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1.
Nat Struct Mol Biol ; 30(5): 629-639, 2023 05.
Article in English | MEDLINE | ID: mdl-36959261

ABSTRACT

N-methyl-D-aspartate (NMDA) receptors are heterotetramers comprising two GluN1 and two alternate GluN2 (N2A-N2D) subunits. Here we report full-length cryo-EM structures of the human N1-N2D di-heterotetramer (di-receptor), rat N1-N2C di-receptor and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a best resolution of 3.0 Å. The bilobate N-terminal domain (NTD) in N2D intrinsically adopts a closed conformation, leading to a compact NTD tetramer in the N1-N2D receptor. Additionally, crosslinking the ligand-binding domain (LBD) of two N1 protomers significantly elevated the channel open probability (Po) in N1-N2D di-receptors. Surprisingly, the N1-N2C di-receptor adopted both symmetric (minor) and asymmetric (major) conformations, the latter further locked by an allosteric potentiator, PYD-106, binding to a pocket between the NTD and LBD in only one N2C protomer. Finally, the N2A and N2C subunits in the N1-N2A-N2C tri-receptor display a conformation close to one protomer in the N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse function in major NMDA receptor subtypes.


Subject(s)
Receptors, N-Methyl-D-Aspartate , Rats , Animals , Humans , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism , Protein Subunits/chemistry , Protein Domains
2.
Glia ; 71(7): 1648-1666, 2023 07.
Article in English | MEDLINE | ID: mdl-36960578

ABSTRACT

Reactive astrocytes can be transformed into new neurons. Vascular endothelial growth factor (VEGF) promotes the transformation of reactive astrocytes into neurons in ischemic brain. Therefore, in this study, the molecular mechanism of VEGF's effect on ischemia/hypoxia-induced astrocyte to neuron transformation was investigated in the models of rat middle cerebral artery occlusion (MCAO) and in astrocyte culture with oxygen and glucose deprivation (OGD). We found that VEGF enhanced ischemia-induced Pax6, a neurogenic fate determinant, expression and Erk phosphorylation in reactive astrocytes and reduced infarct volume of rat brain at 3 days after MCAO, which effects could be blocked by administration of U0126, a MAPK/Erk inhibitor. In cultured astrocytes, VEGF also enhanced OGD-induced Erk phosphorylation and Pax6 expression, which was blocked by U0126, but not wortmannin, a PI3K/Akt inhibitor, or SB203580, a MAPK/p38 inhibitor, suggesting VEGF enhanced Pax6 expression via activation of MAPK/Erk pathway. OGD induced the increase of miR365 and VEGF inhibited the increase of OGD-induced miR365 expression. However, miR365 agonists blocked VEGF-enhanced Pax6 expression in hypoxic astrocytes, but did not block VEGF-enhanced Erk phosphorylation. We further found that VEGF promoted OGD-induced astrocyte-converted to neuron. Interestingly, both U0126 and Pax6 RNAi significantly reduced enhancement of VEGF on astrocytes-to-neurons transformation, as indicated Dcx and MAP2 immunopositive signals in reactive astrocytes. Moreover, those transformed neurons become mature and functional. We concluded that VEGF enhanced astrocytic neurogenesis via the MAPK/Erk-miR-365-Pax6 signal axis. The results also indicated that astrocytes play important roles in the reconstruction of neurovascular units in brain after stroke.


Subject(s)
Astrocytes , Vascular Endothelial Growth Factor A , Rats , Animals , Astrocytes/metabolism , Vascular Endothelial Growth Factor A/metabolism , MAP Kinase Signaling System , Cell Transdifferentiation , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Infarction, Middle Cerebral Artery/metabolism , Protein Kinase Inhibitors/pharmacology , Neurons/metabolism , Glucose/metabolism
3.
Front Pharmacol ; 13: 888308, 2022.
Article in English | MEDLINE | ID: mdl-35754487

