Neuroinflammation induces synaptic scaling through IL-1β-mediated activation of the transcriptional repressor REST/NRSF

AbstractNeuroinflammation is associated with synapse dysfunction and cognitive decline in patients and animal models. One candidate for translating the inflammatory stress into structural and functional changes in neural networks is the transcriptional repressor RE1-silencing transcription factor (REST) that regulates the expression of a wide cluster of neuron-specific genes during neurogenesis and in mature neurons. To study the cellular and molecular pathways activated under inflammatory conditions mimicking the experimental autoimmune encephalomyelitis (EAE) environment, we analyzed REST activity in neuroblastoma cells and mouse cortical neurons treated with activated T cell or microglia supernatant and distinct pro-inflammatory cytokines. We found that REST is activated by a variety of neuroinflammatory stimuli in both neuroblastoma cells and primary neurons, indicating that a vast transcriptional change is triggered during neuroinflammation. While a dual activation of REST and its dominant-negative splicing isoform REST4 was observed in N2a neuroblastoma cells, primary neurons responded with a pure full-length REST upregulation in the absence of changes in REST4 expression. In both cases, REST upregulation was associated with activation of Wnt signaling and increased nuclear translocation of β-catenin, a well-known intracellular transduction pathway in neuroinflammation. Among single cytokines, IL-1β caused a potent and prompt increase in REST transcription and translation in neurons, which promoted a delayed and strong synaptic downscaling specific for excitatory synapses, with decreased frequency and amplitude of spontaneous synaptic currents, decreased density of excitatory synaptic connections, and decreased frequency of action potential-evoked Ca2+ transients. Most important, the IL-1β effects on excitatory transmission were strictly REST dependent, as conditional deletion of REST completely occluded the effects of IL-1β activation on synaptic transmission and network excitability. Our results demonstrate that REST upregulation represents a new pathogenic mechanism for the synaptic dysfunctions observed under neuroinflammatory conditions and identify the REST pathway as therapeutic target for EAE and, potentially, for multiple sclerosis.

Download Full-text

Regulation of Neurite Outgrowth in N1E-115 Cells through PDZ-Mediated Recruitment of Diacylglycerol Kinase ζ

Molecular and Cellular Biology ◽

10.1128/mcb.25.16.7289-7302.2005 ◽

2005 ◽

Vol 25 (16) ◽

pp. 7289-7302 ◽

Cited By ~ 51

Author(s):

Yury Yakubchyk ◽

Hanan Abramovici ◽

Jean-Christian Maillet ◽

Elias Daher ◽

Christopher Obagi ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Neurite Outgrowth ◽

Cortical Neurons ◽

Neuroblastoma Cells ◽

Diacylglycerol Kinase ◽

Dominant Negative ◽

Adult Brain ◽

Binding Motif ◽

Kinase C

ABSTRACT Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase ζ (DGK-ζ), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-ζ and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-ζ in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-ζ kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-ζ mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-ζ promotes neurite outgrowth through association with the GTPase Rac1. DGK-ζ colocalized with Rac1 in neuronal processes and DGK-ζ-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-ζ directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-ζ mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1V12) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-ζ with Rac1V12, suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-ζ binding to active Rac1. Collectively, these findings suggest DGK-ζ, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.

Download Full-text

Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

Cell Death and Disease ◽

10.1038/s41419-021-03752-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Anthony R. Anzell ◽

Garrett M. Fogo ◽

Zoya Gurm ◽

Sarita Raghunayakula ◽

Joseph M. Wider ◽

...

Keyword(s):

Cortical Neurons ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Mitochondrial Fission ◽

Conditional Knockout ◽

Mitochondrial Morphology ◽

Primary Neurons ◽

Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

Download Full-text

Naja mossambica mossambica Cobra Cardiotoxin Targets Mitochondria to Disrupt Mitochondrial Membrane Structure and Function

Toxins ◽

10.3390/toxins11030152 ◽

2019 ◽

Vol 11 (3) ◽

pp. 152 ◽

Cited By ~ 5

Author(s):

Boris Zhang ◽

Feng Li ◽

Zhengyao Chen ◽

Indira Shrivastava ◽

Edward Gasanoff ◽

...

