. Therefore, it is important to study the mechanism of cetuximab resistance and find more potential biomarkers. Methods: We previously established the acquired cetuximab resistant cell lines Caco2-CR and NCI-H508-CR by treating their parental cetuximab sensitive colorectal cancer cell lines, Caco2 and NCI-H508, with continuously increasing concentrations of cetuximab. Microarray analysis was utilized to detect the expression profiling of resistant cells and sensitive cells. Gene expression data of mCRC treated with cetuximab (GSE5851) was obtained from GEO. Patient-derived xenografts (PDX) were established using the tumor tissues from either primary lesions or liver metastases of mCRC patients without previous treatment. The metabolic flux of 13 C labelled glutamine in NCI-H508-CR and its parental cells was analyzed. Findings: Microarray analysis indicated that GLUD1, a key enzyme in glutamine metabolism, was one of the differentially expressed genes between Caco2-CR and Caco2. The expression of GLUD1 in the cetuximab resistant patients from GSE5851 dataset were higher than that in the sensitive ones. The same results were seen in animal PDX models. The glutamine metabolism was stronger in NCI-H508-CR compared with NCI-H508 and inhibition of glutamine metabolism could reduce the cetuximab resistance. GLUD1 could promote cetuximab resistance by generating more α-KG to support tricarboxylic acid (TCA) cycle. Furthermore, the expression of GLUD1 in resistant cells could be promoted by the nucleus translocated FOXO3. Interpretation: In this study，we found that GLUD1 can promote cetuximab resistance in mCRC, and it was transcriptional regulated by FOXO3. Funding: This work was supported by the National Natural Science Foundation of China (81772511). Declaration of Interest: None to declare. Ethical Approval: This study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University

Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain.

In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.

Glutamate dehydrogenase (GLUD1) expression in breast cancer.

Dysregulated cellular metabolism is one of the hallmarks of cancer with some tumours utilising the glutamine metabolism pathway for their sustained proliferation and survival. Glutamate dehydrogenase (GLUD1) is a key enzyme in glutaminolysis converting glutamate to α-ketoglutarate for entry into the TCA cycle. Breast cancer (BC) comprises a heterogeneous group of tumours in terms of molecular biology and clinical behaviour, and we have previously shown that altered glutamine metabolism varies substantially among the different molecular subtypes. We hypothesise that the prognostic value of GLUD1 expression will differ between the BC molecular subtypes and may act as a potential therapeutic target for BC tumours. GLUD1 was assessed at the DNA, mRNA (n = 1980) and protein (n = 1300) levels in large, well-characterised cohorts and correlated with clinicopathological parameters, molecular subtypes, patient outcome, and treatments. There was a correlation between GLUD1 mRNA and GLUD1 protein expression which were highly expressed in low grade luminal/ER + BC (p < 0.01). GLUD1 mRNA and protein was associated with good patient outcome but not in any specific molecular subtypes. However, high GLUD1 protein expression was associated with a better outcome in triple negative (TN) patients treated with chemotherapy (p = 0.03). High GLUD1 mRNA was associated with the glutamine transporter, SLC1A5, and leucine transporter, SLC7A8 as well as mTOR (p < 0.0001). We provide comprehensive data indicating GLUD1 plays an important role in luminal/ER + BC. GLUD1 expression predicts a better patient outcome and we show that it has the potential for predicting response to chemotherapy in TNBC patients.

Glutamate delta-1 receptor regulates cocaine-induced plasticity in the nucleus accumbens

Cocaine exposure induces plasticity of glutamatergic synapses of medium spiny neurons (MSNs) in the nucleus accumbens (NAc), which has been proposed to contribute to its addictive behavior. The mechanisms underlying cocaine-induced plasticity are not fully understood. The orphan glutamate delta-1 (GluD1) receptor is a member of the ionotropic glutamate receptor family but does not function as a typical ligand-gated ion channel. Instead it serves a synaptogenic function by interacting with presynaptic Neurexin protein. Recent neuroanatomical studies have demonstrated enriched expression of GluD1 in the NAc but its role in reward behavior, MSN function, and drug-induced plasticity remains unknown. Using a combination of constitutive and conditional GluD1 KO models, we evaluated the effect of GluD1 ablation on cocaine-conditioned place preference (CPP) and cocaine-induced structural and functional plasticity. GluD1 KO mice showed higher cocaine CPP. Selective ablation of GluD1 from striatal neurons but not cortico-limbic excitatory neurons reproduced higher CPP. Higher cocaine preference in GluD1 KO correlated with an increase in spine density, greater maturation of dendritic spines, and basally upregulated spine-regulating active cofilin. GluD1 loss did not affect basal excitatory neurotransmission or plasticity but masked the generation of cocaine-induced silent synapses. Finally, loss of GluD1 increased the GluN2B subunit contribution to NMDA receptor currents in MSNs and a partial agonist of GluN2B-containing NMDA receptors normalized the higher active cofilin and cocaine preference in GluD1 KO mice. Together, these findings demonstrate a critical role of GluD1 in controlling susceptibility to cocaine preference and cocaine-induced plasticity by modulating NMDA receptor subunit contribution.