ABSTRACT

N-methyl-D-aspartate receptors (NMDARs) are Ca2+-permeable ionotropic glutamate receptors (iGluRs) in the central nervous system and play important roles in neuronal development and synaptic plasticity. Conventional NMDARs, which typically comprise GluN1 and GluN2 subunits, have different biophysical properties than GluN3-containing NMDARs: GluN3-containing NMDARs have smaller unitary conductance, less Ca2+-permeability and lower Mg2+-sensitivity than those of conventional NMDARs. However, there are very few specific modulators for GluN3-containing NMDARs. Here, we developed a cell-based high-throughput calcium assay and identified 3-fluoro-1,2-phenylene bis (3-hydroxybenzoate) (WZB117) as a relatively selective inhibitor of GluN1/GluN3 receptors. The IC50 value of WZB117 on GluN1/GluN3A receptors expressed in HEK-293 cells was 1.15 ± 0.34 µM. Consistently, WZB117 exhibited strong inhibitory activity against glycine-induced currents in the presence of CGP-78608 but only slightly affected the NMDA-, KA- and AMPA-induced currents in the acutely isolated rat hippocampal neurons. Among the four types of endogenous currents, only the first one is primarily mediated by GluN1/GluN3 receptors. Mechanistic studies showed that WZB117 inhibited the GluN1/GluN3A receptors in a glycine-, voltage- and pH-independent manner, suggesting it is an allosteric modulator. Site-directed mutagenesis and chimera construction further revealed that WZB117 may act on the GluN3A pre-M1 region with key determinants different from those of previously identified modulators. Together, our study developed an efficient method to discover modulators of GluN3-containing NMDARs and characterized WZB117 as a novel allosteric inhibitor of GluN1/GluN3 receptors.

4.
Schizophr Res ; 249: 93-97, 2022 11.
Article in English | MEDLINE | ID: mdl-34916095

ABSTRACT

BACKGROUND: Accumulating evidence suggests that the pathology of some psychiatric symptoms may relate to autoantibodies against various neuronal surface antigens, such as NMDA receptors (NMDARs) or inhibitory GABAA receptors (GABAARs). However, it is unclear whether the plasma of patients with schizophrenia contains autoantibodies targeting to NMDARs or GABAARs. METHODS: Serum samples of 293 patients with schizophrenia were analyzed using a combination of live-cell-based assay (CBA) and immunostaining on primary neurons to quantify the positive rate of autoantibodies targeting NMDARs or GABAARs. RESULTS: Only one sample was found positive for anti-NMDAR autoantibodies, and no surface autoantibodies against GABAARs were found. No obvious difference in clinical manifestations was observed between the patients with positive and negative anti-NMDAR autoantibodies. CONCLUSIONS: Our results suggest that autoantibodies against NMDARs or GABAARs may affect only a small group of patients with schizophrenia, and the rates of these autoantibodies are lower than reported in prior work. It would be interesting to perform studies with psychotic disorder instead of schizophrenia to determine whether NMDAR or GABAAR autoantibody can be used as a biomarker to provide a new avenue for immunomodulatory therapy.


Subject(s)
Schizophrenia , Humans , Autoantibodies , Receptors, GABA-A , Receptors, N-Methyl-D-Aspartate , gamma-Aminobutyric Acid
5.
Glia ; 67(7): 1344-1358, 2019 07.
Article in English | MEDLINE | ID: mdl-30883902

ABSTRACT

Astrocytic calcium signaling plays pivotal roles in the maintenance of neural functions and neurovascular coupling in the brain. Vascular endothelial growth factor (VEGF), an original biological substance of vessels, regulates the movement of calcium and potassium ions across neuronal membrane. In this study, we investigated whether and how VEGF regulates glutamate-induced calcium influx in astrocytes. We used cultured astrocytes combined with living cell imaging to detect the calcium influx induced by glutamate. We found that VEGF quickly inhibited the glutamate/hypoxia-induced calcium influx, which was blocked by an AMPA receptor antagonist CNQX, but not D-AP5 or UBP310, NMDA and kainate receptor antagonist, respectively. VEGF increased phosphorylation of PKCα and AMPA receptor subunit GluA2 in astrocytes, and these effects were diminished by SU1498 or calphostin C, a PKC inhibitor. With the pHluorin assay, we observed that VEGF significantly increased membrane insertion and expression of GluA2, but not GluA1, in astrocytes. Moreover, siRNA-produced knockdown of GluA2 expression in astrocytes reversed the inhibitory effect of VEGF on glutamate-induced calcium influx. Together, our results suggest that VEGF reduces glutamate-induced calcium influx in astrocytes via enhancing PKCα-mediated GluA2 phosphorylation, which in turn promotes the membrane insertion and expression of GluA2 and causes AMPA receptors to switch from calcium-permeable to calcium-impermeable receptors, thereby inhibiting astrocytic calcium influx. The present study reveals that excitatory neurotransmitter glutamate-mediated astrocytic calcium influx can be regulated by vascular biological factor via activation of AMPA receptor GluA2 subunit and uncovers a novel coupling mechanism between astrocytes and endothelial cells within the neurovascular unit.


