scholarly journals Brain Region-Specific Neuroprotective Action and Signaling of Corticotropin-Releasing Hormone in Primary Neurons

Endocrinology ◽  
2003 ◽  
Vol 144 (9) ◽  
pp. 4051-4060 ◽  
Author(s):  
Nadhim Bayatti ◽  
Jürgen Zschocke ◽  
Christian Behl
Keyword(s):  
Brain Region ◽  
Cortical Neurons ◽  
Intracellular Signaling ◽  
Glycogen Synthase ◽  
Primary Cultures ◽  
Amyloid Β ◽  
Receptor Expression ◽  
Neuronal Cultures ◽  
Neuroprotective Effects ◽  
Hippocampal Cultures

Abstract CRH regulates the body’s response to stressful stimuli by modulating the activity of the hypothalamic pituitary axis. In primary cultures and cell lines, CRH also acts as a potent neuroprotective factor in response to a number of toxins. Using primary neuronal cultures from the cerebellum, cerebral cortex, and hippocampus, we demonstrate that CRH exerts a brain region-specific neuroprotective effect on amyloid β 25–35 toxicity. At low CRH concentrations (10−8m), neuroprotective effects can be observed only in cerebellar and hippocampal cultures, but a higher CRH concentration (10−7m) additionally led to the protection of cortical neurons. These neuroprotective effects were inhibited by H89, a specific protein kinase A inhibitor. Western blot analysis, carried out using phospho-specific antibodies directed against MAPK, cAMP response element-binding protein (CREB), and glycogen synthase kinase (GSK)3β also resulted in brain legion-specific differences regarding intracellular signaling. Correlating with cell survival, low CRH concentrations resulted in activation of the CREB pathway and inactivation of GSK3β in cerebellar and hippocampal cultures, but higher concentrations additionally resulted in activated CREB and inactivated GSK3β in cortical cultures. In contrast, MAPK activation occurred only in cortical neurons. Differences in signaling were found to be independent of receptor expression levels because RT-PCR analysis indicated no region-specific differences in CRHR1 mRNA expression.

scholarly journals Calcium export from neurons and multi-kinase signaling cascades contribute to ouabain neuroprotection in hyperhomocysteinemia

2020 ◽  
Author(s):  
Maria A. Ivanova ◽  
Arina D. Kokorina ◽  
Polina D. Timofeeva ◽  
Tatiana V. Karelina ◽  
Polina A. Abushik ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Intracellular Signaling ◽  
Primary Cultures ◽  
Membrane Voltage ◽  
Glutamate Excitotoxicity ◽  
Kinase Signaling ◽  
Neuroprotective Effects ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Voltage Loss

Abstract Background: Subnanomolar ouabain binding to the Na,K-ATPase triggers intracellular signaling, prevents an overload of neurons with Ca2+, and their apoptosis caused by glutamate receptor agonists. Elevated plasma homocysteine (HCY), known as hyperhomocysteinemia, represents a risk factor for stroke and can exacerbate many neuronal disorders. HCY acts as a persistent N-methyl-D-aspartate receptor (NMDAR) agonist, which, in contrast to glutamate, desensitizes NMDARs containing GluN2B subunits. Mechanisms of HCY neurotoxicity remain not clearly understood since GluN2B-containing NMDARs provide a major contribution to excitotoxicity among glutamate receptors.Methods: Using fluorescent tools combined with the confocal microscopy, we compared 0.1 - 1 nM ouabain effects on the intracellular Ca2+ signaling, on the mitochondrial inner membrane voltage and the cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. We also studied an apoptosis-related protein expression and the involvement of some kinases in ouabain mediated effects.Results: In short insults HCY was less potent than glutamate as a neurotoxic agent. This amino acid induced the voltage loss (∆φmit) of 0.2 of the total mitochondrial inner membrane voltage (φmit) instead of ∆φmit = 0.7 for glutamate. We have found that subnanomolar ouabain exhibited rapid and postponed neuroprotective effects on neurons and (1) rapidly reduced the Ca2+ overload of neurons and the voltage loss of inner mitochondrial membranes evoked by glutamate and HCY, and (2) prevented neuronal apoptosis during 24 h treatments with glutamate or HCY. Using a set of specific kinase inhibitors such as PKA inhibitor, chelerythrine, and KN93, we demonstrated the role of multi-kinase signaling pathways involving PKC and PKA in neuronal survival caused by ouabain in hyperhomocysteinemia. Conclusions: Subnanomolar ouabain prevents neurodegeneration caused by glutamate and HCY. For both amino acids, ouabain evokes an acceleration of Ca2+ export by sodium-calcium exchangers from neurons preventing the voltage loss by mitochondrial inner membranes that rescue neurons in short insults. In prolonged insults, ouabain triggers intracellular neuroprotective cascades, including activation of PKA and PKC for HCY, but not for glutamate. This suggests that different appropriate pharmacology for hyperhomocysteinemia and glutamate excitotoxicity could be applied for clinical treatments.

