Mechanistic study of the anti-excitatory amino acid toxicity of Bushen Zhichan decoction for Parkinson's disease based on the transcriptional regulation of EAAT1 by YY1.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen Zhichan decoction (BSZCF) is derived from Liuwei Dihuang Pill, a famous Chinese herbal formula recorded in the book Key to Therapeutics of Children's Diseases. It has been widely used as a basic prescription for nourishing and tonifying the liver and kidneys to treat Parkinson's disease (PD), but its mechanism remains to be explored. AIM OF THE STUDY: BSZCF, a Chinese herbal formula comprising five herbs: Rehmannia glutinosa (Gaertn.) DC., Dioscorea oppositifolia L., Cornus officinalis Siebold & Zucc., Fallopia multiflora (Thunb.) Haraldson and Cistanche tubulosa (Schenk) Wight, is used clinically to treat PD. In vivo and in vitro experiments were designed to elucidate the mechanism of BSZCF in the protection of dopamine (DA) neurons and the treatment of PD. The toxicity of excitatory amino acids (EAA) may be attenuated by inhibiting the transcription factor Yin Yang 1 (YY1) and up-regulating the expression of excitatory amino acid transporter 1 (EAAT1). MATERIALS AND METHODS: IN VIVO: After 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was intraperitoneally injected into specific pathogen free (SPF) C57BL/6J mice, model mice were intragastrically given adamantane hydrochloride tablets (AHT) or different doses of BSZCF for 14 days. Both open field and pole-climbing tests were conducted to assess behavioral changes. In vitro: 1-Methyl-4-phe-nylpyridiniumiodide (MPP+)-injured human neuroblastoma cells (SH-SY5Y) were utilized to construct PD cell models. Primary astrocytes were transfected with EAAT1 and YY1 lentiviruses for EAAT1 gene knockout and YY1 gene knockout astrocytes, respectively. The high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of BSZCF was performed to control the quality of blood drugs. The optimal concentration and time of PD cell models treated by BSZCF were determined by the use of Cell Counting Kit-8 (CCK8). Enzyme-linked immunosorbent assay (ELISA) was used for measuring glutamate (Glu) in the peripheral blood and cells of each group. Western blotting (WB) and real-time quantitative polymerase chain reaction (qPCR) were used to detect tyrosine hydroxylase (TH), dopamine transporters (DAT), EAAT1 and YY1 protein and mRNA. After the blockade of EAAT1, immunofluorescence (IF) assay was used to detect the TH protein in each group. RESULTS: In vivo research showed that BSZCF improved the behavioral symptoms of PD mice, and reduced the death of DA neurons and the level of Glu. The mechanism may be related to the decrease of YY1 expression and the increase of EAAT1 levels. In vitro experiments showed that the anti-excitatory amino acid toxicity of BSZCF was achieved by inhibiting YY1 expression and regulating EAAT1. CONCLUSIONS: By inhibiting YY1 to increase the expression of EAAT1 and attenuating the toxicity of Glu, BSZCF exerts the effect of protecting DA neurons and treating PD-like symptoms in mice.

Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6.

Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters GltTk. We show that also in GltTk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.

Yes-associated protein regulates glutamate homeostasis through promoting the expression of excitatory amino acid transporter-2 in astrocytes via ß-catenin signaling.

