Oxidative stress accelerates synaptic glutamate dyshomeostasis and NMDARs disorder during methylmercury‐induced neuronal apoptosis in rat cerebral cortex

Methylmercury (MeHg) is a potent neurotoxin,which leads to a wide range of intracellular effects. The molecular mechanismsassociated to MeHg‐induced neurotoxicity have not been fully understood.Oxidative stress, as well as synaptic glutamate (Glu) dyshomeostasis have beenidentified as two critical mechanisms during MeHg‐mediated cytotoxicity. Here,we developed a rat model of MeHg poisoning to evaluate its neurotoxic effectsby focusing on cellular oxidative stress and synaptic Glu disruption. Inaddition, we investigated the neuroprotective role of alpha‐lipoic acid (α‐LA), a natural antioxidant, todeeply explore the underlying interaction between them. Fifty‐six rats wererandomly divided into four groups: saline control, MeHg treatment (4 or 12μmol/kg MeHg), and α‐LApre‐treatment (35 μmol/kg α‐LA+12μmol/kg MeHg). Rats exposed to 12 μmol/kg MeHg induced neuronal oxidativestress, with ROS accumulation and cellular antioxidant system impairment. Nrf2 andxCT pathways were activated with MeHg treatment. The enzymatic or non‐enzymaticof cellular GSH synthesis were also disrupted by MeHg. On the other hand, the abnormalactivities of GS and PAG disturbed the “Glu‐Gln cycle”, leading to NMDARsover‐activation, Ca2+ overload, and the calpain activation, which acceleratedNMDARs degradation. Meanwhile, the high expressions of phospho‐p44/42 MAPK,phospho‐p38 MAPK, phospho‐CREB, and the high levels of caspase 3 and Bax/Bcl‐2 finallyindicated the neuronal apoptosis after MeHg exposure. Pre‐treatment with α‐LA significantly preventedMeHg‐induced neurotoxicity. In conclusion, the oxidative stress and synapticGlu dyshomeostasis contributed to MeHg‐induced neuronal apoptosis. Alpha‐LAattenuated these toxic effects through mechanisms of anti‐oxidation andindirect Glu dyshomeostasis prevention     

Reverse glial glutamate uptake triggers neuronal cell death through extrasynaptic NMDA receptor activation

Evidence have accumulated that reverse glutamate uptake plays a key role in the pathophysiology of cerebral ischemia. Here, we investigated the effects of glial glutamate transporter dysfunction on neuronal survival using the substrate inhibitor of glutamate transporters, l-trans-pyrrolidine,2-4,dicarboxylate (PDC), that partly mimics reverse glutamate uptake. On mice primary cortical co-cultures of neurons and astrocytes, PDC treatment triggered an elevation of extracellular glutamate concentration, induced neuronal calcium influx and a massive NMDA receptor (NMDAR) mediated-neuronal death without having any direct agonist activity on NMDARs. We investigated the NMDAR subpopulation activated by PDC-induced glutamate release. PDC application led to the activation of both subtypes of NMDARs but the presence of astrocytes was required to activate NMDARs located extra-synaptically. Extrasynaptic NMDAR activation was also confirmed by the loss of neuronal mitochondrial membrane potential and the inhibition of pro-survival p-ERK signalling pathway. These data suggest that reverse glial glutamate uptake may trigger neuronal death through preferential activation of extrasynaptic NMDAR-related pathways.     

Neurogranin phosphorylation fine-tunes long-term potentiation

Learning-related potentiation of synaptic strength at Cornu ammonis subfield 1 (CA1) hippocampal excitatory synapses is dependent on neuronal activity and the activation of glutamate receptors. However, molecular mechanisms that regulate and fine-tune the expression of long-term potentiation (LTP) are not well understood. Recently it has been indicated that neurogranin (Ng), a neuron-specific, postsynaptic protein that is phosphorylated by protein kinase C, potentiates synaptic transmission in an LTP-like manner. Here, we report that a Ng mutant that is unable to be phosphorylated cannot potentiate synaptic transmission in rat CA1 hippocampal neurons and results in a submaximal expression of LTP. Our results provide the first evidence that the phosphorylation of Ng can regulate LTP expression.     