Subject(s)
Astrocytes/metabolism , Calcium Signaling/physiology , Protein Kinase C/metabolism , Receptors, AMPA/metabolism , Vascular Endothelial Growth Factor A/pharmacology , Animals , Animals, Newborn , Astrocytes/drug effects , Calcium/metabolism , Calcium Signaling/drug effects , Cells, Cultured , Excitatory Amino Acid Antagonists/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, AMPA/agonists , Receptors, AMPA/antagonists & inhibitors
6.
Glia ; 66(7): 1346-1362, 2018 07.
Article in English | MEDLINE | ID: mdl-29451327

ABSTRACT

Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte-to-neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6-mediated astrocyte-to-neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv-GFAP-EGFP) to permit cell fate mapping for tracing astrocytes-derived neurons. We verified that miR-365 directly targets to the 3'-UTR of Pax6 by luciferase assay. We found that miR-365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR-365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR-365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte-to-neuron conversion and abolished the effects of miR-365. Our results demonstrate that increase of miR-365 in the ischemic brain inhibits astrocyte-to-neuron conversion by targeting Pax6, whereas knockdown of miR-365 enhances PAX6-mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.


Subject(s)
Astrocytes/metabolism , MicroRNAs/metabolism , Neurogenesis/physiology , Neurons/metabolism , PAX6 Transcription Factor/metabolism , Stroke/metabolism , Animals , Antagomirs/administration & dosage , Astrocytes/pathology , Brain/metabolism , Brain/pathology , Brain Ischemia/metabolism , Brain Ischemia/pathology , Cell Hypoxia/physiology , Cells, Cultured , Disease Models, Animal , Glucose/deficiency , Male , MicroRNAs/antagonists & inhibitors , Neurons/pathology , Rats, Sprague-Dawley , Stroke/pathology
7.
Front Cell Neurosci ; 11: 290, 2017.
Article in English | MEDLINE | ID: mdl-28966577

ABSTRACT

Brain microvascular endothelial cells (BMEC) have been found to guide the migration, promote the survival and regulate the differentiation of neural cells. However, whether BMEC promote development and maturation of immature neurons is still unknown. Therefore, in this study, we used a direct endothelium-neuron co-culture system combined with patch clamp recordings and confocal imaging analysis, to investigate the effects of endothelial cells on neuronal morphology and function during development. We found that endothelial cells co-culture or BMEC-conditioned medium (B-CM) promoted neurite outgrowth and spine formation, accelerated electrophysiological development and enhanced synapse function. Moreover, B-CM treatment induced vascular endothelial growth factor (VEGF) expression and p38 phosphorylation in the cortical neurons. Through pharmacological analysis, we found that incubation with SU1498, an inhibitor of VEGF receptor, abolished B-CM-induced p-p38 upregulation and suppressed the enhancement of synapse formation and transmission. SB203580, an inhibitor of p38 MAPK also blocked B-CM-mediated synaptic regulation. Together these results clearly reveal that the endothelium-neuron interactions promote morphological and functional maturation of neurons. In addition, neurovascular interaction-mediated promotion of neural network maturation relies on activation of VEGF/Flk-1/p38 MAPK signaling. This study provides novel aspects of endothelium-neuron interactions and novel mechanism of neurovascular crosstalk.

8.
Front Mol Neurosci ; 10: 424, 2017.
Article in English | MEDLINE | ID: mdl-29311814

ABSTRACT

Axon-transport plays an important role in neuronal activity and survival. Reduced endogenous VEGF can cause neuronal damage and axon degeneration. It is unknown at this time if VEGF can be transported within the axon or whether it can be released by axonal depolarization. We transfected VEGF-eGFP plasmids in cultured hippocampal neurons and tracked their movement in the axons by live-cell confocal imaging. Then, we co-transfected phVEGF-eGFP and kinesin-1B-DsRed vectors into neurons and combined with immunoprecipitation and two-color imaging to study the mechanism of VEGF axon-trafficking. We found that VEGF vesicles morphologically co-localized and biochemically bounded with kinesin-1B, as well as co-trafficked with it in the axons. Moreover, the capacity for axonal trafficking of VEGF was reduced by administration of nocodazole, an inhibitor of microtubules, or kinesin-1B shRNA. In addition, we found that VEGF could release from the cultured neurons under acute depolarizing stimulation with potassium chloride. Therefore, present findings suggest that neuronal VEGF is stored in the vesicles, actively released, and transported in the axons, which depends on the presence of kinesin-1B and functional microtubules. These results further help us to understand the importance of neuronal VEGF in the maintenance of neuronal activity and survival throughout life.