Keyword(s):

Molecular Mechanism ◽

Cortical Neurons ◽

Neuroblastoma Cells ◽

Docking Studies ◽

Mitochondrial Membranes ◽

Paramagnetic Resonance ◽

Phosphorescence Quenching ◽

In Silico Docking ◽

Membrane Structure And Function ◽

Dynamics Simulations

Cobra venom cardiotoxins (CVCs) can translocate to mitochondria to promote apoptosis by eliciting mitochondrial dysfunction. However, the molecular mechanism(s) by which CVCs are selectively targeted to the mitochondrion to disrupt mitochondrial function remains to be elucidated. By studying cardiotoxin from Naja mossambica mossambica cobra (cardiotoxin VII4), a basic three-fingered S-type cardiotoxin, we hypothesized that cardiotoxin VII4 binds to cardiolipin (CL) in mitochondria to alter mitochondrial structure/function and promote neurotoxicity. By performing confocal analysis, we observed that red-fluorescently tagged cardiotoxin rapidly translocates to mitochondria in mouse primary cortical neurons and in human SH-SY5Y neuroblastoma cells to promote aberrant mitochondrial fragmentation, a decline in oxidative phosphorylation, and decreased energy production. In addition, by employing electron paramagnetic resonance (EPR) and protein nuclear magnetic resonance (1H-NMR) spectroscopy and phosphorescence quenching of erythrosine in model membranes, our compiled biophysical data show that cardiotoxin VII4 binds to anionic CL, but not to zwitterionic phosphatidylcholine (PC), to increase the permeability and formation of non-bilayer structures in CL-enriched membranes that biochemically mimic the outer and inner mitochondrial membranes. Finally, molecular dynamics simulations and in silico docking studies identified CL binding sites in cardiotoxin VII4 and revealed a molecular mechanism by which cardiotoxin VII4 interacts with CL and PC to bind and penetrate mitochondrial membranes.

Download Full-text

Plasminogen activator inhibitor-1 potentiates LPS-induced neutrophil activation through a JNK–mediated pathway

Thrombosis and Haemostasis ◽

10.1160/th05-12-0782 ◽

2006 ◽

Vol 95 (05) ◽

pp. 829-835 ◽

Cited By ~ 31

Author(s):

Sang-Hyun Kwak ◽

Xue-Qing Wang ◽

Qianbin He ◽

Wen-Feng Fang ◽

Sanchayita Mitra ◽

...

Keyword(s):

Plasminogen Activator ◽

Proinflammatory Cytokines ◽

Nuclear Translocation ◽

Plasminogen Activator Inhibitor ◽

Neutrophil Activation ◽

Plasminogen Activator Inhibitor 1 ◽

Inflammatory Conditions ◽

Jnk Activation ◽

Activator Inhibitor ◽

Pai 1

SummaryPlasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily, modulates fibrinolysis by interacting with proteolytic mediators, including urokinase plasminogen activator (uPA). Although the roles of uPA and PAI-1 in plasmin generation and the degradation of fibrin are well known, recent evidence also suggests that they can participate in acute inflammatory conditions that involve neutrophil activation. In the present experiments, we found that the addition of PAI-1 to LPS-stimulated neutrophils resulted in enhanced nuclear translocation of NF-κB and increased production of the proinflammatory cytokines IL-1β,Tnf-α,and Mip-2.uPA and the kringle domain (KD) of uPA potentiated cytokine expression and NF-κB activation by neutrophils cultured with LPS, and had additive effects when combined with PAI-1. The c-Jun N-terminal kinase (JNK) was activated after exposure of resting neutrophils to PAI-1 or the uPA KD. Enhanced JNK activation, but not that of other kinases induced by LPS, was present in neutrophils cocultured with PAI-1 or uPA KD. Inhibition of JNK activation prevented the potentiation of expression of proinflammatory cytokines induced by PAI-1 or uPA KD in LPS stimulated neutrophils. These results demonstrate that PAI-1 and uPA KD enhance LPSinduced neutrophil responses through their effects on JNK mediated pathways.

Download Full-text

Activation of Nuclear Factor κb and bcl-x Survival Gene Expression by Nerve Growth Factor Requires Tyrosine Phosphorylation of IκBα

The Journal of Cell Biology ◽

10.1083/jcb.152.4.753 ◽

2001 ◽

Vol 152 (4) ◽

pp. 753-764 ◽

Cited By ~ 100

Author(s):

Nguyen Truc Bui ◽

Antonia Livolsi ◽

Jean-Francois Peyron ◽

Jochen H.M. Prehn

Keyword(s):

Gene Expression ◽

Tyrosine Phosphorylation ◽

Fluorescent Protein ◽

Nuclear Translocation ◽

Nuclear Factor Κb ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Nuclear Factor ◽