α-Synuclein Oligomers Induce Glutamate Release from Astrocytes and Excessive Extrasynaptic NMDAR Activity in Neurons,Thus Contributing to Synapse Loss

Synaptic and neuronal loss are major neuropathological characteristics of Parkinson''s disease. Misfolded protein aggregates in the form of Lewy bodies, comprised mainly of α-synuclein (αSyn), are associated with disease progression, and have also been linked to other neurodegenerative diseases, including Lewy body dementia, Alzheimer''s disease, and frontotemporal dementia. However, the effects of αSyn and its mechanism of synaptic damage remain incompletely understood. Here, we show that αSyn oligomers induce Ca²⁺-dependent release of glutamate from astrocytes obtained from male and female mice, and that mice overexpressing αSyn manifest increased tonic release of glutamate in vivo. In turn, this extracellular glutamate activates glutamate receptors, including extrasynaptic NMDARs (eNMDARs), on neurons both in culture and in hippocampal slices of αSyn-overexpressing mice. Additionally, in patch-clamp recording from outside-out patches, we found that oligomerized αSyn can directly activate eNMDARs. In organotypic slices, oligomeric αSyn induces eNMDAR-mediated synaptic loss, which can be reversed by the drug NitroSynapsin. When we expose human induced pluripotent stem cell-derived cerebrocortical neurons to αSyn, we find similar effects. Importantly, the improved NMDAR antagonist NitroSynapsin, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from oligomeric αSyn-induced damage in our model systems, thus meriting further study for its therapeutic potential.SIGNIFICANCE STATEMENT Loss of synaptic function and ensuing neuronal loss are associated with disease progression in Parkinson''s disease (PD), Lewy body dementia (LBD), and other neurodegenerative diseases. However, the mechanism of synaptic damage remains incompletely understood. α-Synuclein (αSyn) misfolds in PD/LBD, forming Lewy bodies and contributing to disease pathogenesis. Here, we found that misfolded/oligomeric αSyn releases excessive astrocytic glutamate, in turn activating neuronal extrasynaptic NMDA receptors (eNMDARs), thereby contributing to synaptic damage. Additionally, αSyn oligomers directly activate eNMDARs, further contributing to damage. While the FDA-approved drug memantine has been reported to offer some benefit in PD/LBD (Hershey and Coleman-Jackson, 2019), we find that the improved eNMDAR antagonist NitroSynapsin ameliorates αSyn-induced synaptic spine loss, providing potential disease-modifying intervention in PD/LBD.     

Molecular characterization of N-methyl-d-aspartate receptor from Bemisia tabaci

The N-methyl-d-aspartate receptors (NMDARs) are ionotropic ligand gated channels that are highly permeable to calcium ions. In insects, NMDARs are associated with glutamatergic neurotransmission governing diverse physiological and biological processes like vitellogenesis and ovarian development. Therefore, NMDAR may act as attractive target for insect pest control. In present study, we performed structural and functional characterization of NMDARs in Bemisia tabaci, a highly invasive crop pest and potent virus vector. We identified that NMDAR consists of three subunits each encoded by single gene in whiteflies which are highly conserved among different insect orders. Expression analysis suggests that subunit 1 (BtNR1) and subunit 2 (BtNR2) are the main functional units. External supplementation of NMDAR ligand or BtNRs silencing was lethal to insects, which suggested that NMDAR function is highly balanced in whiteflies.     

Influence of the NR3A subunit on NMDA receptor functions

Various combinations of subunits assemble to form the NMDA-type glutamate receptor (NMDAR), generating diversity in its functions. Here we review roles of the unique NMDAR subunit, NR3A, which acts in a dominant-negative manner to suppress receptor activity. NR3A-containing NMDARs display striking regional and temporal expression specificity, and, unlike most other NMDAR subtypes, they have a low conductance, are only modestly permeable to Ca²⁺, and pass current at hyperpolarized potentials in the presence of magnesium. While glutamate activates triheteromeric NMDARs composed of NR1/NR2/NR3A subunits, glycine is sufficient to activate diheteromeric NR1/NR3A-containing receptors. NR3A dysfunction may contribute to neurological disorders involving NMDARs, and the subunit offers an attractive therapeutic target given its distinct pharmacological and structural properties.     