scholarly journals Ketamine decreases neuronally released glutamate via retrograde stimulation of presynaptic adenosine A1 receptors

2021 ◽  
Author(s):  
Vesna Lazarevic ◽  
Yunting Yang ◽  
Ivana Flais ◽  
Per Svenningsson
Keyword(s):  
Cortical Neurons ◽  
Intracellular Signaling ◽  
Ex Vivo ◽  
Forced Swim Test ◽  
Glutamate Release ◽  
Neuronal Cultures ◽  
Extracellular Glutamate ◽  
Primary Neuronal Cultures ◽  
Adenosine A1 Receptors ◽  
Adenosine A1

AbstractKetamine produces a rapid antidepressant response in patients with major depressive disorder (MDD), but the underlying mechanisms appear multifaceted. One hypothesis, proposes that by antagonizing NMDA receptors on GABAergic interneurons, ketamine disinhibits afferens to glutamatergic principal neurons and increases extracellular glutamate levels. However, ketamine seems also to reduce rapid glutamate release at some synapses. Therefore, clinical studies in MDD patients have stressed the need to identify mechanisms whereby ketamine decreases presynaptic activity and glutamate release. In the present study, the effect of ketamine and its antidepressant metabolite, (2R,6R)-HNK, on neuronally derived glutamate release was examined in rodents. We used FAST methodology to measure depolarization-evoked extracellular glutamate levels in vivo in freely moving or anesthetized animals, synaptosomes to detect synaptic recycling ex vivo and primary cortical neurons to perform functional imaging and to examine intracellular signaling in vitro. In all these versatile approaches, ketamine and (2R,6R)-HNK reduced glutamate release in a manner which could be blocked by AMPA receptor antagonism. Antagonism of adenosine A1 receptors, which are almost exclusively expressed at nerve terminals, also counteracted ketamine’s effect on glutamate release and presynaptic activity. Signal transduction studies in primary neuronal cultures demonstrated that ketamine reduced P-T286-CamKII and P-S9-Synapsin, which correlated with decreased synaptic vesicle recycling. Moreover, systemic administration of A1R antagonist counteracted the antidepressant-like actions of ketamine and (2R,6R)-HNK in the forced swim test. To conclude, by studying neuronally released glutamate, we identified a novel retrograde adenosinergic feedback mechanism that mediate inhibitory actions of ketamine on glutamate release that may contribute to its rapid antidepressant action.

Neuroprotective effects of vitexin by inhibition of NMDA receptors in primary cultures of mouse cerebral cortical neurons

2013 ◽  
Vol 386 (1-2) ◽  
pp. 251-258 ◽  
Author(s):  
Le Yang ◽  
Zhi-ming Yang ◽  
Nan Zhang ◽  
Zhen Tian ◽  
Shui-bing Liu ◽  
...  
Keyword(s):  
Nmda Receptors ◽  
Cortical Neurons ◽  
Primary Cultures ◽  
Neuroprotective Effects ◽  
Cerebral Cortical Neurons

scholarly journals Reelin Signals through Phosphatidylinositol 3-Kinase and Akt To Control Cortical Development and through mTor To Regulate Dendritic Growth

2007 ◽  
Vol 27 (20) ◽  
pp. 7113-7124 ◽  
Author(s):  
Yves Jossin ◽  
André M. Goffinet
Keyword(s):  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Matrix Protein ◽  
Glycogen Synthase ◽  
Extracellular Matrix Protein ◽  
Cortical Plate ◽  
Target Cells ◽  
Neuronal Cultures ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

ABSTRACT Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3β (GSK3β) are not. On the other hand, mTor, but not GSK3β, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt.

scholarly journals Excitotoxic glutamate causes neuronal insulin resistance by inhibiting insulin receptor/Akt/mTOR pathway

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Igor Pomytkin ◽  
Irina Krasil’nikova ◽  
Zanda Bakaeva ◽  
Alexander Surin ◽  
Vsevolod Pinelis
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Cortical Neurons ◽  
Glycogen Synthase ◽  
Primary Cultures ◽  
Biological Response ◽  
Mtor Pathway ◽  
Serine Phosphorylation ◽  
Glutamate Excitotoxicity ◽  
Neuronal Insulin Resistance

