Downregulation of Ambra1 by altered DNA methylation exacerbates dopaminergic neuron damage in a fenpropathrin-induced Parkinson-like mouse model.

Fenpropathrin (Fen), a volatile pyrethroid insecticide, is used widely for agricultural applications and has been reported to increase the risk of Parkinson's disease (PD). However, the molecular basis, underlying mechanisms, and pathophysiology of Fen-exposed Parkinsonism remain unknown. Recent studies have revealed epigenetic mechanisms underlying PD-related pathway regulation, including DNA methylation. Epigenetic mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. After whole-genome bisulfite sequencing (WGBS) of midbrain tissues from a Fen-exposed PD-like mouse model, we performed an association analysis of DNA methylation and gene expression. Then we successfully screened for the DNA methylation differential gene Ambra1, which is closely related to PD. The hypermethylation-low expression Ambra1 gene aggravated DA neuron damage in vitro and in vivo through the Ambra1/Parkin/LC3B-mediated mitophagy pathway. We administered 5-aza-2'-deoxycytidine (5-Aza-dC) to upregulate Ambra1 expression, thereby reducing Ambra1-mediated mitophagy and protecting DA neurons against Fen-induced damage. In conclusion, these findings elucidate the potential function of Ambra1 under the regulation of DNA methylation, suggesting that the inhibition of DNA methylation may alleviate Fen-exposed neuron damage.

Cath-KP, a novel peptide derived from frog skin, prevents oxidative stress damage in a Parkinson's disease model.

Parkinson's disease (PD) is a neurodegenerative condition that results in dyskinesia, with oxidative stress playing a pivotal role in its progression. Antioxidant peptides may thus present therapeutic potential for PD. In this study, a novel cathelicidin peptide (Cath-KP; GCSGRFCNLFNNRRPGRLTLIHRPGGDKRTSTGLIYV) was identified from the skin of the Asiatic painted frog ( Kaloula pulchra). Structural analysis using circular dichroism and homology modeling revealed a unique αßß conformation for Cath-KP. In vitro experiments, including free radical scavenging and ferric-reducing antioxidant analyses, confirmed its antioxidant properties. Using the 1-methyl-4-phenylpyridinium ion (MPP +)-induced dopamine cell line and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice, Cath-KP was found to penetrate cells and reach deep brain tissues, resulting in improved MPP +-induced cell viability and reduced oxidative stress-induced damage by promoting antioxidant enzyme expression and alleviating mitochondrial and intracellular reactive oxygen species accumulation through Sirtuin-1 (Sirt1)/Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway activation. Both focal adhesion kinase (FAK) and p38 were also identified as regulatory elements. In the MPTP-induced PD mice, Cath-KP administration increased the number of tyrosine hydroxylase (TH)-positive neurons, restored TH content, and ameliorated dyskinesia. To the best of our knowledge, this study is the first to report on a cathelicidin peptide demonstrating potent antioxidant and neuroprotective properties in a PD model by targeting oxidative stress. These findings expand the known functions of cathelicidins, and hold promise for the development of therapeutic agents for PD.

miR-100a-5p-enriched exosomes derived from mesenchymal stem cells enhance the anti-oxidant effect in a Parkinson's disease model via regulation of Nox4/ROS/Nrf2 signaling.

BACKGROUND: The pathogenesis of Parkinson's disease (PD) has not been fully elucidated, and there are no effective disease-modifying drugs for the treatment of PD. Mesenchymal stem cells have been used to treat several diseases, but are not readily available. METHODS: Here, we used phenotypically uniform trophoblast stage-derived mesenchymal stem cells (T-MSCs) from embryonic stem cells, which are capable of stable production, and their exosomes (T-MSCs-Exo) to explore the molecular mechanisms involved in dopaminergic (DA) neuron protection in PD models using experimental assays (e.g., western blotting, immunofluorescence and immunohistochemistry staining). RESULTS: We assessed the levels of DA neuron injury and oxidative stress in MPTP-induced PD mice and MPP+-induced MN9D cells after treating them with T-MSCs or T-MSCs-Exo. Furthermore, T-MSCs-Exo miRNA sequencing analysis revealed that miR-100-5p-enriched T-MSCs-Exo directly targeted the 3' UTR of NOX4, which could protect against the loss of DA neurons, maintain nigro-striatal system function, ameliorate motor deficits, and reduce oxidative stress via the Nox4-ROS-Nrf2 axis in PD models. CONCLUSIONS: The study suggests that miR-100-5p-enriched T-MSCs-Exo may be a promising biological agent for the treatment of PD. Schematic summary of the mechanism underlying the neuroprotective actions of T-MSCs-Exo in PD. T-MSCs Exo may inhibit the expression level of the target gene NOX4 by delivering miR-100-5p, thereby reducing ROS production and alleviating oxidative stress via the Nox4-ROS-Nrf2 axis, thus improving DA neuron damage in PD.

