Glutamate transporters regulate synaptic concentrations of this neurotransmitter by coupling its flux to that of sodium and other cations. Available crystal structures of an archeal homologue of these transporters, GltPh, resemble an extracellular-facing state, in which the bound substrate is occluded only by a small helical hairpin segment called HP2. However, a pathway to the cytoplasmic side of the membrane is not clearly apparent. We previously modeled an alternate state of a transporter from the neurotransmitter:sodium symporter family, which has an entirely different fold, solely on the presence of inverted-topology structural repeats. In GltPh, we identified two distinct sets of inverted-topology repeats and used these repeats to model an inward-facing conformation of the protein. To test this model, we introduced pairs of cysteines into the neuronal glutamate transporter EAAC1, at positions that are >27 Å apart in the crystal structures of GltPh, but Ϸ10 Å apart in the inward-facing model. Transport by these mutants was activated by pretreatment with the reducing agent dithithreitol. Subsequent treatment with the oxidizing agent copper(II)(1,10-phenantroline) 3 abolished this activation. The inhibition of transport was potentiated under conditions thought to promote the inward-facing conformation of the transporter. By contrast, the inhibition was reduced in the presence of the nontransportable substrate analogue D,L-threo-␤-benzyloxyaspartate, which favors the outward-facing conformation. Other conformation-sensitive accessibility measurements are also accommodated by our inward-facing model. These results suggest that the inclusion of inverted-topology repeats in transporters may provide a general solution to the requirement for two symmetry-related states in a single protein.alternating access ͉ conformationally sensitive cross-linking ͉ homology modeling ͉ neurotransmitter ͉ secondary transport

Glutamate transporters play a key role in glutamate clearance and protect the central nervous system from glutamate excitotoxicity. Dysfunctional glutamate transporters contribute to the pathogenesis of Parkinson's disease (PD); however, the mechanisms that underlie the regulation of glutamate transporters in PD are still not well characterized. Here we report that Nedd4-2 mediates the ubiquitination of glutamate transporters in 1-methyl-4- phenylpyridinium (MPP+)-treated astrocytes and in the midbrain of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-constructed PD model mice. Nedd4-2-mediated ubiquitination induces abnormal glutamate transporter trafficking between the membrane and cytoplasm and consequently decreases the expression and function of glutamate transporters in the membrane. Conversely, Nedd4-2 knockdown decreases glutamate transporter ubiquitination, promotes glutamate uptake and increases glutamate transporter expression in vitro and in vivo. We report for the first time that Nedd4-2 knockdown ameliorates movement disorders in PD mice and increases tyrosine hydroxylase expression in the midbrain and striatum of PD mice; Nedd4-2 knockdown also attenuates astrogliosis and reactive microgliosis in the MPTP model that may be associated with glutamate excitotoxicity. Furthermore, the SGK/PKC pathway is regulated downstream of Nedd4-2 in MPTP-treated mice. These findings indicate that Nedd4-2 may serve as a potential therapeutic target for the treatment of PD.

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.

Rapamycin protects mice against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons, which is an established model for Parkinson's disease. We demonstrated that rapamycin preserves astrocytic expression of glutamate transporters and glutamate reuptake. The protective effect was also observed in astrocyte cultures, indicating that rapamycin acts directly on astrocytes. In the MPTP model, rapamycin caused reduced expression of the E3 ubiquitin ligase Nedd4-2 (neuronal precursor cell expressed developmentally downregulated 4-2) and reduced colocalization of glutamate transporters with ubiquitin. Rapamycin increased interleukin-6 (IL-6) expression, which was associated with reduced expression of inflammatory cytokines, indicating anti-inflammatory properties of IL-6 in the MPTP model. NF-κB was shown to be a key mediator for rapamycin, whereas Janus kinase 2, signal transducer and activator of transcription 3, phosphoinositide 3-kinase, and Akt partially mediated rapamycin effects in astrocytes. These results demonstrate for the first time in a Parkinson's disease animal model that the neuroprotective effects of rapamycin are associated with glial and anti-inflammatory effects.

Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. Their transport cycle consists of two separate translocation steps, namely cotransport of glutamic acid with three Na ؉ followed by countertransport of K ؉ . Two Tl ؉ binding sites, presumed to serve as sodium sites, were observed in the crystal structure of a related archeal homolog and the side chain of a conserved aspartate residue contributed to one of these sites. We have mutated the corresponding residue of the eukaryotic glutamate transporters GLT-1 and EAAC1 to asparagine, serine, and cysteine. Remarkably, these mutants exhibited significant sodium-dependent radioactive acidic amino acid uptake when expressed in HeLa cells. Reconstitution experiments revealed that net uptake by the mutants in K ؉ -loaded liposomes was impaired. However, with Na ؉ and unlabeled L-aspartate inside the liposomes, exchange levels were around 50 -90% of those by wild-type. In further contrast to wild-type, where either substrate or K ؉ stimulated the anion conductance by the transporter, substrate but not K ؉ modulated the anion conductance of the mutants expressed in oocytes. Both with wild-type EAAC1 and EAAC1-D455N, not only sodium but also lithium could support radioactive acidic amino acid uptake. In contrast, with D455S and D455C, radioactive uptake was only observed in the presence of sodium. Thus the conserved aspartate is required for transporter-cation interactions in each of the two separate translocation steps and likely participates in an overlapping sodium and potassium binding site.cation binding site ͉ obligate exchange mutant ͉ sodium selectivity G lutamate transporters are key elements in the termination of the synaptic actions of the neurotransmitter and keep its synaptic concentrations below neurotoxic levels. Glutamate transport is an electrogenic process (1, 2) consisting of two sequential translocation steps: (i) Cotransport of the neurotransmitter with three sodium ions and a proton (3, 4) and (ii) the countertransport of one potassium ion (5-7). The mechanism involving two half-cycles (Fig. 1A) is supported by the fact that mutants impaired in the interaction with potassium are ''locked'' in an obligatory exchange mode (7,8). Glutamate transporters mediate two distinct types of substrate-induced steady-state current: An inward-rectifying or ''coupled'' current, reflecting electrogenic sodium-coupled glutamate translocation, and an ''uncoupled'' sodium-dependent current, which is carried by chloride ions and further activated by substrates of the transporter (9-11). Nontransportable substrate analogs, expected to ''lock'' the transporter in an outward-facing conformation (stippled part of Fig. 1 A), are not only competitive inhibitors of the two types of substrate-induced current, but also inhibit the basal sodium-dependent anion conductance (12,13).Recently a high-resolution crystal structure of a glutamate transporter homolog, Glt Ph , from the archeon Pyrococcus horikoshii was pu...