Abstract Aim An impaired biological response to insulin in the brain, known as central insulin resistance, was identified during stroke and traumatic brain injury, for which glutamate excitotoxicity is a common pathogenic factor. The exact molecular link between excitotoxicity and central insulin resistance remains unclear. To explore this issue, the present study aimed to investigate the effects of glutamate-evoked increases in intracellular free Ca2+ concentrations [Ca2+]i and mitochondrial depolarisations, two key factors associated with excitotoxicity, on the insulin-induced activation of the insulin receptor (IR) and components of the Akt/ mammalian target of rapamycin (mTOR) pathway in primary cultures of rat cortical neurons. Methods Changes in [Ca2+]i and mitochondrial inner membrane potentials (ΔΨm) were monitored in rat cultured cortical neurons, using the fluorescent indicators Fura-FF and Rhodamine 123, respectively. The levels of active, phosphorylated signalling molecules associated with the IR/Akt/mTOR pathway were measured with the multiplex fluorescent immunoassay. Results When significant mitochondrial depolarisations occurred due to glutamate-evoked massive influxes of Ca2+ into the cells, insulin induced 48% less activation of the IR (assessed by IR tyrosine phosphorylation, pY1150/1151), 72% less activation of Akt (assessed by Akt serine phosphorylation, pS473), 44% less activation of mTOR (assessed by mTOR pS2448), and 38% less inhibition of glycogen synthase kinase β (GSK3β) (assessed by GSK3β pS9) compared with respective controls. These results suggested that excitotoxic glutamate inhibits signalling via the IR/Akt/mTOR pathway at multiple levels, including the IR, resulting in the development of acute neuronal insulin resistance within minutes, as an early pathological event associated with excitotoxicity.

scholarly journals Loss of function of the mitochondrial peptidase PITRM1 induces proteotoxic stress and Alzheimer’s disease-like pathology in human cerebral organoids

2020 ◽  
Author(s):  
Dina Ivanyuk ◽  
María José Pérez ◽  
Vasiliki Panagiotakopoulou ◽  
Gabriele Di Napoli ◽  
Dario Brunetti ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Death ◽  
Cortical Neurons ◽  
Amyloid Β ◽  
Neuronal Cell ◽  
Protein Aggregates ◽  
Neuronal Cultures ◽  
Loss Of Function ◽  
Proteotoxic Stress

AbstractMutations in pitrilysin metallopeptidase 1 (PITRM1), a mitochondrial protease involved in mitochondrial precursor processing and degradation, result in a slow-progressive syndrome, characterized by cerebellar ataxia, psychotic episodes and obsessive behavior as well as cognitive decline. To investigate the pathogenetic mechanisms of mitochondrial presequence processing, we employed cortical neurons and cerebral organoids generated from PITRM1 knockout human induced pluripotent stem cells (iPSCs). PITRM1 deficiency strongly induced mitochondrial unfolded protein response (UPRmt) and enhanced mitochondrial clearance in iPSC-derived neurons. Furthermore, we observed increased levels of amyloid precursor protein and amyloid β in PITRM1 knockout neurons. However, neither cell death nor protein aggregates were observed in 2D iPSC-derived cortical neuronal cultures. On the contrary, cerebral organoids generated from PITRM1 knockout iPSCs spontaneously developed over time pathological features of Alzheimer’s disease (AD), including accumulation of protein aggregates, tau pathology, and neuronal cell death. Importantly, we provide evidence for a protective role of UPRmt and mitochondrial clearance against impaired mitochondrial presequence processing and proteotoxic stress. In summary, we propose a novel concept of PITRM1-linked neurological syndrome whereby defects of mitochondrial presequence processing induce an early activation of UPRmt that, in turn, modulates cytosolic quality control pathways. Thus our work supports a mechanistic link between mitochondrial function and common neurodegenerative proteinopathies.

scholarly journals Quantifying Amyloid Beta (Aβ)–Mediated Changes in Neuronal Morphology in Primary Cultures

2012 ◽  
Vol 17 (6) ◽  
pp. 835-842 ◽  
Author(s):  
Lan Nguyen ◽  
Sarah Wright ◽  
Mike Lee ◽  
Zhao Ren ◽  
John-Michael Sauer ◽  
...  
Keyword(s):  
Amyloid Beta ◽  
Cell Injury ◽  
Primary Cultures ◽  
Neuronal Morphology ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Blocking Antibody ◽  
Cortical Cultures ◽  
Hippocampal Cultures

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in the brain. Herein, the authors quantify Aβ-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aβ caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aβ-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aβ-mediated cytotoxicity. Finally, we showed that Aβ challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aβ-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.

