scholarly journals Excitatory pathway engaging glutamate, calcineurin, and NFAT upregulates IL-4 in ischemic neurons to polarize microglia

2019 ◽  
Vol 40 (3) ◽  
pp. 513-527 ◽  
Author(s):  
Shun-Ming Ting ◽  
Xiurong Zhao ◽  
Xueping Zheng ◽  
Jaroslaw Aronowski
Keyword(s):  
Cortical Neurons ◽  
Culture Medium ◽  
Calcineurin Inhibitor ◽  
Glucose Deprivation ◽  
Interleukin 4 ◽  
Activated T Cells ◽  
Injury Model ◽  
Rat Cortical Neurons ◽  
Anti Inflammatory Cytokine

Excitotoxicity and microglia/macrophage over-activation are the important pathogenic steps in brain damage caused by ischemic stroke. Recent studies from our group suggest that the neurons in ischemic penumbra generate an anti-inflammatory cytokine, interleukin-4 (IL-4). This neuron-produced IL-4 could subsequently convert surrounding microglia/macrophages to a reparative (M2)-phenotype. The present study was designed to establish the mechanisms by which neurons under transient ischemic condition produce/secrete IL-4. We employed primary rat cortical neurons and a validated in vitro ischemic injury model involving transient oxygen–glucose deprivation (OGD). We discovered that only sublethal OGD induces IL-4 production/secretion by neurons. We then showed that excitotoxic stimulus (an integral component of OGD-mediated damage) involving N-methyl-D-aspartate (NMDA), and not kainate receptor, triggers neuronal IL-4 production/release. Of note, oxidative stress or pro-apoptotic stimuli did not induce IL-4 production by neurons. Next, using the calcineurin inhibitor FK506, we implicated this phosphatase in activation of the nuclear factor of activated T-cells (NFAT; a transcription factor activated through calcineurin-mediated dephosphorylation) and propose that this pathway is involved in transcriptional upregulation of the IL-4 synthesis in NMDA-treated neurons. Finally, using a transfer of culture medium from NMDA-conditioned neuron to microglia, we showed that the neuronal IL-4 can polarize microglia toward a restorative, phagocytic phenotype.

scholarly journals Comparative Proteomics Unveils LRRFIP1 as a New Player in the DAPK1 Interactome of Neurons Exposed to Oxygen and Glucose Deprivation

Antioxidants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1202
Author(s):  
Núria DeGregorio-Rocasolano ◽  
Verónica Guirao ◽  
Jovita Ponce ◽  
Marc Melià-Sorolla ◽  
Alicia Aliena-Valero ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Primary Cultures ◽  
Lipid Peroxides ◽  
Glucose Deprivation ◽  
Redox Status ◽  
Protein Levels ◽  
Neuronal Fate ◽  
Death Associated Protein Kinase ◽  
Rat Cortical Neurons

Death-associated protein kinase 1 (DAPK1) is a pleiotropic hub of a number of networked distributed intracellular processes. Among them, DAPK1 is known to interact with the excitotoxicity driver NMDA receptor (NMDAR), and in sudden pathophysiological conditions of the brain, e.g., stroke, several lines of evidence link DAPK1 with the transduction of glutamate-induced events that determine neuronal fate. In turn, DAPK1 expression and activity are known to be affected by the redox status of the cell. To delineate specific and differential neuronal DAPK1 interactors in stroke-like conditions in vitro, we exposed primary cultures of rat cortical neurons to oxygen/glucose deprivation (OGD), a condition that increases reactive oxygen species (ROS) and lipid peroxides. OGD or control samples were co-immunoprecipitated separately, trypsin-digested, and proteins in the interactome identified by high-resolution LC-MS/MS. Data were processed and curated using bioinformatics tools. OGD increased total DAPK1 protein levels, cleavage into shorter isoforms, and dephosphorylation to render the active DAPK1 form. The DAPK1 interactome comprises some 600 proteins, mostly involving binding, catalytic and structural molecular functions. OGD up-regulated 190 and down-regulated 192 candidate DAPK1-interacting proteins. Some differentially up-regulated interactors related to NMDAR were validated by WB. In addition, a novel differential DAPK1 partner, LRRFIP1, was further confirmed by reverse Co-IP. Furthermore, LRRFIP1 levels were increased by pro-oxidant conditions such as ODG or the ferroptosis inducer erastin. The present study identifies novel partners of DAPK1, such as LRRFIP1, which are suitable as targets for neuroprotection.