The homeostasis of glutamate is mainly regulated by the excitatory amino acid transporters (EAATs), especially by EAAT2 in astrocytes. Excessive glutamate in the synaptic cleft caused by dysfunction or dysregulation of EAAT2 can lead to excitotoxicity, neuronal death and cognitive dysfunction. However, it remains unclear about the detailed regulation mechanism of expression and function of astrocytic EAAT2. In this study, first, we found increased neuronal death and impairment of cognitive function in YAPGFAP -CKO mice (conditionally knock out Yes-associated protein [YAP] in astrocytes), and identified EAAT2 as a downstream target of YAP through RNA sequencing. Second, the expression of EAAT2 was decreased in cultured YAP-/- astrocytes and the hippocampus of YAPGFAP -CKO mice, and glutamate uptake was reduced in YAP-/- astrocytes, but increased in YAP-upregulated astrocytes. Third, further investigation of the mechanism showed that the mRNA and protein levels of ß-catenin were decreased in YAP-/- astrocytes and increased in YAP-upregulated astrocytes. Wnt3a activated YAP signaling and up-regulated EAAT2 through ß-catenin. Furthermore, over-expression or activation of ß-catenin partially restored the downregulation of EAAT2, the impairment of glutamate uptake, neuronal death and cognitive decline that caused by YAP deletion. Finally, activation of EAAT2 also rescued neuronal death and cognitive decline in YAPGFAP -CKO mice. Taken together, our study identifies an unrecognized role of YAP signaling in the regulation of glutamate homeostasis through the ß-catenin/EAAT2 pathway in astrocytes, which may provide novel insights into the pathogenesis of brain diseases that closely related to the dysfunction or dysregulation of EAAT2, and promote the development of clinical strategy.

Selective Clearance of Senescent Chondrocytes in Osteoarthritis by Targeting Excitatory Amino Acid Transporter Protein 1 to Induce Ferroptosis.

Aims: This study aimed at exploring the mechanism of ferroptosis (an iron-dependent form of nonapoptotic cell death) resistance in senescent chondrocytes (SenChos). Results: In this study, by utilizing metabolomics and single-cell RNA sequencing, we found that hyperactivation of ferroptosis metabolism was one of the most prominent metabolic features in SenChos. Interestingly, however, SenChos were able to survive in this state and were resistant to ferroptosis-induced cell death. Next, we elucidated that this survival mechanism of SenChos could be primarily attributed to overexpression of the membrane protein excitatory amino acid transporter protein 1 (EAAT1), which can increase intracellular glutamate (Glu) levels and activate the glutathione system to counteract ferroptosis. In addition, 2-amino-5,6,7,8-tetrahydro-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-4H-chromene-3-carbonitrile (UCPH-101) (a specific inhibitor of EAAT1) and siRNA-EAAT1 were able to substantially increase the sensitivity of SenChos to ferroptosis and to induce cell death, with no apparent effects on the normal cells. Administration of an intraarticular injection of UCPH-101 caused inhibition of EAAT1 selectively, cleared SenChos from cartilage, improved the cartilage homeostasis, and significantly delayed the progression of osteoarthritis (OA). Innovation: This work supports a relevant role for EAAT1 in ferroptosis resistance mechanism for SenChos, revealing a potential therapeutic target of OA. Conclusions: EAAT1-Glu-glutathione peroxidase 4 anti-ferroptosis axis is a key survival mechanism for SenChos, and EAAT1 is an effective and specific target for anti-senescence therapy in OA. Antioxid. Redox Signal. 39, 262-277.

EAAT1-dependent slc1a3 Transcriptional Control depends on the Substrate Translocation Process.

SUMMARY STATEMENT: EAAT1/GLAST down-regulates its expression and function at the transcriptional level by activating a signaling pathway that includes PI3K, PKC and NF-κB, favoring the notion of an activity-dependent fine-tuning of glutamate recycling and its synaptic transactions through glial cells.

Glutamate Uptake Is Not Impaired by Hypoxia in a Culture Model of Human Fetal Neural Stem Cell-Derived Astrocytes.

Hypoxic ischemic injury to the fetal and neonatal brain is a leading cause of death and disability worldwide. Although animal and culture studies suggest that glutamate excitotoxicity is a primary contributor to neuronal death following hypoxia, the molecular mechanisms, and roles of various neural cells in the development of glutamate excitotoxicity in humans, is not fully understood. In this study, we developed a culture model of human fetal neural stem cell (FNSC)-derived astrocytes and examined their glutamate uptake in response to hypoxia. We isolated, established, and characterized cultures of FNSCs from aborted fetal brains and differentiated them into astrocytes, characterized by increased expression of the astrocyte markers glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 1 (EAAT1) and EAAT2, and decreased expression of neural stem cell marker Nestin. Differentiated astrocytes were exposed to various oxygen concentrations mimicking normoxia (20% and 6%), moderate and severe hypoxia (2% and 0.2%, respectively). Interestingly, no change was observed in the expression of the glutamate transporter EAAT2 or glutamate uptake by astrocytes, even after exposure to severe hypoxia for 48 h. These results together suggest that human FNSC-derived astrocytes can maintain glutamate uptake after hypoxic injury and thus provide evidence for the possible neuroprotective role of astrocytes in hypoxic conditions.