Human Neuroblastoma ◽

Tnf Α

NGF has been shown to support neuron survival by activating the transcription factor nuclear factor-κB (NFκB). We investigated the effect of NGF on the expression of Bcl-xL, an anti–apoptotic Bcl-2 family protein. Treatment of rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, or primary rat hippocampal neurons with NGF (0.1–10 ng/ml) increased the expression of bcl-xL mRNA and protein. Reporter gene analysis revealed a significant increase in NFκB activity after treatment with NGF that was associated with increased nuclear translocation of the active NFκB p65 subunit. NGF-induced NFκB activity and Bcl-xL expression were inhibited in cells overexpressing the NFκB inhibitor, IκBα. Unlike tumor necrosis factor-α (TNF-α), however, NGF-induced NFκB activation occurred without significant degradation of IκBs determined by Western blot analysis and time-lapse imaging of neurons expressing green fluorescent protein–tagged IκBα. Moreover, in contrast to TNF-α, NGF failed to phosphorylate IκBα at serine residue 32, but instead caused significant tyrosine phosphorylation. Overexpression of a Y42F mutant of IκBα potently suppressed NFG-, but not TNF-α–induced NFκB activation. Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-α–, but not NGF-induced NFκB activation. We conclude that NGF and TNF-α induce different signaling pathways in neurons to activate NFκB and bcl-x gene expression.

Download Full-text

Abstract 3115: Low-Level Laser Therapy Protects Against OGD-induced Neurotoxicity Of Primary Cultured Mouse Cortical Neurons

Stroke ◽

10.1161/str.43.suppl_1.a3115 ◽

2012 ◽

Vol 43 (suppl_1) ◽

Author(s):

Zhanyang Yu ◽

Ning Liu ◽

Eng H Lo ◽

Thomas J McCarthy ◽

Xiaoying Wang

Keyword(s):

Laser Therapy ◽

Mitochondrial Function ◽

Cortical Neurons ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Low Level Laser Therapy ◽

Primary Neurons ◽

Novel Therapy ◽

Low Level ◽

Mtt Reduction

Background: Low level light (or laser) therapy (LLLT) has been studied and practiced for promoting wound healing, reducing pain, inflammation, and ischemic tissue damage. Recently, a series of experimental and clinical investigations have suggested that LLLT may be a novel therapy against hypoxic/ischemic brain damage. A clinical trial of LLLT therapy for ischemic stroke is now on going. However, the molecular mechanism of LLLT-conferred neuroprotection remains poorly defined. In this study, we tested our hypothesis that LLLT may attenuate impairments of mitochondrial function induced by hypoxic/ischemic insults in primary cultured mouse cortical neurons. Method: At day 9 of culture, primary neurons were subjected to 4 hr OGD followed by reoxygenation. One 810-nm LLLT treatment was applied for 2 minutes at 2 hr after reoxygenation. Neurotoxicity was measured after 20 hr after reoxygenation by LDH release assay. We also measured MTT reduction and mitochondria membrane potential (MMP) at 2 hr after LLLT treatment as markers of mitochondrial function. Results: The neurotoxicity study showed that 4 hr OGD plus 20 hr reoxygenation caused 33.8± 3.4% neuronal cell death, while LLLT treatment significantly reduced the neuronal death rate to 23.6± 2.9% (30.2% reduction, n=6, p smaller than 0.05). Mitochondrial functional assays showed OGD decreased MTT reduction to 75.9± 2.68%, but LLLT treatment significantly rescued MTT reduction to 87.6±4.55% (15.4% improvement, n=6, p smaller than 0.05). Furthermore, after OGD, MMP was reduced to 48.9±4.39%, while LLLT treatment significantly ameliorated this reduction to 89.6± 13.9% (83% improvement, n=4, p smaller than 0.05) compared to normoxic controls. Conclusion: The present study suggests that LLLT treatment is protective against OGD-induced neurotoxicity of primary neurons and that this protection may be conferred through preservation or rescue of mitochondrial function.

Download Full-text

All-Trans Retinoic Acid induces synaptic plasticity in human cortical neurons

10.1101/2020.09.04.267104 ◽

2020 ◽

Author(s):

Maximilian Lenz ◽

Pia Kruse ◽

Amelie Eichler ◽

Julia Muellerleile ◽

Jakob Straehle ◽

...

