Glycine Tranporter-1 Blockade Potentiates NMDA-Mediated Responses in Rat Prefrontal Cortical Neurons In Vitro and In Vivo

The N-methyl-d-aspartate (NMDA) receptor (NMDA-R) has pivotal roles in neural development, learning, memory, and synaptic plasticity. Functional impairment of NMDA-R has been implicated in schizophrenia. NMDA-R activation requires glycine to act on the glycine-B (GlyB) site of the NMDA-R as an obligatory co-agonist with glutamate. Extracellular glycine near NMDA-R is regulated effectively by a glial glycine transporter (GlyT1). Using whole-cell voltage-clamp recordings in prefrontal cortex (PFC) slices, we have shown that exogenous GlyB site agonists glycine and d-serine, or a specific GlyT1 inhibitor N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS) in the presence of exogenous glycine (10 μM), potentiated synaptically evoked NMDA excitatory postsynaptic currents (EPSCs) in vitro. Furthermore, in urethan-anesthetized rats, microiontophoretic NMDA pulses excite single PFC neurons. When these responses were blocked by approximately 50% to approximately 90% on continuous iontophoretic application of the GlyB site, antagonist (+)HA-966, intravenous NFPS (5 mg/kg), or a GlyB site agonist d-serine (50 mg/kg iv) reversed this (+)HA-966 block. NFPS may elevate endogenous glycine levels sufficiently to displace (+)HA-966 from the GlyB sites of the NMDA-R, thus enabling reactivation of the NMDA-Rs by iontophoretic NMDA applications. d-Serine (50–100 mg/kg iv) or NFPS (1–2 mg/kg iv) alone also augmented NMDA-evoked excitatory responses. These data suggest that direct GlyB site stimulation byd-serine, or blockade of GLYT1 to elevate endogenous glycine to act on unsaturated GlyB sites on NMDA-Rs, potentiated NMDA-R-mediated firing responses in rat PFC. Hence, blockade of GlyT1 to elevate glycine near the NMDA-R may activate hypofunctional NMDA-R, which has been implicated to play a critical role in the pathophysiology of schizophrenia.

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Regionally Selective Blockade of GABAergic Inhibition by Zinc in the Thalamocortical System: Functional Significance

Journal of Neurophysiology ◽

10.1152/jn.2000.83.3.1510 ◽

2000 ◽

Vol 83 (3) ◽

pp. 1510-1521 ◽

Cited By ~ 14

Author(s):

John W. Gibbs III ◽

Yun-Fu Zhang ◽

Melissa D. Shumate ◽

Douglas A. Coulter

Keyword(s):

Cortical Neurons ◽

Cell Voltage ◽

Clonic Seizure ◽

Gabaergic Inhibition ◽

Extracellular Medium ◽

Selective Blockade ◽

Zinc Release ◽

Tightly Coupled

The thalamocortical (TC) system is a tightly coupled synaptic circuit in which GABAergic inhibition originating from the nucleus reticularis thalami (NRT) serves to synchronize oscillatory TC rhythmic behavior. Zinc is colocalized within nerve terminals throughout the TC system with dense staining for zinc observed in NRT, neocortex, and thalamus. Whole cell voltage-clamp recordings of GABA-evoked responses were conducted in neurons isolated from ventrobasal thalamus, NRT, and somatosensory cortex to investigate modulation of the GABA-mediated chloride conductance by zinc. Zinc blocked GABA responses in a regionally specific, noncompetitive manner within the TC system. The regional levels of GABA blockade efficacy by zinc were: thalamus > NRT > cortex. The relationship between clonazepam and zinc sensitivity of GABAA-mediated responses was examined to investigate possible presence or absence of specific GABAAreceptor (GABAR) subunits. These properties of GABARs have been hypothesized previously to be dependent on presence or absence of the γ2 subunit and seem to display an inverse relationship. In cross-correlation plots, thalamic and NRT neurons did not show a statistically significant relationship between clonazepam and zinc sensitivity; however, a statistically significant correlation was observed in cortical neurons. Spontaneous epileptic TC oscillations can be induced in vitro by perfusion of TC slices with an extracellular medium containing no added Mg2+. Multiple varieties of oscillations are generated, including simple TC burst complexes (sTBCs), which resemble spike-wave discharge activity. A second variant was termed a complex TC burst complex (cTBC), which resembled generalized tonic clonic seizure activity. sTBCs were exacerbated by zinc, whereas cTBCs were blocked completely by zinc. This supported the concept that zinc release may modulate TC rhythms in vivo. Zinc interacts with a variety of ionic conductances, including GABAR currents, N-methyl-d-aspartate (NMDA) receptor currents, and transient potassium (A) currents.d−2-amino-5-phosphonovaleric acid and 4-aminopyridine blocked both s- and cTBCs in TC slices. Therefore NMDA and A current-blocking effects of zinc are insufficient to explain differential zinc sensitivity of these rhythms. This supports a significant role of zinc-induced GABAR modulation in differential TC rhythm effects. Zinc is localized in high levels within the TC system and appears to be released during TC activity. Furthermore application of exogenous zinc modulates TC rhythms and differentially blocks GABARs within the TC system. These data are consistent with the hypothesis that endogenously released zinc may have important neuromodulatory actions impacting generation of TC rhythms, mediated at least in part by effects on GABARs.