Ketamine Inhibits Glutamate-, N-Methyl-D-Aspartate-, and Quisqualate-stimulated cGMP Production in Cultured Cerebral Neurons 

1995 ◽  
Vol 82 (1) ◽  
pp. 205-213 ◽  
Author(s):  
Jerry M. Gonzales ◽  
Alex L. Loeb ◽  
Peter S. Reichard ◽  
Steven Irvine
Keyword(s):  
Cortical Neurons ◽  
Primary Cultures ◽  
Neuronal Cultures ◽  
No Production ◽  
Mk 801 ◽  
Cgmp Production ◽  
Glutamatergic Signaling ◽  
Cerebral Neurons ◽  
Synthase Inhibitor ◽  
Stimulation Of

Background Glutamatergic signaling has been linked to the recently discovered neurotransmitter/neuromodulator nitric oxide (NO), and several classes of anesthetics block some step in glutamatergic signaling. This study was designed to determine whether or not ketamine would prevent NO-dependent cGMP production stimulated by glutamate (GLU) and the GLU analogs NMDA, quisqualate (QUIS), and kainate (KAIN). Methods Primary cultures of cortical neurons and glia (prepared from 16-day gestational rat fetuses) were used after 12-16 days in culture. Reactions were carried out in magnesium-free buffer containing 100 microM 3-isobutyl-1-methylxanthine, and cGMP content of cultures was used as a bioassay of NO production. Results Cyclic GMP production stimulated by sodium nitroprusside (100 microM) occurred predominately in neurons and not in glia. Neurons were spontaneously active in these cultures; basal cGMP production was decreased by 50% in the presence of 1 microM tetrodotoxin (TTX). Glutamate (100 microM), NMDA (100 microM), QUIS (300 microM), and KAIN (100 microM) each increased cGMP content of neuronal cultures. L-NMMA (100 microM), a NO synthase inhibitor, prevented the stimulation of cGMP production by GLU or its analogs. Pretreatment with MK-801 (1 microM) or ketamine (10-100 microM) inhibited GLU-, NMDA-, and QUIS-stimulated cGMP production. Quisqualate-stimulated responses were the most sensitive to inhibition by ketamine and NMDA-stimulated responses were the least sensitive to inhibition. MK-801 and ketamine did not significantly inhibit KAIN-stimulated cGMP production. CNQX (10 microns) blocked KAIN-stimulated cGMP production only. Conclusions The authors' data demonstrate that ketamine inhibited NO synthesis stimulated by NMDA- and non-NMDA-receptor specific analogs. Our findings indicate that blockade of QUIS- as well as NMDA-subtypes of GLU- receptor may be important in the development of ketamine-induced anesthesia.

scholarly journals Calcium Export from Neurons and Multi-Kinase Signaling Cascades Contribute to Ouabain Neuroprotection in Hyperhomocysteinemia

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1104
Author(s):  
Maria A. Ivanova ◽  
Arina D. Kokorina ◽  
Polina D. Timofeeva ◽  
Tatiana V. Karelina ◽  
Polina A. Abushik ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Primary Cultures ◽  
Glutamate Excitotoxicity ◽  
Neuroprotective Effects ◽  
Mediated Effects ◽  
Mitochondrial Inner Membrane ◽  
Rat Cortical Neurons ◽  
Proteinkinase C ◽  
Apoptosis Related Protein

Pathological homocysteine (HCY) accumulation in the human plasma, known as hyperhomocysteinemia, exacerbates neurodegenerative diseases because, in the brain, this amino acid acts as a persistent N-methyl-d-aspartate receptor agonist. We studied the effects of 0.1–1 nM ouabain on intracellular Ca2+ signaling, mitochondrial inner membrane voltage (φmit), and cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. In addition, apoptosis-related protein expression and the involvement of some kinases in ouabain-mediated effects were evaluated. In short insults, HCY was less potent than glutamate as a neurotoxic agent and induced a 20% loss of φmit, whereas glutamate caused a 70% decrease of this value. Subnanomolar ouabain exhibited immediate and postponed neuroprotective effects on neurons. (1) Ouabain rapidly reduced the Ca2+ overload of neurons and loss of φmit evoked by glutamate and HCY that rescued neurons in short insults. (2) In prolonged 24 h excitotoxic insults, ouabain prevented neuronal apoptosis, triggering proteinkinase A and proteinkinase C dependent intracellular neuroprotective cascades for HCY, but not for glutamate. We, therefore, demonstrated here the role of PKC and PKA involving pathways in neuronal survival caused by ouabain in hyperhomocysteinemia, which suggests existence of different appropriate pharmacological treatment for hyperhomocysteinemia and glutamate excitotoxicity.

Sign in / Sign up

Export Citation Format

Share Document