Engineered Selenium/Human Serum Albumin Nanoparticles for Efficient Targeted Treatment of Parkinson's Disease via Oral Gavage.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopamine (DA) neurons in the midbrain substantia nigra pars compacta (SNpc). While existing therapeutic strategies can alleviate PD symptoms, they cannot inhibit DA neuron loss. Herein, a tailor-made human serum albumin (HSA)-based selenium nanosystem (HSA/Se nanoparticles, HSA/Se NPs) to treat PD that can overcome the intestinal epithelial barrier (IEB) and blood-brain barrier (BBB) is described. HSA, a transporter for drug delivery, has superior biological characteristics that make it an ideal potential drug delivery substance. Findings reveal that HSA/Se NPs have lower toxicity and higher efficacy than other selenium species and the ability to overcome the IEB and BBB to enrich DA neurons, which then protect MN9D cells from MPP+-induced neurotoxicity and ameliorate both behavioral deficits and DA neuronal death in MPTP-model mice. Thus, a therapeutic drug delivery system composed of orally gavaged HSA/Se NPs for the treatment of PD is described.

Fenpropathrin Induces GLT-1 Ubiquitination and Increases IL-6 Secretion through the Mdm2-p53 Pathway.

Human exposure to fenpropathrin, a widely used pesticide, is linked to Parkinson's-like symptoms in the body. However, a specific pathogenic mechanism is still unclear. This study found that fenpropathrin increased the expression of murine double minute 2 (Mdm2) and reduced the expression of p53. Fenpropathrin stimulated the expression of neural precursor cell expressed, developmentally down-regulated 4-like (Nedd4L) and promoted the secretion of the inflammatory cytokine interleukin-6 (IL-6) through the Mdm2-p53 pathway. Nedd4L, a ubiquitin ligase, mediated the ubiquitination degradation of glutamate transporter 1 (GLT-1), resulting in glutamate accumulation and excitotoxicity aggravation. Our findings elucidate part of the pathogenic mechanism of fenpropathrin toxicity and provide scientific evidence to help develop guidance for pesticide control and environmental protection.

Functional investigation of SLC1A2 variants associated with epilepsy.

Epilepsy is a common neurological disorder and glutamate excitotoxicity plays a key role in epileptic pathogenesis. Astrocytic glutamate transporter GLT-1 is responsible for preventing excitotoxicity via clearing extracellular accumulated glutamate. Previously, three variants (G82R, L85P, and P289R) in SLC1A2 (encoding GLT-1) have been clinically reported to be associated with epilepsy. However, the functional validation and underlying mechanism of these GLT-1 variants in epilepsy remain undetermined. In this study, we reported that these disease-linked mutants significantly decrease glutamate uptake, cell membrane expression of the glutamate transporter, and glutamate-elicited current. Additionally, we found that these variants may disturbed stromal-interacting molecule 1 (STIM1)/Orai1-mediated store-operated Ca2+ entry (SOCE) machinery in the endoplasmic reticulum (ER), in which GLT-1 may be a new partner of SOCE. Furthermore, knock-in mice with disease-associated variants showed a hyperactive phenotype accompanied by reduced glutamate transporter expression. Therefore, GLT-1 is a promising and reliable therapeutic target for epilepsy interventions.

Baicalin Induces a Potent Innate Immune Response to Inhibit Respiratory Syncytial Virus Replication via Regulating Viral Non-Structural 1 and Matrix RNA.