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Antibody against Microbial Neuraminidases Recognizes Human Sialidase 3 (NEU3): the Neuraminidase/Sialidase Superfamily Revisited

mBio ◽

10.1128/mbio.00078-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 4

Author(s):

Chiguang Feng ◽

Jihong Li ◽

Greg Snyder ◽

Wei Huang ◽

Simeon E. Goldblum ◽

...

Keyword(s):

Influenza Virus ◽

Clostridium Perfringens ◽

Neural Development ◽

Catalytic Domain ◽

Critical Role ◽

Microbial Pathogens ◽

Human Neutrophils ◽

Sialidase Activity

ABSTRACT Neuraminidases (NAs) are critical virulence factors for several microbial pathogens. With a highly conserved catalytic domain, a microbial NA “superfamily” has been proposed. We previously reported that murine polymorphonuclear leukocyte (PMN) sialidase activity was important in leukocyte trafficking to inflamed sites and that antibodies to Clostridium perfringens NA recognized a cell surface molecule(s), presumed to be a sialidase of eukaryotic origin on interleukin-8-stimulated human and murine PMNs. These antibodies also inhibited cell sialidase activity both in vitro and, in the latter instance, in vivo . We therefore hypothesized that mammalian sialidases share structural homology and epitopes with microbial NAs. We now report that antibodies to one of the isoforms of C. perfringens NA, as well as anti-influenza virus NA serum, recognize human NEU3 but not NEU1 and that antibodies to C. perfringens NA inhibit NEU3 enzymatic activity. We conclude that the previously described microbial NA superfamily extends to human sialidases. Strategies designed to therapeutically inhibit microbial NA may need to consider potential compromising effects on human sialidases, particularly those expressed in cells of the immune system. IMPORTANCE We previously reported that sialidase activity of human neutrophils plays a critical role in the host inflammatory response. Since the catalytic domains of microbial neuraminidases are highly conserved, we hypothesized that antibodies against Clostridium perfringens neuraminidase might inhibit mammalian sialidase activity. Before the recognition of four mammalian sialidase ( Neu ) isoforms, we demonstrated that anti- C. perfringens neuraminidase antibodies inhibited human and murine sialidase activity in vivo and in vitro . We now show that the antibodies to microbial neuraminidase ( C. perfringens and influenza virus) recognize human NEU3, which is important for neural development and cell signaling. Since many microbes that infect mucosal surfaces express neuraminidase, it is possible that the use of sialidase inhibitors (e.g., zanamivir), might also compromise human sialidase activity critical to the human immune response. Alternatively, sialidase inhibitors may prove useful in the treatment of hyperinflammatory conditions.

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The AMPK-related kinase NUAK1 controls cortical axons branching though a local modulation of mitochondrial metabolic functions

10.1101/2020.05.18.102582 ◽

2020 ◽

Author(s):

Marine Lanfranchi ◽

Géraldine Meyer-Dilhet ◽

Raphael Dos Reis ◽

Audrey Garcia ◽

Camille Blondet ◽

...