[Effects of GLAST gene knockout on phenotype and hearing in mice].

OBJECTIVE: To investigate the effects of glutamate aspartate transporter (GLAST)deletion on the normal auditory function of mice. METHODS: We hybridized GLAST+/- mice with C57BL/6J background and identified the genotypes of their offspring by agarose gel electrophoresis. 9-10-week-old mice were selected to detect the expression of GLAST protein in the cochlea by immunofluorescence staining and to verify the knockout results(n=3). The changes in weight from 7 days to 30 days after birth and the 30-day body length of male and female mice were compared(n=8). The auditory brainstem response(ABR) was used to detect the auditory threshold and the amplitude of wave I in 9-10-week-old male and female mice(n=5). RESULTS: Male GLAST-/- mice had shown significantly lower weight and body length compared to male GLAST+/+ and GLAST+/- mice(P＜0.01), and male GLAST-/- mice showed significant differences compared to GLAST+/+ from P7 to P30 statistical time. Male GLAST-/- mice exhibited a significant reduction in weight after P15 compared to male GLAST+/- mice. In contrast, no significant differences in weight and body length were observed in female GLAST-/- mice compared with female GLAST+/+ and GLAST+/- mice. There was no difference in the hearing threshold detected by ABR between the three genotypes in both male and female mice, but the amplitude of wave I in GLAST-/- mice was significantly lower than that in male GLAST+/+ mice(P＜0.01). In contrast, the amplitude of wave I in females was reduced throughout the stimulus intensity but was most significant only at high-intensity stimulation (e.g.80 dB, 90 dB) (P＜0.05). CONCLUSION: GLAST knockout affects the normal growth and development of male mice, and decreases the amplitude of wave I, but do not change the threshold, suggesting that GLAST knockout may lead to synaptic pathological changes, and there are gender differences in this effect.

Excitatory Amino Acid Transporter EAAT5 Improves Temporal Resolution in the Retina.

Excitatory amino acid transporters (EAATs) remove glutamate from the synaptic cleft. In the retina, EAAT1 and EAAT2 are considered the major glutamate transporters. However, it has not yet been possible to determine how EAAT5 shapes the retinal light responses because of the lack of a selective EAAT5 blocker or EAAT5 knock-out (KO) animal model. In this study, EAAT5 was found to be expressed in a punctate manner close to release sites of glutamatergic synapses in the mouse retina. Light responses from retinae of wild-type (WT) and of a newly generated model with a targeted deletion of EAAT5 (EAAT5-/-) were recorded in vitro using multielectrode arrays (MEAs). Flicker resolution was considerably lower in EAAT5-/- retinae than in WT retinae. The close proximity to the glutamate release site makes EAAT5 an ideal tool to improve temporal information processing in the retina by controlling information transfer at glutamatergic synapses.

Evidence for the interaction of COX-2 with mGluR5 in the regulation of EAAT1 and EAAT3 protein levels in the mouse hippocampus. The influence of oxidative stress mechanisms.