Respiratory syncytial virus (RSV) infection is the most frequent cause of hospitalization in pediatric patients. Current systemic treatment and vaccines are not curative and re-infection is often associated with a more drastic incidence of the disease. Baicalin is a flavonoid isolated from Scutellaria baicalensis with potent anti-viral characteristics, namely against RSV. However, its precise mechanism of action remains unclear. Here, using in vitro methods and an in vivo murine model of RSV infection, we showed that baicalin inhibits RSV replication induces translational upregulation of type I interferons (IFNs), IFN-α and IFN-ß, and reverses epithelial thickening in lung tissues. Moreover, baicalin inhibits transcription of the RSV non-structural proteins NS1 and NS2. Molecular docking and surface plasmon resonance-based affinity analysis showed that baicalin also binds to the α3 helix of the NS1 protein with an affinity constant of 1.119 × 10-5 M. Polysome profiling showed that baicalin inhibits translation of the RSV matrix protein (M) RNA. Baicalin mediates increased release of the ribosomal protein L13a from the large ribosomal subunit, where the extra ribosomal subunit L13a inhibits M RNA translation. These results comprehensively establish the multiple mechanisms by which baicalin induces a potent innate immune response against RSV infection.

An anti-inflammatory and anti-fibrotic proprietary Chinese medicine nasal spray designated as Allergic Rhinitis Nose Drops (ARND) with potential to prevent SARS-CoV-2 coronavirus infection by targeting RBD (Delta)- angiotensin converting enzyme 2 (ACE2) binding.

BACKGROUND: Since the outbreak of COVID-19 has resulted in over 313,000,000 confirmed cases of infection and over 5,500,000 deaths, substantial research work has been conducted to discover agents/ vaccines against COVID-19. Undesired adverse effects were observed in clinical practice and common vaccines do not protect the nasal tissue. An increasing volume of direct evidence based on clinical studies of traditional Chinese medicines (TCM) in the treatment of COVID-19 has been reported. However, the safe anti-inflammatory and anti-fibrotic proprietary Chinese medicines nasal spray, designated as Allergic Rhinitis Nose Drops (ARND), and its potential of re-purposing for suppressing viral infection via SARS-CoV-2 RBD (Delta)- angiotensin converting enzyme 2 (ACE2) binding have not been elucidated. PURPOSE: To characterize ARND as a potential SARS-CoV-2 entry inhibitor for its possible preventive application in anti-virus hygienic agent. METHODS: Network pharmacology analysis of ARND was adopted to asacertain gene targets which were commonly affected by COVID-19. The inhibitory effect of ARND on viral infection was determined by an in vitro pseudovirus assay. Furthermore, ARND was confirmed to have a strong binding affinity with ACE2 and SARS-CoV-2 spike-RBD (Delta) by ELISA. Finally, inflammatory and fibrotic cell models were used in conjunction in this study. RESULTS: The results suggested ARND not only inhibited pseudovirus infection and undermined the binding affinity between ACE2 and the Spike protein (Delta), but also attenuated the inflammatory response upon infection and may lead to a better prognosis with a lower risk of pulmonary fibrosis. The data in this study also provide a basis for further development of ARND as an antiviral hygienic product and further investigations on ARND in the live virus, in vivo and COVID-19 patients. ARND holds promise for use in the current COVID-19 outbreak as well as in future pandemics. CONCLUSION: ARND could be considered as a safe anti-SARS-CoV-2 agent with potential to prevent SARS-CoV-2 coronavirus infection.

A Comparison of Epileptogenic Effect of Status Epilepticus Treated With Diazepam, Midazolam, and Pentobarbital in the Mouse Pilocarpine Model of Epilepsy.