Keyword(s):

Cortical Neurons ◽

Metabolic Regulation ◽

Critical Role ◽

Dual Function ◽

Axon Branching ◽

Cellular Mechanisms ◽

Peripheral Neurons ◽

Branch Formation

ABSTRACTThe precise regulation of the cellular mechanisms underlying axonal morphogenesis is essential to the formation of functional neuronal networks. We previously identified the autism-candidate kinase NUAK1 as a central regulator of axon branching in mouse cortical neurons through the control of mitochondria trafficking. How does local mitochondrial position or function regulate axon branching during development? Here, we characterized the metabolic regulation in the developing axon and report a marked metabolic decorrelation between axon elongation and collateral branching. We next solved the cascade of event leading to presynaptic clustering and mitochondria recruitment during spontaneous branch formation. Interestingly and contrary to peripheral neurons, mitochondria are recruited after but not prior to branch formation in cortical neurons. Using flux metabolomics and fluorescent biosensors, we observed that NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration. Upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Altogether, our results indicate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondria distribution and activity, and suggest that a mitochondrial-dependent remodeling of local metabolic homeostasis plays a critical role during axon morphogenesis.

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Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X

10.1101/2022.01.09.475579 ◽

2022 ◽

Author(s):

Jinli Geng ◽

Wenxiang Li ◽

Yingjun Tang ◽

Yunming Gao ◽

Yitong Lu ◽

...

Keyword(s):

Neural Development ◽

Cortical Neurons ◽

Intracellular Signaling ◽

Sensory Response ◽

Imaging Data ◽

Membrane Excitability ◽

Sensory Evoked

Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. sensory response), and also mediate intracellular signaling cascades normally of longer time scales (e.g., Ca2+- dependent neuritogenesis). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate that the recently developed GCaMP-X outperforms GCaMP in long-term probe expression and/or chronic Ca2+ imaging. GCaMP-X shows much improved compatibility with neurons and thus more reliable than GCaMP as demonstrated in vivo by acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations over an extended time frame. Chronic Ca2+ imaging data (≥1 month) are acquired from the same set of cultured cortical neurons, unveiling that spontaneous/local Ca2+ activities would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length or soma size, the major metrics of oscillatory Ca2+, including rate, amplitude, synchrony among different neurons or organelles have also been examined along with the developmental stages. Both neuritogenesis and Ca2+ signals are dysregulated by GCaMP in virus-infected or transgenic neurons, in direct contrast to GCaMP-X without any noticeable side-effect. Such in vitro data altogether consolidate the unique importance of oscillatory Ca2+ to activity-dependent neuritogenesis, as one major factor responsible for the distinctions between GCaMP vs GCaMP-X in vivo. For the first time with GCaMP-X of long-term expression in neurons, spontaneous and sensory-evoked Ca2+ activities are imaged and evaluated both in vitro and in vivo, providing new opportunities to monitor neural development or other chronic processes concurrently with Ca2+ dynamics.

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Fast Rhythmic Bursting Can Be Induced in Layer 2/3 Cortical Neurons by Enhancing Persistent Na+ Conductance or by Blocking BK Channels

Journal of Neurophysiology ◽

10.1152/jn.00573.2002 ◽

2003 ◽

Vol 89 (2) ◽

pp. 909-921 ◽

Cited By ~ 97

Author(s):

Roger D. Traub ◽

Eberhard H. Buhl ◽

Tengis Gloveli ◽

Miles A. Whittington

Keyword(s):