9.
Neuroscience ; 334: 275-282, 2016 Oct 15.
Article in English | MEDLINE | ID: mdl-27531855

ABSTRACT

This study examined the effect of neuron-endothelial coupling on the survival of neurons after ischemia and the possible mechanism underlying that effect. Whole-cell patch-clamp experiments were performed on cortical neurons cultured alone or directly cocultured with brain microvascular endothelial cells (BMEC). Propidium iodide (PI) and NeuN staining were performed to examine neuronal death following oxygen and glucose deprivation (OGD). We found that the neuronal transient outward potassium currents (IA) decreased in the coculture system, whereas the outward delayed-rectifier potassium currents (IK) did not. Sodium nitroprusside, a NO donor, enhanced BMEC-induced IA inhibition and nitro-l-arginine methylester, a NOS inhibitor, partially prevented this inhibition. Moreover, the neurons directly cocultured with BMEC showed more resistance to OGD-induced injury compared with the neurons cultured alone, and that neuroprotective effect was abolished by treatment with NS5806, an activator of the IA. These results indicate that vascular endothelial cells assist neurons to prevent hypoxic injury via inhibiting neuronal IA by production of NO in the direct neuron-BMEC coculture system. These results further provide direct evidence of functional coupling between neurons and vascular endothelial cells. This study clearly demonstrates that vascular endothelial cells play beneficial roles in the pathophysiological processes of neurons after hypoxic injury, suggesting that the improvement of neurovascular coupling or functional remodeling may become an important therapeutic target for preventing brain injury.


Subject(s)
Cell Hypoxia/physiology , Endothelium/metabolism , Glucose/deficiency , Neurons/metabolism , Neuroprotection/physiology , Neurovascular Coupling/physiology , Animals , Cell Hypoxia/drug effects , Cell Survival/drug effects , Cell Survival/physiology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Coculture Techniques , Endothelium/drug effects , Endothelium/pathology , Microvessels/drug effects , Microvessels/metabolism , Microvessels/pathology , Neurons/drug effects , Neurons/pathology , Neuroprotection/drug effects , Neurovascular Coupling/drug effects , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Patch-Clamp Techniques , Potassium/metabolism , Potassium Channels/metabolism , Rats, Sprague-Dawley
10.
Brain Res ; 1599: 32-43, 2015 Mar 02.
Article in English | MEDLINE | ID: mdl-25511996

ABSTRACT

PURPOSE: Astrocytes can be reactivated after cerebral ischemia by expressing nestin and other characteristic markers of neural stem cells (NSCs). However, the epigenetic features of reactive astrocytes are not well known. Methyl-CpG-binding protein 2 (MeCP2) is a vital transcriptional modulator in brain development. Although the expression and function of some phosphorylated MeCP2 isoforms have been clarified, phospho-serine 292 (pS292) MeCP2 has not yet drawn much attention. In this study, we used western blot analysis and immunohistochemical and immunofluorescent staining to reveal the expressive features of pS292 MeCP2 and MeCP2 in the adult rat striatum following transient middle cerebral artery occlusion (MCAO). RESULTS: We first discovered that the ischemia-induced expression of cytoplasmic pS292 MeCP2 is primarily accumulated in nestin-positive reactive astrocytes in the stroke-injured striatum. Moreover, the enhancement of astrocytic pS292 MeCP2 was correlated with the augmentation of VEGF in astrocytes, as determined by the substantial co-localization of pS292 MeCP2 and VEGF after stroke. Finally, the exogenous overproduction of VEGF further promoted the expression of pS292 MeCP2 in reactive astrocytes, and this effect was accompanied by a marked increase in reactive astrocytes. On the contrary, MeCP2 was predominantly expressed in the neuronal nucleus, and the level of this protein was not significantly altered after ischemic injury and VEGF overproduction. CONCLUSION: Our data provide the first demonstration that overexpression of VEGF enhances the accumulation of pS292 MeCP2 in reactive astrocytes in the ischemic-injured rat striatum, implicating a pS292 MeCP2-related epigenetic role of exogenous VEGF in reactive astrocytes following cerebral ischemia.


Subject(s)
Brain Ischemia/metabolism , Corpus Striatum/metabolism , Methyl-CpG-Binding Protein 2/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Astrocytes/metabolism , Astrocytes/pathology , Brain Ischemia/pathology , Cell Nucleus/metabolism , Cell Nucleus/pathology , Corpus Striatum/pathology , Cytoplasm/metabolism , Cytoplasm/pathology , Disease Models, Animal , Infarction, Middle Cerebral Artery , Male , Phosphorylation , Random Allocation , Rats, Sprague-Dawley
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