Status epilepticus (SE) is a medical emergency associated with acute severe systemic damage and high mortality. Moreover, symptomatic SE is one of the highest risk factors for epileptogenesis. While the antiepileptic drugs (AEDs) are chosen in favor of acute control of SE, the potential short-term and long-term effects of such AEDs have been ignored in clinics. In this study, we hypothesized that AEDs that are used to control acute SE might affect the feasibility for the chronic development of epileptogenesis after SE. Therefore, we sought to compare the epileptogenic effects of SE that are terminated by three AEDs, i.e., diazepam, midazolam, and pentobarbital, which are widely used as first-line anti-SE AEDs. For this purpose, we used a mouse model of SE induced by intraperitoneal (i.p.) injection of lithium chloride (LiCl)-pilocarpine. The pilocarpine-induced SE was terminated with diazepam, midazolam, or pentobarbital. Then we compared short-term and long-term effects of SE with different AED treatments by examining SE-associated mortality and behavioral spontaneous recurrent seizures (SRSs) and by using magnetic resonance imaging (MRI) and immunohistochemistry to evaluate pathological and cellular alterations of mice in the different treatment groups. We found that i.p. injections of diazepam (5 mg/kg), midazolam (10 mg/kg), and pentobarbital (37.5 mg/kg) were able to terminate acute pilocarpine-SE effectively, while pentobarbital treatment showed less neuroprotective action against lethality in the short phase following SE. Long-term evaluation following SE revealed that SE treated with midazolam had resulted in relatively less behavioral SRS, less hippocampal atrophy, and milder neuronal loss and gliosis. Our data revealed an obvious advantage of midazolam vs. diazepam or pentobarbital in protecting the brain from epileptogenesis. Therefore, if midazolam provides as strong action to quench SE as other AEDs in clinics, midazolam should be the first choice of anti-SE AEDs as it provides additional benefits against epileptogenesis.

Overexpression of miR-132-3p contributes to neuronal protection in in vitro and in vivo models of Alzheimer's disease.

One of the neuropathological hallmarks of Alzheimer's disease (AD) is accumulation and deposition of amyloid-beta (Aß1-42) plaques in the hippocampus. Recently, microRNAs (miRNAs), have been demonstrated to play an essential role in AD. We have previously demonstrated that miR-132-3p exerts neuroprotection via regulating histone deacetylase 3 (HDAC3) in a mouse model of AD. In the present study, we further unveiled neuroprotective roles of miR-132-3p in transgenic amyloid precursor protein/presenilin 1 (APP/PS1) mice compared with those in age-matched wild-type C57BL/6 mice. Lentiviral-mediated inhibition or overexpression of miR-132-3p in the hippocampus of APP/PS1 mice was used to explore the contributions of hippocampal miR-132-3p in spatial memory, amyloid burden, apoptosis, and the number of hippocampal cells in a mouse model of AD. Overexpression of hippocampal miR-132-3p ameliorated spatial memory deficits in the Morris water maze, reduced both Aß1-42 accumulation and apoptosis, and promoted the numbers of hippocampal cells in the brains of APP/PS1 mice. Furthermore, trichostatin A (TSA) promoted the expression of miR-132-3p in Aß1-42-burdened neurons while increasing the expression levels of synaptic proteins. Taken together, our results suggest that miR-132-3p may represent a promising therapeutic target for the treatment of AD.

The Conformationally Sensitive Spatial Distance Between the TM3-4 Loop and Transmembrane Segment 7 in the Glutamate Transporter Revealed by Paired-Cysteine Mutagenesis.

Excitatory amino acid transporters can maintain extracellular glutamate concentrations lower than neurotoxic levels by transferring neurotransmitters from the synaptic cleft into surrounding glial cells and neurons. Previous work regarding the structural studies of Glt Ph , Glt TK , excitatory amino acid transporter 1 (EAAT1), EAAT3 and alanine serine cysteine transporter 2 described the transport mechanism of the glutamate transporter in depth. However, much remains unknown about the role of the loop between transmembrane segment 3 and 4 during transport. To probe the function of this loop in the transport cycle, we engineered a pair of cysteine residues between the TM3-TM4 loop and TM7 in cysteine-less EAAT2. Here, we show that the oxidative cross-linking reagent CuPh inhibits transport activity of the paired mutant L149C/M414C, whereas DTT inhibits the effect of CuPh on transport activity of L149C/M414C. Additionally, we show that the effect of cross-linking in the mutant is due to the formation of the disulfide bond within the molecules of EAAT2. Further, L-glutamate or KCl protect, and D,L-threo-ß-benzyloxy-aspartate (TBOA) increases, CuPh-induced inhibition in the L149C/M414 mutant, suggesting that the L149C and M414C cysteines are closer or farther away in the outward- or inward-facing conformations, respectively. Together, our findings provide evidence that the distance between TM3-TM4 loop and TM7 alter when substrates are transported.