Cortical Neurons ◽

Firing Pattern ◽

Critical Role ◽

Sensory Stimulation ◽

Bk Channels ◽

Bk Channel ◽

Neocortical Neurons ◽

Layer 2

Fast rhythmic bursting (or “chattering”) is a firing pattern exhibited by selected neocortical neurons in cats in vivo and in slices of adult ferret and cat brain. Fast rhythmic bursting (FRB) has been recorded in certain superficial and deep principal neurons and in aspiny presumed local circuit neurons; it can be evoked by depolarizing currents or by sensory stimulation and has been proposed to depend on a persistent g Na that causes spike depolarizing afterpotentials. We constructed a multicompartment 11-conductance model of a layer 2/3 pyramidal neuron, containing apical dendritic calcium-mediated electrogenesis; the model can switch between rhythmic spiking (RS) and FRB modes of firing, with various parameter changes. FRB in this model is favored by enhancing persistent g Na and also by measures that reduce [Ca2+]i or that reduce the conductance of g K(C) (a fast voltage- and Ca2+-dependent conductance). Axonal excitability plays a critical role in generating fast bursts in the model. In vitro experiments in rat layer 2/3 neurons confirmed (as shown previously by others) that RS firing could be switched to fast rhythmic bursting, either by buffering [Ca2+]i or by enhancing persistent g Na. In addition, our experiments confirmed the model prediction that reducing g KC (with iberiotoxin) would favor FRB. During the bursts, fast prepotentials (spikelets) could occur that did not originate in apical dendrites and that appear to derive from the axon. We suggest that modulator-induced regulation of [Ca2+] dynamics or of BK channel conductance, for example via protein kinase A, could play a role in determining the firing pattern of neocortical neurons; specifically, such modulation could play a role in regulating whether neurons respond to strong stimulation with fast rhythmic bursts.

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High current optogenetic channels for stimulation and inhibition of primary rat cortical neurons

10.1101/145441 ◽

2017 ◽

Author(s):

Lei Jin ◽

Eike Frank Joest ◽

Wenfang Li ◽

Shiqiang Gao ◽

Andreas Offenhäusser ◽

...

Keyword(s):

Neural Development ◽

Cortical Neurons ◽

Single Channel ◽

Chloride Concentration ◽

Neuronal Firing ◽

High Current ◽

Neuronal Inhibition ◽

Rat Cortical Neurons

AbstractChR2-XXL and GtACR1 are currently the cation and anion ends of the optogenetic single channel current range. These were used in primary rat cortical neurons in vitro to manipulate neuronal firing patterns. ChR2-XXL provides high cation currents via elevated light sensitivity and a prolonged open state. Stimulating ChR2-XXL expressing putative presynaptic neurons induced neurotransmission. Moreover, stable depolarisation block could be generated in single neurons using ChR2-XXL, proving that ChR2-XXL is a promising candidate for in vivo applications of optogenetics, for example to treat peripheral neuropathic pain. We also addressed an anion channelrhodopsin (GtACR1) for the next generation of optogenetic neuronal inhibition in primary rat cortical neurons. GtACR1‘s light-gated chloride conduction was verified in primary neurons and the efficient photoinhibition of action potentials, including spontaneous activity, was shown. Our data also implies that the chloride concentration in neurons decreases during neural development. In both cases, we find surprising applications of these high current channels. For ChR2-XXL inhibition and stimulation are possible, while for GtACR1 the role of Cl−during neural development becomes a new optogenetic target.

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RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽

10.1038/s41388-021-01877-4 ◽

2021 ◽

Author(s):

Jiuna Zhang ◽

Xiaoyu Jiang ◽

Jie Yin ◽

Shiying Dou ◽

Xiaoli Xie ◽

...

Keyword(s):

Colorectal Cancer ◽

Plasma Membrane ◽

Tumor Growth ◽

Cancer Progression ◽

Critical Role ◽

Ring Finger ◽

Immunohistochemical Analysis ◽

Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

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Emerging Roles for Ion Channels in Ovarian Cancer: Pathomechanisms and Pharmacological Treatment

Cancers ◽

10.3390/cancers13040668 ◽

2021 ◽

Vol 13 (4) ◽

pp. 668

Author(s):

Concetta Altamura ◽

Maria Raffaella Greco ◽

Maria Rosaria Carratù ◽

Rosa Angela Cardone ◽

Jean-François Desaphy

Keyword(s):

Ovarian Cancer ◽

Ion Channels ◽

Transient Receptor Potential ◽

Critical Role ◽

Gynecologic Cancer ◽

Receptor Potential ◽

Transient Receptor Potential Channels ◽

Platinum Resistance

Ovarian cancer (OC) is the deadliest gynecologic cancer, due to late diagnosis, development of platinum resistance, and inadequate alternative therapy. It has been demonstrated that membrane ion channels play important roles in cancer processes, including cell proliferation, apoptosis, motility, and invasion. Here, we review the contribution of ion channels in the development and progression of OC, evaluating their potential in clinical management. Increased expression of voltage-gated and epithelial sodium channels has been detected in OC cells and tissues and shown to be involved in cancer proliferation and invasion. Potassium and calcium channels have been found to play a critical role in the control of cell cycle and in the resistance to apoptosis, promoting tumor growth and recurrence. Overexpression of chloride and transient receptor potential channels was found both in vitro and in vivo, supporting their contribution to OC. Furthermore, ion channels have been shown to influence the sensitivity of OC cells to neoplastic drugs, suggesting a critical role in chemotherapy resistance. The study of ion channels expression and function in OC can improve our understanding of pathophysiology and pave the way for identifying ion channels as potential targets for tumor diagnosis and treatment.