Keyword(s):

Retinoic Acid ◽

Synaptic Plasticity ◽

Cortical Neurons ◽

Mrna Translation ◽

Dependent Manner ◽

Human Cortex ◽

Functional Changes ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Structural And Functional Properties

ABSTRACTA defining feature of the brain is its ability to adapt structural and functional properties of synaptic contacts in an experience-dependent manner. In the human cortex direct experimental evidence for synaptic plasticity is currently missing. Here, we probed plasticity in human cortical slices using the vitamin A derivative all-trans retinoic acid, which has been suggested as medication for the treatment of neuropsychiatric disorders, e.g., Alzheimer’s disease. Our experiments demonstrate coordinated structural and functional changes of excitatory synapses of superficial (layer 2/3) pyramidal neurons in the presence of all-trans retinoic acid. This synaptic adaptation is accompanied by ultrastructural remodeling of the calcium-storing spine apparatus organelle and requires mRNA-translation. We conclude that all-trans retinoic acid is a potent mediator of synaptic plasticity in the adult human cortex.

Download Full-text

Rab7 Reduces α-Synuclein Toxicity in Rats and Primary Neurons

10.21203/rs.3.rs-277472/v1 ◽

2021 ◽

Author(s):

Éva M. Szegõ ◽

Eva M. Szegö ◽

Chris Van den Haute ◽

Lennart Höfs ◽

Veerle Baekelandt ◽

...

Keyword(s):

Dna Damage ◽

Rat Brain ◽

Neuronal Degeneration ◽

Degradation Pathway ◽

Alpha Synuclein ◽

Dominant Negative ◽

Primary Neurons ◽

Vicious Cycle ◽

Axon Terminals

Abstract BackgroundDuring the pathogenesis of Parkinson’s disease (PD), aggregation of alpha-synuclein (αSyn) induces a vicious cycle of cellular impairments that lead to neurodegeneration. Consequently, removing toxic αSyn aggregates constitutes a plausible strategy against PD. In this work, we tested whether stimulating the autolysosomal degradation of αSyn aggregates through the Ras-related in brain 7 (Rab7) pathway can reverse αSyn-induced cellular impairment and prevent neurodegeneration in vivo.MethodsThe disease-related A53T mutant of αSyn was expressed in primary neurons and in dopaminergic neurons of the rat brain simultaneously with wild type (WT) Rab7 or its dominant-negative T22N mutant as a control. The cellular integrity was quantified by morphological and biochemical analyses.ResultsIn primary neurons, WT Rab7 rescued the αSyn -induced loss of neurons and neurites. Furthermore, Rab7 decreased the amount of reactive oxygen species and the amount of Triton X-100 insoluble αSyn. In rat brain, WT Rab7 reduced αSyn -induced loss of dopaminergic axon terminals in the striatum and the loss of dopaminergic dendrites in the substantia nigra pars reticulata. Further, WT Rab7 lowered αSyn pathology as quantified by phosphorylated αSyn staining. Finally, WT Rab7 attenuated αSyn-induced DNA damage in primary neurons and rat brain.ConclusionRab7 reduced αSyn-induced pathology, ameliorated αSyn-induced neuronal degeneration, oxidative stress and DNA damage. These findings indicate that Rab7 is able to disrupt the vicious cycle of cellular impairment, αSyn pathology and neurodegeneration present in PD. Stimulation of Rab7 and the autolysosomal degradation pathway could therefore constitute a beneficial strategy for PD.

Download Full-text

Dimerization of the cellular prion protein inhibits propagation of scrapie prions

Journal of Biological Chemistry ◽

10.1074/jbc.ra117.000990 ◽

2018 ◽

Vol 293 (21) ◽

pp. 8020-8031 ◽

Cited By ~ 2

Author(s):

Anna D. Engelke ◽

Anika Gonsberg ◽

Simrika Thapa ◽

Sebastian Jung ◽

Sarah Ulbrich ◽

...

Keyword(s):

Transgenic Mice ◽

Prion Protein ◽

Conformational Transition ◽

Prion Diseases ◽

Neuroblastoma Cells ◽

Direct Interaction ◽

Cellular Prion Protein ◽

Dominant Negative ◽

Dimerization Domain ◽

Hydrophobic Domain

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrPC) into the scrapie isoform, denoted PrPSc. Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrPC and PrPSc; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrPC is converted in scrapie-infected cells, suggesting that not all PrPC species are suitable substrates for the conversion. On the basis of the observation that PrPC can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrPC, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrPC. Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrPC dimerization completely blocked its conversion into PrPSc in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrPC dimers had a dominant-negative inhibition effect on the conversion of monomeric PrPC. Our findings suggest that PrPC monomers are the major substrates for PrPSc propagation and that it may be possible to halt prion formation by stabilizing PrPC dimers.

Download Full-text