Since we found that inhibition of cyclooxygenase-2 (COX-2) with concomitant application of a metabotropic glutamate receptor subtype 5 (mGluR5) antagonist (MTEP) down-regulates mGluR7 in the hippocampus (HC) and changes behavior of mice, our team decided to investigate the mechanism responsible for the observed changes. The amino acid glutamate (Glu) is a major excitatory neurotransmitter in the brain. Glu uptake is regulated by excitatory amino acid transporters (EAAT). There are five transporters with documented expression in neurons and glia in the central nervous system (CNS). EAATs, maintain the correct transmission of the Glu signal and prevent its toxic accumulation by removing Glu from the synapse. It has been documented that the toxic level of Glu is one of the main causes of mental and cognitive abnormalities. Given the above mechanisms involved in the functioning of the Glu synapse, we hypothesized modification of Glu uptake, involving EAATs as the cause of the observed changes. This study investigated the level of selected EAATs in the HC after chronic treatment with mGluR5 antagonist MTEP, NS398, and their combination using Western blot. Concomitant MTEP treatment with NS398 or a single administration of the above causes changes in LTP and modulation of EAAT levels in mouse HC. As EAATs are cellular markers of oxidative stress mechanisms, the E. coli lipopolysaccharide (LPS) challenge was performed. The modified Barnes maze test (MBM) revealed alterations in the mouse spatial learning abilities. This study reports an interaction between the mGluR5 and COX-2 in the HC, with EAAT1 and EAAT3 involvement.

Deficiency in MT5-MMP Supports Branching of Human iPSCs-Derived Neurons and Reduces Expression of GLAST/S100 in iPSCs-Derived Astrocytes.

For some time, it has been accepted that the ß-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the ß-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer's disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100ß expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology.

Identification of 3'-UTR single nucleotide variants and prediction of select protein imbalance in mesial temporal lobe epilepsy patients.

The genetic influence in epilepsy, characterized by unprovoked and recurrent seizures, is through variants in genes critical to brain development and function. We have carried out variant calling in Mesial Temporal Lobe Epilepsy (MTLE) patients by mapping the RNA-Seq data available at SRA, NCBI, USA onto human genome assembly hg-19. We have identified 1,75,641 SNVs in patient samples. These SNVs are distributed over 14700 genes of which 655 are already known to be associated with epilepsy. Large number of variants occur in the 3'-UTR, which is one of the regions involved in the regulation of protein translation through binding of miRNAs and RNA-binding proteins (RBP). We have focused on studying the structure-function relationship of the 3'-UTR SNVs that are common to at-least 10 of the 35 patient samples. For the first time we find SNVs exclusively in the 3'-UTR of FGF12, FAR1, NAPB, SLC1A3, SLC12A6, GRIN2A, CACNB4 and FBXO28 genes. Structural modelling reveals that the variant 3'-UTR segments possess altered secondary and tertiary structures which could affect mRNA stability and binding of RBPs to form proper ribonucleoprotein (RNP) complexes. Secondly, these SNVs have either created or destroyed miRNA-binding sites, and molecular modeling reveals that, where binding sites are created, the additional miRNAs bind strongly to 3'-UTR of only variant mRNAs. These two factors affect protein production thereby creating an imbalance in the amounts of select proteins in the cell. We suggest that in the absence of missense and nonsense variants, protein-activity imbalances associated with MTLE patients can be caused through 3'-UTR variants in relevant genes by the mechanisms mentioned above. 3'-UTR SNV has already been identified as causative variant in the neurological disorder, Tourette syndrome. Inhibition of these miRNA-mRNA bindings could be a novel way of treating drug-resistant MTLE patients. We also suggest that joint occurrence of these SNVs could serve as markers for MTLE. We find, in the present study, SNV-mediated destruction of miRNA binding site in the 3'-UTR of the gene encoding glutamate receptor subunit, and, interestingly, overexpression of one of this receptor subunit is also associated with Febrile Seizures.

Auditory synaptopathy in mice lacking the glutamate transporter GLAST and its impact on brain activity.