Apocynum venetum leaf extract protects rat cortical neurons from injury induced by oxygen and glucose deprivation in vitro

2010 ◽  
Vol 88 (9) ◽  
pp. 907-917 ◽  
Author(s):  
Jun Xiang ◽  
Yu-Ping Tang ◽  
Zi-Yi Zhou ◽  
Pin Wu ◽  
Zhong Wang ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Leaf Extract ◽  
Caspase 3 ◽  
Cell Injury ◽  
Glucose Deprivation ◽  
Oxygen And Glucose Deprivation ◽  
Bax Protein ◽  
Rat Cortical Neurons ◽  
Apocynum Venetum

This study aimed to investigate the protective effect of Apocynum venetum leaf extract (AVLE) on an in vitro model of ischemia–reperfusion induced by oxygen and glucose deprivation (OGD) and further explored the possible mechanisms underlying protection. Cell injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage; cell viability was measured by XTT reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3, -8, and -9 activation and Bcl-2/Bax protein expression were determined by Western blot analysis. We report that treatment with AVLE (5 and 50 µg/mL) effectively reduced neuronal cell death and relieved cell injury induced by OGD. Moreover, AVLE decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspase-3 and -8 and Bax, and inhibited the reduction of Bcl-2 expression. These findings indicate that AVLE protects against OGD-induced injury by inhibiting apoptosis in rat cortical neurons by down-regulating caspase-3 activation and modulating the Bcl-2/Bax ratio.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals The Carbamate, Physostigmine does not Impair Axonal Transport in Rat Cortical Neurons

2021 ◽  
Vol 16 ◽  
pp. 263310552110202
Author(s):  
Sean X Naughton ◽  
Wayne D Beck ◽  
Zhe Wei ◽  
Guangyu Wu ◽  
Peter W Baas ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Cortical Neurons ◽  
Acetylcholinesterase Inhibitors ◽  
Neurological Deficits ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rat Cortical Neurons ◽  
Toxicological Mechanism

Among the various chemicals that are commonly used as pesticides, organophosphates (OPs), and to a lesser extent, carbamates, are most frequently associated with adverse long-term neurological consequences. OPs and the carbamate, pyridostigmine, used as a prophylactic drug against potential nerve agent attacks, have also been implicated in Gulf War Illness (GWI), which is often characterized by chronic neurological symptoms. While most OP- and carbamate-based pesticides, and pyridostigmine are relatively potent acetylcholinesterase inhibitors (AChEIs), this toxicological mechanism is inadequate to explain their long-term health effects, especially when no signs of acute cholinergic toxicity are exhibited. Our previous work suggests that a potential mechanism of the long-term neurological deficits associated with OPs is impairment of axonal transport (AXT); however, we had not previously evaluated carbamates for this effect. Here we thus evaluated the carbamate, physostigmine (PHY), a highly potent AChEI, on AXT using an in vitro neuronal live imaging assay that we have previously found to be very sensitive to OP-related deficits in AXT. We first evaluated the OP, diisopropylfluorophosphate (DFP) (concentration range 0.001-10.0 µM) as a reference compound that we found previously to impair AXT and subsequently evaluated PHY (concentration range 0.01-100 nM). As expected, DFP impaired AXT in a concentration-dependent manner, replicating our previously published results. In contrast, none of the concentrations of PHY (including concentrations well above the threshold for impairing AChE) impaired AXT. These data suggest that the long-term neurological deficits associated with some carbamates are not likely due to acute impairments of AXT.

scholarly journals Scapinin-induced Inhibition of Axon Elongation Is Attenuated by Phosphorylation and Translocation to the Cytoplasm

2011 ◽  
Vol 286 (22) ◽  
pp. 19724-19734 ◽  
Author(s):  
Hovik Farghaian ◽  
Yu Chen ◽  
Ada W. Y. Fu ◽  
Amy K. Y. Fu ◽  
Jacque P. K. Ip ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Brain Cortex ◽  
Inhibitory Effect ◽  
Adult Brain ◽  
Actin Binding ◽  
Mutant Form ◽  
Axon Elongation ◽  
Rat Cortical Neurons ◽  
The Brain