Glutamate Dehydrogenase-Deficient Mice Display Schizophrenia-Like Behavioral Abnormalities and CA1-Specific Hippocampal Dysfunction.

Brain imaging has revealed that the CA1 subregion of the hippocampus is hyperactive in prodromal and diagnosed patients with schizophrenia (SCZ), and that glutamate is a driver of this hyperactivity. Strikingly, mice deficient in the glutamate synthetic enzyme glutaminase have CA1 hypoactivity and a SCZ-resilience profile, implicating glutamate-metabolizing enzymes. To address this further, we examined mice with a brain-wide deficit in the glutamate-metabolizing enzyme glutamate dehydrogenase (GDH), encoded by Glud1, which should lead to glutamate excess due to reduced glutamate metabolism in astrocytes. We found that Glud1-deficient mice have behavioral abnormalities in the 3 SCZ symptom domains, with increased baseline and amphetamine-induced hyperlocomotion as a positive symptom proxy, nest building and social preference as a negative symptom proxy, and reversal/extradimensional set shifting in the water T-maze and contextual fear conditioning as a cognitive symptom proxy. Neuroimaging of cerebral blood volume revealed hippocampal hyperactivity in CA1, which was associated with volume reduction. Parameters of hippocampal synaptic function revealed excess glutamate release and an elevated excitatory/inhibitory balance in CA1. Finally, in a direct clinical correlation using imaging-guided microarray, we found a significant SCZ-associated postmortem reduction in GLUD1 expression in CA1. These findings advance GLUD1 deficiency as a driver of excess hippocampal excitatory transmission and SCZ symptoms, and identify GDH as a target for glutamate modulation pharmacotherapy for SCZ. More broadly, these findings point to the likely involvement of alterations in glutamate metabolism in the pathophysiology of SCZ.

Imbalance of excitatory/inhibitory synaptic protein expression in iPSC-derived neurons from FOXG1(+/-) patients and in foxg1(+/-) mice.

Rett syndrome (RTT) is a severe neurodevelopmental disorder associated with mutations in either MECP2, CDKL5 or FOXG1. The precise molecular mechanisms that lead to the pathogenesis of RTT have yet to be elucidated. We recently reported that expression of GluD1 (orphan glutamate receptor δ-1 subunit) is increased in iPSC-derived neurons obtained from patients with mutations in either MECP2 or CDKL5. GluD1 controls synaptic differentiation and shifts the balance between excitatory and inhibitory synapses toward the latter. Thus, an increase in GluD1 might be a critical factor in the etiology of RTT by affecting the excitatory/inhibitory balance in the developing brain. To test this hypothesis, we generated iPSC-derived neurons from FOXG1(+/-) patients. We analyzed mRNA and protein levels of GluD1 together with key markers of excitatory and inhibitory synapses in these iPSC-derived neurons and in Foxg1(+/-) mouse fetal (E11.5) and adult (P70) brains. We found strong correlation between iPSC-derived neurons and fetal mouse brains, where GluD1 and inhibitory synaptic markers (GAD67 and GABA AR-α1) were increased, whereas the levels of a number of excitatory synaptic markers (VGLUT1, GluA1, GluN1 and PSD-95) were decreased. In adult mice, GluD1 was decreased along with all GABAergic and glutamatergic markers. Our findings further the understanding of the etiology of RTT by introducing a new pathological event occurring in the brain of FOXG1(+/-) patients during embryonic development and its time-dependent shift toward a general decrease in brain synapses.

Glutamate receptors of the delta family are widely expressed in the adult brain.