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Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer’s disease

Cell Death and Differentiation ◽

10.1038/s41418-020-00685-9 ◽

2021 ◽

Author(s):

Wen-Dai Bao ◽

Pei Pang ◽

Xiao-Ting Zhou ◽

Fan Hu ◽

Wan Xiong ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Memory Impairment ◽

Iron Homeostasis ◽

Critical Role ◽

Gene Set Enrichment Analysis ◽

Molecular Characteristics ◽

Intracellular Iron

AbstractIron homeostasis disturbance has been implicated in Alzheimer’s disease (AD), and excess iron exacerbates oxidative damage and cognitive defects. Ferroptosis is a nonapoptotic form of cell death dependent upon intracellular iron. However, the involvement of ferroptosis in the pathogenesis of AD remains elusive. Here, we report that ferroportin1 (Fpn), the only identified mammalian nonheme iron exporter, was downregulated in the brains of APPswe/PS1dE9 mice as an Alzheimer’s mouse model and Alzheimer’s patients. Genetic deletion of Fpn in principal neurons of the neocortex and hippocampus by breeding Fpnfl/fl mice with NEX-Cre mice led to AD-like hippocampal atrophy and memory deficits. Interestingly, the canonical morphological and molecular characteristics of ferroptosis were observed in both Fpnfl/fl/NEXcre and AD mice. Gene set enrichment analysis (GSEA) of ferroptosis-related RNA-seq data showed that the differentially expressed genes were highly enriched in gene sets associated with AD. Furthermore, administration of specific inhibitors of ferroptosis effectively reduced the neuronal death and memory impairments induced by Aβ aggregation in vitro and in vivo. In addition, restoring Fpn ameliorated ferroptosis and memory impairment in APPswe/PS1dE9 mice. Our study demonstrates the critical role of Fpn and ferroptosis in the progression of AD, thus provides promising therapeutic approaches for this disease.

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TRAF3 mediates neuronal apoptosis in early brain injury following subarachnoid hemorrhage via targeting TAK1-dependent MAPKs and NF-κB pathways

Cell Death and Disease ◽

10.1038/s41419-020-03278-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yan Zhou ◽

Tao Tao ◽

Guangjie Liu ◽

Xuan Gao ◽

Yongyue Gao ◽

...

Keyword(s):

Subarachnoid Hemorrhage ◽

Brain Injury ◽

Therapeutic Target ◽

Cortical Neurons ◽

Neuronal Apoptosis ◽

Early Brain Injury ◽

Neurological Deficits ◽

Human Brain Tissue

AbstractNeuronal apoptosis has an important role in early brain injury (EBI) following subarachnoid hemorrhage (SAH). TRAF3 was reported as a promising therapeutic target for stroke management, which covered several neuronal apoptosis signaling cascades. Hence, the present study is aimed to determine whether downregulation of TRAF3 could be neuroprotective in SAH-induced EBI. An in vivo SAH model in mice was established by endovascular perforation. Meanwhile, primary cultured cortical neurons of mice treated with oxygen hemoglobin were applied to mimic SAH in vitro. Our results demonstrated that TRAF3 protein expression increased and expressed in neurons both in vivo and in vitro SAH models. TRAF3 siRNA reversed neuronal loss and improved neurological deficits in SAH mice, and reduced cell death in SAH primary neurons. Mechanistically, we found that TRAF3 directly binds to TAK1 and potentiates phosphorylation and activation of TAK1, which further enhances the activation of NF-κB and MAPKs pathways to induce neuronal apoptosis. Importantly, TRAF3 expression was elevated following SAH in human brain tissue and was mainly expressed in neurons. Taken together, our study demonstrates that TRAF3 is an upstream regulator of MAPKs and NF-κB pathways in SAH-induced EBI via its interaction with and activation of TAK1. Furthermore, the TRAF3 may serve as a novel therapeutic target in SAH-induced EBI.

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