Neurotransmission of acoustic signals from the hair cells to the auditory nerve relies on a tightly controlled communication between pre-synaptic ribbons and post-synaptic glutamatergic terminals. After noise overexposure, de-afferentation occurs as a consequence of excessive glutamate release. What maintains synaptic integrity in the cochlea is poorly understood. The objective of this study is to evaluate the role of GLAST in maintaining synaptic integrity in the cochlea in absence or presence of noise, and its impact on sound-evoked brain activity using manganese-enhanced MRI (MeMRI). The glutamate aspartate transporter GLAST is present in supporting cells near the afferent synapse and its genetic deletion leads to greater synaptic swelling after noise overexposure. At baseline, GLAST knockout (GLAST KO) mice displayed two-fold lower wave 1 amplitude of the auditory brainstem response (ABR) when compared to their wild-type littermates in spite of similar ABR and distortion product otoacoustic emissions (DPOAE) thresholds. While the abundance of ribbons was not affected by the loss of GLAST function, the number of paired synapses was halved in GLAST KO mice, suggestive of a pre-existing auditory synaptopathy. Immediately after the noise exposure ABR thresholds rose by 41-62dB to a similar degree in GLAST WT and KO mice and DPOAE remained unaffected. In the acute phase following noise exposure, GLAST KO mice showed near complete de-afferentation unlike WT mice which maintained four to seven paired synapses per IHC. Brain activity using MeMRI found noise exposure to cause greater activity in the inferior colliculus in GLAST KO but not in WT mice. No changes in brain activity was found in GLAST KO mice at baseline in spite of affected afferent synapses, suggesting that auditory synaptopathy may not be sufficient to alter brain activity in the absence of noise exposure.

Glutamate transporters: Critical components of glutamatergic transmission.

Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Once released, it binds to specific membrane receptors and transporters activating a wide variety of signal transduction cascades, as well as its removal from the synaptic cleft in order to avoid its extracellular accumulation and the overstimulation of extra-synaptic receptors that might result in neuronal death through a process known as excitotoxicity. Although neurodegenerative diseases are heterogenous in clinical phenotypes and genetic etiologies, a fundamental mechanism involved in neuronal degeneration is excitotoxicity. Glutamate homeostasis is critical for brain physiology and Glutamate transporters are key players in maintaining low extracellular Glutamate levels. Therefore, the characterization of Glutamate transporters has been an active area of glutamatergic research for the last 40 years. Transporter activity its regulated at different levels: transcriptional and translational control, transporter protein trafficking and membrane mobility, and through extensive post-translational modifications. The elucidation of these mechanisms has emerged as an important piece to shape our current understanding of glutamate actions in the nervous system.

IκB kinase inhibition remodeled connexins, pannexin-1, and excitatory amino-acid transporters expressions to promote neuroprotection of galantamine and morphine.

Inflammatory pathway and disruption in glutamate homeostasis join at the level of the glia, resulting in various neurological disorders. In vitro studies have provided evidence that membrane proteins connexions (Cxs) are involved in glutamate release, meanwhile, excitatory amino-acid transporters (EAATs) are crucial for glutamate reuptake (clearance). Moreover, pannexin-1 (Panx-1) activation is more detrimental to neurons. Their expression patterns during inflammation and the impacts of IκB kinase (IKK) inhibition, morphine, and galantamine on the inflammatory-associated glutamate imbalance remain elusive. To investigate this, rats were injected with saline or lipopolysaccharide. Thereafter, vehicles, morphine, galantamine, and BAY-117082 were administered in different groups of animals. Subsequently, electroencephalography, enzyme-linked immunosorbent assay, western blot, and histopathological examinations were carried out and various indicators of inflammation and glutamate level were determined. Parallel analysis of Cxs, Panx-1, and EAAts in the brain was performed. Our findings strengthen the concept that unregulated expressions of Cxs, Panx-1, and EAATs contribute to glutamate accumulation and neuronal cell loss. Nuclear factor-kB (NF-κB) pathway can significantly contribute to glutamate homeostasis via modulating Cxs, Panx-1, and EAATs expressions. BAY-117082, via inhibition of IkK, promoted the anti-inflammatory effects of morphine as well as galantamine. We concluded that NF-κB is an important component of reshaping the expressions of Cxs, panx-1, and EAATs and the development of glutamate-induced neuronal degeneration.

Chronic optogenetic stimulation of Bergman glia leads to dysfunction of EAAT1 and Purkinje cell death, mimicking the events caused by expression of pathogenic ataxin-1.