Protective Effect of An-Gong-Niu-Huang Wan Pre-treatment Against Experimental Cerebral Ischemia Injury via Regulating GSK-3ß/HO-1 Pathway.

An-Gong-Niu-Huang Wan (AGNHW), a famous formula in traditional Chinese medicine, has been clinically used for centuries for treating cerebral diseases, but the protective effects of pre-treatment with AGNHW on cerebral ischemia have not yet been reported. The present study aimed to test such protective effects and elucidate the underlying mechanisms on cerebral ischemia in rats by phenotypic approaches (i.e. including the neurological functional score, cerebral infarct area, neuron apoptosis, and brain oxidative stress status) and target-based approaches (i.e. involving the GSK-3ß/HO-1 pathway). AGNHW was administered orally at the doses of 386.26, 772.52, and 1545.04 mg/kg respectively for 7 days to male Sprague-Dawley rats and then cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 1.5 h. Pre-treatment with AGNHW significantly ameliorated ischemic damage to the brain in a dose-dependent manner, including reduction of the neurological deficit score and infarct area. AGNHW pre-treatment increased the number of Nissl+ cells, NeuN+ and DCX+ cells, and decreased the number of Tunel+ cells. Moreover, AGNHW reversed the up-regulation of ROS and MDA induced by cerebral ischemia. AGNHW pre-treatment increased the expression of p-GSK-3ß(Ser9)/GSK-3ß (glycogen synthase kinase-3ß) ratio and heme oxygenase-1 (HO-1). These results firstly revealed that short-term pre-treatment of AGNHW could significantly protect the rats from injury caused by cerebral ischemia-reperfusion, which support further clinical studies for disease prevention. The in vivo protective effect of AGNWH pre-treatment could be associated with its antioxidant properties by the activation of GSK-3ß-mediated HO-1 pathway.

MiR-30a-5p Regulates GLT-1 Function via a PKCα-Mediated Ubiquitin Degradation Pathway in a Mouse Model of Parkinson's Disease.

Glutamate excitotoxicity is caused by dysfunctional glutamate transporters and plays an important role in the pathogenesis of Parkinson's disease (PD); however, the mechanisms that underlie the regulation of glutamate transporters in PD are still not fully elucidated. MicroRNAs(miRNA), which are abundant in astrocytes and neurons, have been reported to play key roles in regulating the translation of glutamate-transporter mRNA. In this study, we hypothesized that the miR-30a-5p contributes to the pathogenesis of PD by regulating the ubiquitin-mediated degradation of glutamate transporter 1 (GLT-1). We demonstrated that short-hairpin RNA-mediated knockdown of miR-30a-5p ameliorated motor deficits and pathological changes like astrogliosis and reactive microgliosis in a mouse model of PD (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice). Western blotting and immunofluorescent labeling revealed that miR-30a-5p suppressed the expression and function of GLT-1 in MPTP-treated mice and specifically in astrocytes treated with 1-methyl-4-phenylpyridinium (MPP+) (cell model of PD). Both in vitro and in vivo, we found that miR-30a-5p knockdown promoted glutamate uptake and increased GLT-1 expression by hindering GLT-1 ubiquitination and subsequent degradation in a PKCα-dependent manner. Therefore, we conclude that miR-30a-5p represents a potential therapeutic target for the treatment of PD.

Effects of α-Synuclein-Associated Post-Translational Modifications in Parkinson's Disease.

α-Synuclein (α-syn), a small highly conserved presynaptic protein containing 140 amino acids, is thought to be the main pathological hallmark in related neurodegenerative disorders. Although the normal function of α-syn is closely involved in the regulation of vesicular neurotransmission in these diseases, the underlying mechanisms of post-translational modifications (PTMs) of α-syn in the pathogenesis of Parkinson's disease (PD) have not been fully characterized. The pathological accumulation of misfolded α-syn has a critical role in PD pathogenesis. Recent studies of factors contributing to α-syn-associated aggregation and misfolding have expanded our understanding of the PD disease process. In this Review, we summarize the structure and physiological function of α-syn, and we further highlight the major PTMs (namely phosphorylation, ubiquitination, nitration, acetylation, truncation, SUMOylation, and O-GlcNAcylation) of α-syn and the effects of these modifications on α-syn aggregation, which may elucidate mechanisms for PD pathogenesis and lay a theoretical foundation for clinical treatment of PD.