美金刚对急性脊髓损伤大鼠运动功能的影响

目的:探讨美金刚对急性脊髓损伤大鼠运动功能恢复的影响及相关作用机制。方法:80只健康成年雄性SD大鼠,采用改良的Allen打击法(10 g×2.5 cm)建立大鼠T12脊髓损伤动物模型,并随机分为假手术组(A组)、模型组(B组)、美金刚组(C组)及MK-801组(D组),每组20只。术前及术后24 h、72 h、5 d、7 d进行运动功能BBB评分,并采用黄嘌呤氧化法和硫代巴比妥酸法检测损伤脊髓节段组织匀浆超氧化物歧化酶(SOD)和丙二醛(MDA)的水平;HE、尼氏染色观察损伤脊髓组织的病理学改变及Tunel法检测神经细胞的凋亡。结果:与模型组比较,美金刚组及MK-801组大鼠伤后各时间点的脊髓组织SOD活性显著升高、MDA含量显著下调、BBB运动功能评分增加、神经细胞凋亡率降低,差异均具有统计学意义(P0.05)。HE及尼氏染色观察可见美金刚组及MK-801组大鼠较模型组灰质出血量、白质结构紊乱、炎性细胞浸润程度等都有明显改善。结论:美金刚可以通过阻断过度激活的NMDA受体功能,减轻大鼠脊髓损伤后氧化应激反应,减少脊髓损伤处神经细胞的凋亡,促进脊髓损伤大鼠运动功能的恢复。     

Glutamatergic mechanisms in the dyskinesias induced by pharmacological dopamine replacement and deep brain stimulation for the treatment of Parkinson's disease

Dyskinesias represent a major complication of dopamine replacement therapy in Parkinson's disease (PD) and have prompted a search for alternative treatments. The most radical advances in this field have been provided by surgical manipulations of the deep basal ganglia nuclei, and particularly by deep brain stimulation (DBS) of the subthalamic nucleus (STN). Although being very effective, high-frequency stimulation (HFS) of the STN is a poorly understood treatment. Besides its anti-akinetic activity, it can be pro-dyskinetic above a certain stimulation intensity. Accumulating evidence indicates that dyskinesias induced by STN-HFS and dopamine replacement therapy are linked to dysregulation of glutamate transmission in the basal ganglia. In rat models of PD, both types of dyskinesia are associated with increased concentrations of extracellular glutamate and altered expression of glutamate transporters in the substantia nigra pars reticulata and the striatum. Furthermore, a vast and ever growing literature has revealed changes in the expression, phosphorylation state, and/or subcellular distribution of specific subtypes of glutamate receptors in these dyskinetic conditions. Both types of dyskinesias are linked to an increased phosphorylation of NR2B-containing NMDA receptors in critical basal ganglia circuits. We conclude that disruption of glutamate homeostasis and activation of perisynaptic and extra-synaptic glutamate receptors are an important pathophysiological component of these treatment-induced dyskinesias in PD. These findings lay the ground for therapeutic development initiatives targeting dysfunctional components of glutamate transmission in the basal ganglia.     

Emerging themes in GABAergic synapse development

Glutamatergic synapse development has been rigorously investigated for the past two decades at both the molecular and cell biological level yet a comparable intensity of investigation into the cellular and molecular mechanisms of GABAergic synapse development has been lacking until relatively recently. This review will provide a detailed overview of the current understanding of GABAergic synapse development with a particular emphasis on assembly of synaptic components, molecular mechanisms of synaptic development, and a subset of human disorders which manifest when GABAergic synapse development is disrupted. An unexpected and emerging theme from these studies is that glutamatergic and GABAergic synapse development share a number of overlapping molecular and cell biological mechanisms that will be emphasized in this review.