Bergmann glia (BG) are highly specialized radial astrocytes of the cerebellar cortex, which play a key role in the uptake of synaptic glutamate via the excitatory amino acid transporter EAAT1. Multiple lines of evidence suggest that in cerebellar neurodegenerative diseases reactive BG has a negative impact on neuronal function and survival through compromised EAAT activity. A family of such diseases are those caused by expansion of CAG repeats in genes of the ataxin family, resulting in spinocerebellar ataxias (SCA). We investigated the contribution of BG to the pathogenesis of cerebellar neurodegeneration in a model of SCA1, which was induced by expression of a polyglutamine mutant of ataxin-1 (ATXN1[Q85]) in BG specifically. We compared the outcomes with a novel model where we triggered excitotoxicity by a chronic optogenetic activation of BG with channelrhodopsin-2 (ChR2). In both cases we detected evidence of reduced glutamate uptake manifested by prolongation of excitatory postsynaptic currents in Purkinje cells which is consistent with documented reduction of expression and/or function of EAAT1. In both models we detected astroglyosis and Purkinje cells atrophy. Finally, the same pattern was detected in a knock-in mouse which expresses a polyglutamine mutant ataxin-1 ATXN1[Q154] in a non-cell-selective manner. Our results suggest that ATXN1[Q85] and ChR2-induced insult targeted to BG closely mimics SCA1 pathology, where excessive glutamate signaling appears to be a common feature likely being an important contributor to cerebellar neurodegeneration.

Slc1a3-2A-CreERT2 mice reveal unique features of Bergmann glia and augment a growing collection of Cre drivers and effectors in the 129S4 genetic background.

Genetic variation is a primary determinant of phenotypic diversity. In laboratory mice, genetic variation can be a serious experimental confounder, and thus minimized through inbreeding. However, generalizations of results obtained with inbred strains must be made with caution, especially when working with complex phenotypes and disease models. Here we compared behavioral characteristics of C57Bl/6-the strain most widely used in biomedical research-with those of 129S4. In contrast to 129S4, C57Bl/6 demonstrated high within-strain and intra-litter behavioral hyperactivity. Although high consistency would be advantageous, the majority of disease models and transgenic tools are in C57Bl/6. We recently established six Cre driver lines and two Cre effector lines in 129S4. To augment this collection, we genetically engineered a Cre line to study astrocytes in 129S4. It was validated with two Cre effector lines: calcium indicator gCaMP5g-tdTomato and RiboTag-a tool widely used to study cell type-specific translatomes. These reporters are in different genomic loci, and in both the Cre was functional and astrocyte-specific. We found that calcium signals lasted longer and had a higher amplitude in cortical compared to hippocampal astrocytes, genes linked to a single neurodegenerative disease have highly divergent expression patterns, and that ribosome proteins are non-uniformly expressed across brain regions and cell types.

Retinoprotective effect of agmatine in streptozotocin-induced diabetic rat model: avenues for vascular and neuronal protection : Agmatine in diabetic retinopathy.

Diabetic retinopathy (DR) is the most common diabetic neurovascular complication, and the leading cause of preventable blindness among working-age individuals. Recently, agmatine, the endogenous decarboxylated L-arginine, has gained attention as a pleiotropic agent that modulates the diabetes-associated decline in quality of life, and exhibited varied protective biological effects. Diabetes was induced by a single streptozotocin (STZ, 50 mg/kg, i.p.) injection. When diabetes was verified, the animals were randomly allocated into three groups (16 rat each); diabetic, agmatine-treated diabetic (1 mg/kg, daily, for 12 weeks), and control group. Blood glucose homeostasis, retinal redox status, apoptotic parameters, nitric oxide synthase (NOS), nitric oxide (NO), vascular endothelial growth factor (VEGF), glutamate, glutamine, glutamine synthase (GS) activity, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein kinase (MAPKs) pathways were assayed biochemically. Retinal vascular permeability was measured. Retinal morphology was evaluated by hematoxylin and eosin staining. Retinal N-methyl-D-aspartic acid receptor1 (NMDAR1) and glutamate aspartate transporter (GLAST) mRNA were quantified. Glucose transporter 1, pro-caspase3, and glial fibrillary acidic protein (GFAP) expression were quantified by immunohistochemistry. Chronic agmatine treatment abrogated STZ-induced retinal neurodegeneration features including gliosis, and neuronal apoptosis, restored retinal vascular permeability, mostly through antioxidant, anti-apoptotic capacity, abolishing glutamate excitotoxicity, modulating the activity of NMDARs, MAPKs/NFκB, and NOS/NO pathways. By restoring the molecular and functional background of retinal neurovascular homeostatic balance, agmatine would be appropriate therapeutic option acting upstream of the DR, impeding its progression.