Role of autophagy and oxidative stress to astrocytes in fenpropathrin-induced Parkinson-like damage.

Fenpropathrin is an insecticide that is widely used in agriculture. It remains unknown whether fenpropathrin exposure increases the risk of Parkinson's disease. We found that fenpropathrin increased oxidative stress both in vitro and in vivo. Additionally, fenpropathrin increased production of ROS, NOS2, and HO-1, and decreased SOD and GSH in astrocytes. We further found that fenpropathrin-mediated oxidative stress might inhibit autophagic flow, including decreased expression of LC3A/B and enhanced expression of SQSTM1 via down-regulation of CDK-5, an upstream marker of autophagy. In mice, autophagy was slightly different from that found in astrocytes, as reflected in the increased expressions of LC3A/B and SQSTM1. Our findings elucidate the toxicological phenomena and pathogenic mechanisms of fenpropathrin and may provide guidance for improved pesticide control and environmental protection.

Critical amino acids in the TM2 of EAAT2 are essential for membrane-bound localization, substrate binding, transporter function and anion currents.

Excitatory amino acid transporter 2 (EAAT2), the gene of which is known as solute carrier family 1 member 2 (SLC1A2), is an important membrane-bound transporter that mediates approximately 90% of the transport and clearance of l-glutamate at synapses in the central nervous system (CNS). Transmembrane domain 2 (TM2) of EAAT2 is close to hairpin loop 2 (HP2) and far away from HP1 in the inward-facing conformation. In the present study, 14 crucial amino acid residues of TM2 were identified via alanine-scanning mutations. Further analysis in EAAT2-transfected HeLa cells in vitro showed that alanine substitutions of these residues resulted in a decrease in the efficiency of trafficking/targeting to the plasma membrane and/or reduced functionality of membrane-bound, which resulted in impaired transporter activity. After additional mutations, the transporter activities of some alanine-substitution mutants recovered. Specifically, the P95A mutant decreased EAAT2-associated anion currents. The Michaelis constant (Km ) values of the mutant proteins L85A, L92A and L101A were increased significantly, whereas R87 and P95A were decreased significantly, indicating that the mutations L85A, L92A and L101A reduced the affinity of the transporter and the substrate, whereas R87A and P95A enhanced this affinity. The maximum velocity (Vmax) values of all 14 alanine mutant proteins were decreased significantly, indicating that all these mutations reduced the substrate transport rate. These results suggest that critical residues in TM2 affect not only the protein expression and membrane-bound localization of EAAT2, but also its interactions with substrates. Additionally, our findings elucidate that the P95A mutant decreased EAAT2-related anion currents. Our results indicate that the TM2 of EAAT2 plays a vital role in the transport process. The key residues in TM2 affect protein expression in the membrane, substrate transport and the anion currents of EAAT2.

Conformationally Sensitive Proximity Between the TM3-4 Loop and Hairpin Loop 2 of the Glutamate Transporter EAAT2 Revealed by Paired-Cysteine Mutagenesis.

Excitatory amino acid transporters (EAATs) serve to maintain extracellular neurotransmitter concentrations below neurotoxic levels by transporting glutamate from the synaptic cleft into apposed glia and neurons. Although the crystal structures of the archaeal EAAT homologue from Pyrococcus horikoshii, GltPh, and the human glutamate transporter, EAAT1cryst, have been resolved, the transport mechanism of the transmembrane 3-4 (TM3-4) loop and its structural rearrangement during transport have remained poorly understood. In order to explore the spatial position and function of the TM3-4 loop in the transport cycle, we engineered a pair of cysteine residues between the TM3-4 loop and hairpin loop 2 (HP2) in cysteine-less EAAT2 (CL-EAAT2). We observed that the oxidative cross-linking reagent Cu(II)(1,10-phenanthroline)3 (CuPh) had a significant inhibitory effect on transport in the disubstituted A167C/G437C mutant, whereas dl-dithiothreitol (DTT) reversed the effect of cross-linking A167C/G437C on transport activity, as assayed by d-[3H]-aspartate uptake. Furthermore, we found that the effect of CuPh in this mutant was due to the formation of disulfide bonds in the transporter molecule. Moreover, dl-threo-ß-benzyloxyaspartic acid (TBOA) attenuated, while l-glutamate or KCl enhanced, the CuPh-mediated inhibitory effect in the A167C/G437C mutant, suggesting that the A167C and G437C cysteines were farther apart in the outward-facing configuration and closer in the inward-facing configuration. Taken together, our findings provide evidence that the TM3-4 loop and HP2 change spatial proximity during the transport cycle.