Scapinin is an actin- and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain.

scholarly journals Modulator of apoptosis-1 is a potential therapeutic target in acute ischemic injury

2018 ◽  
Vol 39 (12) ◽  
pp. 2406-2418 ◽  
Author(s):  
Su Jing Chan ◽  
Hui Zhao ◽  
Kazuhide Hayakawa ◽  
Chou Chai ◽  
Chong Teik Tan ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Infarct Volume ◽  
Neuronal Loss ◽  
Ischemic Injury ◽  
Glucose Deprivation ◽  
Artery Occlusion ◽  
In Vivo Models ◽  
The Brain

Modulator of apoptosis 1 (MOAP-1) is a Bax-associating protein highly enriched in the brain. In this study, we examined the role of MOAP-1 in promoting ischemic injuries following a stroke by investigating the consequences of MOAP-1 overexpression or deficiency in in vitro and in vivo models of ischemic stroke. MOAP-1 overexpressing SH-SY5Y cells showed significantly lower cell viability following oxygen and glucose deprivation (OGD) treatment when compared to control cells. Consistently, MOAP-1−/− primary cortical neurons were observed to be more resistant against OGD treatment than the MOAP-1+/+ primary neurons. In the mouse transient middle cerebral artery occlusion (tMCAO) model, ischemia triggered MOAP-1/Bax association, suggested activation of the MOAP-1-dependent apoptotic cascade. MOAP-1−/− mice were found to exhibit reduced neuronal loss and smaller infarct volume 24 h after tMCAO when compared to MOAP-1+/+ mice. Correspondingly, MOAP-1−/− mice also showed better integrity of neurological functions as demonstrated by their performance in the rotarod test. Therefore, both in vitro and in vivo data presented strongly support the conclusion that MOAP-1 is an important apoptotic modulator in ischemic injury. These results may suggest that a reduction of MOAP-1 function in the brain could be a potential therapeutic approach in the treatment of acute stroke.

Abstract TP101: Intra-arterial IL-1α is Well Tolerated and Neuroprotective After Experimental Ischemic Stroke

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Kathleen E Salmeron ◽  
Michael E Maniskas ◽  
Amanda Trout ◽  
Emmanuel Pinteaux ◽  
Justin F Fraser ◽  
...  
Keyword(s):  
Side Effects ◽  
Cortical Neurons ◽  
Glucose Deprivation ◽  
Cresyl Violet ◽  
Current Standard ◽  
Preclinical Research ◽  
Vessel Occlusion ◽  
Post Stroke ◽  
Primary Mouse

Endovascular thrombectomy and t-PA are the only current standard of care treatments for emergent large vessel occlusion (ELVO) stroke. Despite rising recanalization rates, stroke remains the leading cause of long-term disability worldwide suggesting that additional therapies are needed. Severe stroke morbidity may be due, in part, to the acute and sustained inflammatory stroke response. Preclinical research has supported anti-inflammatory agents in limiting brain injury and improving functional outcome; however, the post-stroke inflammatory cascade appears to have both beneficial and deleterious effects, necessitating careful therapeutic translation. We have recently demonstrated that delayed (3 day) post-stroke intravenous (IV) administration of the interleukin (IL)-1α (one of the two major isoforms of the pro-inflammatory family of cytokine IL-1), promoted, rather than suppressed, post-stroke angiogenesis in the transient middle cerebral artery occlusion (MCAo) mouse model. In this study, we aimed to show a therapeutic efficacy of IL-1α in neuroprotection. We investigated the potential for IL-1α, administered acutely IV or intra-arterial (IA) (n=5) after mouse MCAo, to also be neuroprotective. We noted that IV IL-1α (1 ng) is neuroprotective (as measured by cresyl violet stained infarct volumes) with mild, transient side effects (blunted hypertension and bradycardia) that were well tolerated, and with better functional recovery in free motion behavioral tests. IA IL-1α (0.1 ng) administration was even more neuroprotective without the systemic changes seen with IV treatment. Additionally, we noted that IL-1α is directly neuroprotective of primary mouse cortical neurons exposed to oxygen and glucose deprivation conditions in vitro . Taken together, these results suggest that IL-1α could be therapeutic after stroke when administered IV or IA, and the latter may eliminate potentially harmful hemodynamic side effects.

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