Glutamate transporters have a chloride channel with two hydrophobic gates.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity1. The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2-5. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport6-8. However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family.

Hashimoto's Thyroiditis Induces Hippocampus-Dependent Cognitive Alterations by Impairing Astrocytes in Euthyroid Mice.

Background: Although studies have reported an increased risk for cognitive disorders in Hashimoto's thyroiditis (HT) patients, even in the euthyroid state, the mechanisms involved remain unclear. The hippocampus is a classic brain region associated with cognitive function, among which the formation of long-term potentiation (LTP) in the Schaffer collateral-CA1 pathway plays an important role in the process of learning and memory. Therefore, this study established a euthyroid HT model in mice and investigated whether and how HT itself has the ability to trigger LTP alterations accompanied by learning and memory abnormality. Methods: An experimental euthyroid HT model was established in NOD mice through immunization with porcine thyroglobulin (Tg). Morris water maze was measured to determine mice spatial learning and memory. We investigated the effect of HT on synaptic transmission and high-frequency stimulation-induced LTP in the Schaffer collateral-CA1 synapse of mice hippocampus in vivo. Then, animals were sacrificed for thyroid-related parameter measure as well as detection of cellular and molecular events associated with the induction of LTP. Results: HT mice showed intrathyroidal lymphocyte infiltration and rising serum thyroid autoantibody levels accompanied by normal thyroid function. The HT mice had poorer performance in Morris water maze than controls. These alterations were mirrored by abnormalities in synaptic plasticity in the Schaffer collateral-CA1 synapses of the hippocampus in vivo. The integrity of the synaptic structure is the premise for the production of LTP. As detected by transmission electron microscopy, the ultrastructure of synapse and astrocyte in the hippocampus were impaired in euthyroid HT mice. Additionally, Western blot and real-time polymerase chain reaction analyses confirmed that in HT mice, GS, GLAST, and GLT-1, key elements in glutamate-glutamine circulation located in astrocyte, were downregulated, accompanied by elevated levels of glutamate in the hippocampus, which impaired the material basis for LTP induction. NMDR2B expression in the hippocampus was also downregulated. Conclusion: HT can induce damage of LTP in the hippocampal Schaffer collateral-CA1 pathway in the euthyroid state, and this can be attributed, at least partly, to astrocytes impairment, which may underlie the deleterious effects of HT itself on hippocampal-dependent learning and memory function.

Research on the Relationship Between Schizophrenia and Excitatory Amino Acid Transporter 1 Gene Based on Nanogold Amplification Technology.

Schizophrenia is one of the most common central nervous system diseases, which is caused by abnormal discharge of neurons in the brain. Its occurrence and development are affected by both genetic and environmental factors. The variation of gene level can affect the development of schizophrenia and the treatment of prognosis by affecting the susceptibility, clinical phenotype and drug response. At present, the research results of susceptibility genes screened by candidate gene association research are not consistent. The method of gene recognition on DNA was studied by QCM and nano gold composite. By using this method, the enantioselective recognition of cysteine on cyclodextrin self-assembled membrane was studied. In this study, EAAT1 gene, which is highly expressed in astrocytes, was used as a candidate gene to analyze the relationship between polymorphism and schizophrenia. The experimental results show that the introduction of nano gold can significantly improve the sensing signal, detection sensitivity and gene differentiation. In addition, this study suggested that EAAT1 gene might be a susceptibility gene of schizophrenia in the population. The results showed that a common SNP allele rs1030239-g was the risk factor (83.8% vs. 79.2%, P = 0.00067, or = 1.35, 95% CI = 1.08-1.69). The results showed that A-T-G increased the risk of schizophrenia.