MTEP, a Selective mGluR5 Antagonist, Had a Neuroprotective Effect but Did Not Prevent the Development of Spontaneous Recurrent Seizures and Behavioral Comorbidities in the Rat Lithium–Pilocarpine Model of Epilepsy

Metabotropic glutamate receptors (mGluRs) are expressed predominantly on neurons and glial cells and are involved in the modulation of a wide range of signal transduction cascades. Therefore, different subtypes of mGluRs are considered a promising target for the treatment of various brain diseases. Previous studies have demonstrated the seizure-induced upregulation of mGluR5; however, its functional significance is still unclear. In the present study, we aimed to clarify the effect of treatment with the selective mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) on epileptogenesis and behavioral impairments in rats using the lithium–pilocarpine model. We found that the administration of MTEP during the latent phase of the model did not improve survival, prevent the development of epilepsy, or attenuate its manifestations in rats. However, MTEP treatment completely prevented neuronal loss and partially attenuated astrogliosis in the hippocampus. An increase in excitatory amino acid transporter 2 expression, which has been detected in treated rats, may prevent excitotoxicity and be a potential mechanism of neuroprotection. We also found that MTEP administration did not prevent the behavioral comorbidities such as depressive-like behavior, motor hyperactivity, reduction of exploratory behavior, and cognitive impairments typical in the lithium–pilocarpine model. Thus, despite the distinct neuroprotective effect, the MTEP treatment was ineffective in preventing epilepsy.

Structural insights into human excitatory amino acid transporter EAAT2

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity, that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. This is the first characterization of molecular features of EAAT2 and the selective inhibition mechanism, underlying structure-based drug design for EAAT2.

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 GltPh, GltTK, 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-β-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.

Abstract P235: Reduced Uterine Perfusion Pressure Impairs Seizure-induced Downregulation Of Neurotransmitter Transporters In Mice

Preeclampsia, a hypertensive disorder of pregnancy, can advance to eclampsia, if new onsetseizures occur. Previous work showed increased susceptibility to chemically-induced seizures inthe reduced uterine perfusion pressure (RUPP) rat model of preeclampsia; however, theunderlying mechanisms are unknown. Because seizures occur due to neurotransmitter activityimbalance, we hypothesized that RUPP mice have elevated excitatory and reduced inhibitoryactivity and that seizures exacerbate this imbalance.Timed-pregnant SMA-GFP mice (n=5-6 per group/treatment) were subjected to sham or RUPPsurgery on gestational day (GD) 13.5 and seizures were induced using 40mg/kg pentylenetetrazolon GD18.5. Tissues were harvested 30 minutes post-seizure induction. Maximum seizure scoreswere similar in sham (4.7±0.3) and RUPP (4.5±0.3) mice; p=0.37. Fluorometric assay showsseizures increased [F (1, 16) = 5.99, p=0.03], while RUPP had no effect [F (1, 16) = 1.15, p=0.3]on hippocampal glutamate concentration. No pairwise differences were observed within the shamand RUPP groups exposed to seizures (p>0.05). Seizures increased [F (1, 16) = 6.96, p=0.02],while RUPP had no effect [F (1, 16) = 0.61, p=0.45] on GABA concentration, with no significantpairwise difference in GABA concentration (p>0.05).Western blot analysis shows seizures significantly reduced hippocampal NMDAR1 (1.0±0.5 vs0.6±0.96, p=0.02; 1.0±0.1 vs 0.6±0.0, p=0.04) and GABAAR receptor expression (1.0±0.4 vs0.35±0.1, p<0.5; 1.05±0.3 vs 0.3±0.1, p<0.05) in sham and RUPP mice. Following seizureexposure, vesicular glutamate transporter (VGLUT1: sham: 1.0±0.3 vs 0.5±0.1; p<0.01, RUPP:0.8±0.2 vs 0.6±0.1; p=0.11), excitatory amino acid transporter 1 (EAAT1: sham: 1.0±0.4 vs0.6±0.2; p=0.02, RUPP: 0.9±0.1 vs 0.6±0.2; p=0.17) and GABA transporter (GAT1: sham: 1.0±0.5vs 0.4±0.1, p=0.01; RUPP: 0.8±0.2 vs 0.5±0.1, p=0.12) was reduced in sham mice, but not RUPPmice.Although RUPP does not change baseline GABA or glutamate related receptor or transporterexpression, our findings suggest seizure-induced reductions in vesicular and astrocyticneurotransmitter transporters is impaired by the RUPP procedure. Ongoing studies assesswhether other receptors and transporters are affected.

Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80% of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinson's disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In Lrrk2 G2019S knockin mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane, causing its intracellular relocalization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

Protective Effect of Memantine on Bergmann Glia and Purkinje Cells Morphology in Optogenetic Model of Neurodegeneration in Mice

Spinocerebellar ataxias are a family of fatal inherited diseases affecting the brain. Although specific mutated proteins are different, they may have a common pathogenetic mechanism, such as insufficient glutamate clearance. This function fails in reactive glia, leading to excitotoxicity and overactivation of NMDA receptors. Therefore, NMDA receptor blockers could be considered for the management of excitotoxicity. One such drug, memantine, currently used for the treatment of Alzheimer’s disease, could potentially be used for the treatment of other forms of neurodegeneration, for example, spinocerebellar ataxias (SCA). We previously demonstrated close parallels between optogenetically induced cerebellar degeneration and SCA1. Here we induced reactive transformation of cerebellar Bergmann glia (BG) using this novel optogenetic approach and tested whether memantine could counteract changes in BG and Purkinje cell (PC) morphology and expression of the main glial glutamate transporter—excitatory amino acid transporter 1 (EAAT1). Reactive BG induced by chronic optogenetic stimulation presented increased GFAP immunoreactivity, increased thickness and decreased length of its processes. Oral memantine (~90 mg/kg/day for 4 days) prevented thickening of the processes (1.57 to 1.81 vs. 1.62 μm) and strongly antagonized light-induced reduction in their average length (186.0 to 150.8 vs. 171.9 μm). Memantine also prevented the loss of the key glial glutamate transporter EAAT1 on BG. Finally, memantine reduced the loss of PC (4.2 ± 0.2 to 3.2 ± 0.2 vs. 4.1 ± 0.3 cells per 100 μm of the PC layer). These results identify memantine as potential neuroprotective therapeutics for cerebellar ataxias.

Modulation of Glutamate Transporter EAAT1 and Inward-Rectifier Potassium Channel Kir4.1 Expression in Cultured Spinal Cord Astrocytes by Platinum-Based Chemotherapeutics

Platinum-based chemotherapeutics still play an essential role in cancer treatment. Despite their high effectiveness, severe side effects such as chemotherapy-induced neuropathy (CIPN) occur frequently. The pathophysiology of CIPN by platinum-based chemotherapeutics is not fully understood yet, but primarily the disturbance of dorsal root ganglion cells is discussed. However, there is increasing evidence of central nervous system involvement with activation of spinal cord astrocytes after treatment with chemotherapeutics. We investigated the influence of cis- or oxaliplatin on the functionality of cultured rat spinal cord astrocytes by using immunocytochemistry and patch-clamp electrophysiology. Cis- or oxaliplatin activated spinal astrocytes and led to downregulation of the excitatory amino acid transporter 1 (EAAT1) expression. Furthermore, the expression and function of potassium channel Kir4.1 were modulated. Pre-exposure to a specific Kir4.1 blocker in control astrocytes led to a reduced immune reactivity (IR) of EAAT1 and a nearly complete block of the current density. When spinal astrocytes were pre-exposed to antibiotic minocycline, all effects of cis- or oxaliplatin were abolished. Taken together, the modulation of Kir4.1 and EAAT1 proteins in astrocytes could be linked to the direct impact of cis- or oxaliplatin, identifying spinal astrocytes as a potential target in the prevention and treatment of chemotherapy-induced neuropathy.

Constitutive Endocytosis of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter-3 Requires ARFGAP1

The eukaryotic endocytic pathway regulates protein levels available at the plasma membrane by recycling them into specific endosomal compartments. ARFGAP1 is a component of the coat protein I (COPI) complex but it also plays a role in promoting adapter protein-2 (AP-2) mediated endocytosis. The excitatory amino acid transporter-3 (EAAT3) mediates the reuptake of glutamate from the synaptic cleft to achieve rapid termination of synaptic transmission at glutamatergic synapses. In this study, we identified two interacting proteins of EAAT3 by mass spectrometry (MS) ARFGAP1 and ARF6. We explored the role of ARFGAP1 and ARF6 in the endocytosis of EAAT3. Our data revealed that ARFGAP1 plays a role in the recycling of EAAT3, by utilizing its GTPase activating protein (GAP) activity and ARF6 acting as the substrate. ARFGAP1 promotes cargo sorting of EAAT3 via a single phenylalanine residue (F508) located at the C-terminus of the transporter. ARFGAP1-promoted AP-2 dependent endocytosis is abolished upon neutralizing F508. We utilized a heterologous expression system to identify an additional motif in the C-terminus of EAAT3 that regulates its endocytosis. Impairment in endocytosis did not affect somatodendritic targeting in cultured hippocampal neurons. Our findings support a model where endocytosis of EAAT3 is a multifactorial event regulated by ARFGAP1, occurring via the C-terminus of the transporter, and is the first study to examine the role of ARFGAP1 in the endocytosis of a transport protein.