Molecular Mechanisms of Glutamate Toxicity in Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disease, the pathological features of which include the presence of Lewy bodies and the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta. However, until recently, research on the pathogenesis and treatment of PD have progressed slowly. Glutamate and dopamine are both important central neurotransmitters in mammals. A lack of enzymatic decomposition of extracellular glutamate results in glutamate accumulating at synapses, which is mainly absorbed by excitatory amino acid transporters (EAATs). Glutamate exerts its physiological effects by binding to and activating ligand-gated ion channels [ionotropic glutamate receptors (iGluRs)] and a class of G-protein-coupled receptors [metabotropic glutamate receptors (mGluRs)]. Timely clearance of glutamate from the synaptic cleft is necessary because high levels of extracellular glutamate overactivate glutamate receptors, resulting in excitotoxic effects in the central nervous system. Additionally, increased concentrations of extracellular glutamate inhibit cystine uptake, leading to glutathione depletion and oxidative glutamate toxicity. Studies have shown that oxidative glutamate toxicity in neurons lacking functional N-methyl-D-aspartate (NMDA) receptors may represent a component of the cellular death pathway induced by excitotoxicity. The association between inflammation and excitotoxicity (i.e., immunoexcitotoxicity) has received increased attention in recent years. Glial activation induces neuroinflammation and can stimulate excessive release of glutamate, which can induce excitotoxicity and, additionally, further exacerbate neuroinflammation. Glutamate, as an important central neurotransmitter, is closely related to the occurrence and development of PD. In this review, we discuss recent progress on elucidating glutamate as a relevant neurotransmitter in PD. Additionally, we summarize the relationship and commonality among glutamate excitotoxicity, oxidative toxicity, and immunoexcitotoxicity in order to posit a holistic view and molecular mechanism of glutamate toxicity in PD.

Pretreatment of Ascorbic Acid Inhibits MPTP-Induced Astrocytic Oxidative Stress through Suppressing NF-κB Signaling.

Astrocytes are a major constituent of the central nervous system (CNS). Astrocytic oxidative stress contributes to the development of Parkinson's disease (PD). Maintaining production of antioxidant and detoxification of reactive oxygen and nitrogen species (ROS/RNS) in astrocytes is critical to prevent PD. Study has illuminated that ascorbic acid (AA) stimulates dopamine synthesis and expression of tyrosine hydroxylase in human neuroblastoma cells. However, the role and regulatory mechanisms of AA on detoxification of astrocytes are still unclear. The purpose of our study is in-depth study of the regulatory mechanism of AA on detoxification of astrocytes. We found that AA pretreatment decreased the expression of ROS and inducible nitric oxide synthase (iNOS) in MPP+-treated astrocytes. In contrast, the expression levels of antioxidative substances-including superoxide dismutase (SOD), glutathione (GSH), and glutamate-cysteine ligase modifier (GCLM) subunit-were upregulated after AA pretreatment in MPP+-treated astrocytes. However, inhibition of NF-κB prevented such AA induced increases in antioxidative substances following MPP+ treatment in astrocytes, suggesting that AA improved antioxidative function of astrocytes through inhibiting NF-κB-mediated oxidative stress. Furthermore, in vivo studies revealed that AA preadministration also suppressed NF-κB and upregulated the expression levels of antioxidative substances in the midbrain of MPTP-treated mice. Additionally, pretreatment of AA alleviated MPTP-induced PD-like pathology in mice. Taken together, our results demonstrate that preadministration of AA improves the antioxidative function of astrocytes through suppressing NF-κB signaling, following alleviated the pathogenesis of PD which induced by MPTP. Hence, our findings elucidate a novel protective mechanism of AA in astrocytes.