Recent reports point to critical roles of glutamate receptor subunit delta2 (GluD2) at excitatory synapses and link GluD1 gene alteration to schizophrenia but the expression patterns of these subunits in the brain remain almost uncharacterized. We examined the distribution of GluD1-2 mRNAs and proteins in the adult rodent brain, focusing mainly on GluD1. In situ hybridization revealed widespread neuronal expression of the GluD1 mRNA, with higher levels occurring in several forebrain regions and lower levels in cerebellum. Quantitative RT-PCR assessed differential GluD1 expression in cortex and cerebellum, and revealed GluD2 expression in cortex, albeit at markedly lower level than in cerebellum. Likewise, a high GluD1/GluD2 mRNA ratio was observed in cortex and a low ratio in cerebellum. GluD1 and GluD2 mRNAs were co-expressed in single cortical and hippocampal neurons, with a large predominance of GluD1. Western blots using GluD1- and GluD2-specific antibodies showed expression of both subunits in various brain structures, but not in non-nervous tissues examined. Both delta subunits were upregulated during postnatal development. Widespread neuronal expression of the GluD1 protein was confirmed using immunohistochemistry. Examination at the electron microscopic level in the hippocampus revealed that GluD1 was mainly localized at postsynaptic density of excitatory synapses on pyramidal cells. Control experiments performed using mice carrying deletion of the GluD1- or the GluD2-encoding gene confirmed the specificity of the present mRNA and protein analyses. Our results support a role for the delta family of glutamate receptors at excitatory synapses in neuronal networks throughout the adult brain.

Enriched Expression of GluD1 in Higher Brain Regions and Its Involvement in Parallel Fiber–Interneuron Synapse Formation in the Cerebellum

Of the two members of the δ subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knock-out mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminished. Therefore, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex; that is, GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function.

Induction of excitatory and inhibitory presynaptic differentiation by GluD1

The δ subfamily of ionotropic glutamate receptor subunits consists of GluD1 and GluD2. GluD2, which is selectively expressed in cerebellar Purkinje neurons, has been shown to contribute to the formation of synapses between granule neurons and Purkinje neurons through interaction with Cbln1 (cerebellin precursor protein1) and presynaptic Neurexin. On the other hand, the synaptogenic activity of GluD1, which is expressed not in the cerebellum but in the hippocampus, remains to be characterized. Here, we report that GluD1 expressed in non-neuronal HEK cells, induced presynaptic differentiation of granule neurons through its N-terminal domain in co-cultures with cerebellar neurons, similarly to GluD2. We also show that GluD1 rescued the defect of synapse formation in GluD2-knockout Purkinje neurons, indicating the functional similarity of GluD1 and GluD2. In contrast, GluD1 expression alone did not induce presynaptic differentiation in co-cultures of HEK cells with hippocampal neurons. However, when Cbln1 was exogenously added to the culture medium, GluD1 induced presynaptic differentiation of not only glutamatergic presynaptic terminals but also GABAergic ones. Cbln1 is not expressed in hippocampal neurons but is expressed in entorhinal cortical neurons projecting to the hippocampus. In co-cultures of HEK cells expressing GluD1 and entorhinal cortical neurons, both glutamatergic and GABAergic presynaptic terminals were formed on the HEK cells without exogenous application of Cbln1. These results suggest that GluD1 might contribute to the formation of specific synapses in the hippocampus such as those formed by the projecting neurons of the entorhinal cortex.

Glutamate dehydrogenase activity in lymphocytes of B-cell chronic lymphocytic leukaemia patients

Objectives: To investigate the pattern of glutamate dehydrogenase (GLDH) activity, GLUD1 and GLUD2 expressions in peripheral blood mononuclear cells (PBMC) of untreated B-chronic lymphocytic leukemia (B-CLL) in healthy individuals (HI) and patients with infectious mononucleosis (IM). Design and methods: GLDH activity was determined in a supernatant obtained from pelleted PBMC. GLUD1 and GLUD2 mRNA expression was determined using a quantitative real-time polymerase chain reaction. CD19 + B cells from PBMC were purified by using positive selection. Results: The highest GLDH activity was found in PBMC of the B-CLL group followed by the HI group and IM group. The PBMC GLDH activity was higher in 60% of the B-CLL patients according to the established reference interval for our HI (2.17–5.70 μkat/g protein). The greater GLDH activity was also found in the CD19 + cell preparation of the B-CLL patients (two of the three) but not in HI ( n = 3). The median value of GLUD1 expression was highest in the IM group ( n = 11), followed by the HI ( n = 14) and B-CLL groups ( n = 59) (median 4.69/3.78, P < 0.005 and 4.69/2.91, P < 0.0005, respectively). GLUD2 expression was not significantly different between groups. Conclusions: The increased GLDH activity is specific for the PBMC of B-CLL patients. The GLUD1 but not the GLUD2 gene expression pattern is different between the PBMC of IM and B-CLL patients.