Parkinson's disease (PD) is the most common movement disorder disease in the elderly and is characterized by degeneration of dopamine neurons and formation of Lewy bodies. Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). If glutamate is not removed promptly in the synaptic cleft, it will excessively stimulate the glutamate receptors and induce excitotoxic effects on the CNS. With lack of extracellular enzyme to decompose glutamate, glutamate uptake in the synaptic cleft is mainly achieved by the excitatory amino acid transporters (EAATs, also known as high-affinity glutamate transporters). Current studies have confirmed that decreased expression and function of EAATs appear in PD animal models. Moreover, single unilateral administration of EAATs inhibitor in the substantia nigra mimics several PD features and this is a solid evidence supporting that decreased EAATs contribute to the process of PD. Drugs or treatments promoting the expression and function of EAATs are shown to attenuate dopamine neurons death in the substantia nigra and striatum, ameliorate the behavior disorder, and improve cognitive abilities in PD animal models. EAATs are potential effective drug targets in treatment of PD and thus study of relationship between EAATs and PD has predominant medical significance currently.

In this study, we conducted a clinical analysis of lymphocyte subtypes in 268 patients with Parkinson's disease (PD) to assess their clinical impact as a potential marker of advanced PD in Chinese patients. The participants comprised 268 sporadic PD patients and 268 healthy controls. The numbers of natural killer (NK) cells and CD3+, CD3+CD4+, CD3+CD8+, and CD19+ lymphocytes from peripheral blood were determined by immunostaining and flow cytometric analysis and the percentages of these CD+ T cells were calculated. The ratio of regulatory T (Treg)/helper T 17 (Th17) lymphocytes from 64 PD patients and 46 controls was determined by flow cytometric analysis. The results showed that the percentage of NK cells was higher in advanced PD patients than in controls (22.92% ± 10.08% versus 19.76% ± 10.09%, P = 0.006), while CD3+ T cells are decreased (62.93% ± 9.27% versus 65.75% ± 9.13%, P = 0.005). The percentage of CD19+ B cells in male patients was lower (P = 0.021) than in female patients, whereas NK cells were increased (P < 0.0001). The scores on the Unified Parkinson's Disease Rating Scale (UPDRS) and the Non-Motor Symptoms Scale in late-onset PD patients were significantly higher than those in early-onset patients (P = 0.024 and P = 0.007, respectively). The percentage of CD19+ B cells in patients with UPDRS scores >24 was lower than in those with scores <24 (10.17% ± 4.19% versus 12.22% ± 5.39%, P = 0.009). In addition, the Treg/Th17 ratio in female patients was higher than that in female controls (13.88 ± 6.32 versus 9.94 ± 4.06, P = 0.042). These results suggest that the percentages of NK cells, CD3+ T cells, and CD19+ B cells along with the Treg/Th17 ratio in peripheral blood may be used to predict the risk of PD in Chinese individuals and provide fresh avenues for novel diagnostic biomarkers and therapeutic designs.

Ceftriaxone has been shown to attenuate the dopaminergic neuron death and alleviate behavioral disorders in Parkinson's disease models via upregulation of glutamate transporter-1 (GLT-1) and decreases in extracellular glutamate. However, details of how this neuroprotection occurs are uncertain. We hypothesized that cytoprotection by ceftriaxone in astrocytes exposed to 1-methyl-4-phenylpyridinium (MPP(+)) involves suppression of the NF-κB/JNK/c-Jun signaling pathway. Here, we observed a protective effect of ceftriaxone in primary astrocytes exposed to MPP(+). Ceftriaxone enhanced glutamate uptake and promoted primary astrocyte viability after MPP(+) exposure. Ceftriaxone enhances glutamate uptake via upregulation of GLT-1 in the plasma membrane, and alleviates MPP(+)-induced neurotoxicity via suppression of NF-κB/JNK/c-Jun signaling. Collectively, our data offer evidence that increased expression and function of GLT-1 are involved in the protective mechanism of ceftriaxone in astrocytes exposed to MPP(+) in vitro, and we offer insight into the potential therapeutic role of ceftriaxone in treatment of Parkinson's disease.