Nuclear factor-κB in rat brain: Enhanced DNA-binding activity following convulsant-induced seizures

doi:10.1016/0304-3940(94)90260-7

Neuroscience Letters

Volume 170, Issue 1, 28 March 1994, Pages 145-148

https://doi.org/10.1016/0304-3940(94)90260-7 Get rights and content

Abstract

The DNA-binding protein nuclear factor-κB (NF-κB) is a pleiotropic transcription factor which regulates the transcription of specific target genes such as cytokines. The existence of NF-κB has not been reported in brain tissue. This is the first report demonstrating the expression of NF-κB in the rat brain. After pentylene tetrazole (s.c)-induced clonic-tonic seizures at an LD₅₀ dose of 85 mg/kg, we have shown a gradual increase in NF-κB expression reaching a maximum at 24 h, a decrease at 48 h and again increased at 96 and 120 h. A similar time-dependent pattern was observed for the NF-κB subunit p50 expression. The NF-κB subunit p65 was not expressed at all. These data suggest a possible underlying mechanism of signal transduction and transcriptional regulation of late-response genes after perturbations in the CNS milieu.

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The NF-κB pathway plays key roles in the regulation of immune and inflammatory responses, including regulating the activity of many cytokines, chemokines, and immunoreceptors [39,40]. Additionally, NF-κB has been implicated in the neuropathological processes of seizures and epilepsy and is essential for a number of physiological CNS functions [41]. Some studies have illustrated that NF-κB is involved in seizure susceptibility, and inhibiting NF-κB activation could enhance the efficacy of AEDs [42].
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The results showed that BAFF and BAFFR expression levels were markedly up-regulated in intractable TLE patients and TLE rats. Moreover, BAFF and BAFFR proteins mainly highly expressed in the membranes and cytoplasms of the dendritic marker MAP2 in the cortex and hippocampus.
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Rapid induction of IEGs results from activation of primary stress pathways including the NF-κB pathway (Baeuerle, 1991). Indeed, activation of NF-κB has been observed in response to a number of stimuli such as increased neuronal activity and chemically and electrically induced seizures (Prasad et al., 1994; Rong and Baudry, 1996). A significant amount of NF-κB in neurons resides at the level of the synapse conferring a direct link from synapse to nucleus for rapid induction of gene expression upon synaptic stimulation (Kaltschmidt et al., 1993).
Predicting seizurogenic properties of pharmacologically active compounds is difficult due to the complex nature of the mechanisms involved and because of the low sensitivity and high variability associated with current behavioral-based methods. To identify early neuronal signaling events predictive of seizure, we exposed transgenic NF-κB/EGFP reporter mice to multiple low doses of kainic acid (KA), postulating that activation of the stress-responsive NF-κB pathway could be a sensitive indicator of seizurogenic potential. The sub-threshold dose level proximal to the induction of seizure was determined as 2.5 mg/kg KA, using video EEG monitoring. Subsequent analysis of reporter expression demonstrated significant increases in NF-κB activation in the CA3 and CA1 regions of the hippocampus 24 h after a single dose of 2.5 mg/kg KA. This response was primarily observed in pyramidal neurons with little non-neuronal expression. Neuronal NF-κB/EGFP expression was observed in the absence of glial activation, indicating a lack of neurodegeneration-induced neuroinflammation. Protein expression of the immediate-early gene, Nurr1, increased in neurons in parallel to NF-κB activation, supporting that the sub-threshold doses of KA employed directly caused neuronal stress. Lastly, KA also stimulated NF-κB activation in organotypic hippocampal slice cultures established from NF-κB/EGFP reporter mice. Collectively, these data demonstrate the potential advantages of using genetically encoded stress pathway reporter models in the screening of seizurogenic properties of new pharamacologically active compounds.
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Microglial cells, known to play key roles in neuroinflammation, can be immunotoxically eliminated from hippocampal slice cultures by treatment with saporin coupled to the microglial receptor Mac1. Considering microglial cells as a target for anti-inflammatory treatment we studied the effects of microglial depletion on anti-inflammatory treatment of mouse hippocampal slice cultures subjected to ischemia-like neurodegeneration, induced by oxygen-glucose deprivation (OGD).
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Subjection of regular cultures to OGD for 30 min induced a significant increase in PI uptake in the CA1 pyramidal cell layer, compared to control cultures. The presence of 100 μM triflusal during OGD protected against OGD-induced neurodegeneration, and reduced the number of OGD-induced NFkB positive-cells correspondingly. Cultures treated with the Mac1-saporin complex for 7 days displayed an almost total loss of microglial cells. When subjected to OGD after microglial depletion, these cultures displayed a significant increase in OGD-induced PI uptake compared to non-depleted cultures. The presence of triflusal during OGD of these cultures reduced neurodegeneration of the irrespective absence of microglia. In accordance with that, the presence of triflusal during OGD significantly inhibited the increase in the number of reactive microglia and proliferative cells in the CA1 pyramidal and dentate granule cell layers.
We conclude that immunotoxic microglia depletion significantly increases the susceptibility of CA1 pyramidal cells to neurodegeneration and that the anti-inflammatory drug triflusal still can exert its neuroprotective role following depletion of microglia.
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To explore the molecular mechanism of brain tissue injury induced by lipopolysaccharide (LPS), we studied the effects of endotoxic shock on rat brain cortex NF-κB and the effects of dexamethasone on these changes. Rats were randomly divided into LPS, LPS + dexamethasone, and control groups. The DNA-binding activity of NF-κB was observed using electrophoretic mobility shift assay (EMSA). Protein expression in nuclear extracts was studied using Western blots, and nuclear translocation was observed using immunohistochemistry. These indices were assayed at 1 h and 4 h after intravenous injection of LPS (4 mg·kg⁻¹). EMSA showed significantly increased NF-κB DNA-binding activity in nuclear extracts from the LPS group at both 1 h and 4 h after LPS injection, compared with the control group (P < 0.01). For the LPS group, the NF-κB DNA-binding activity was greater at 1 h than at 4 h (P < 0.05). The expression of p65 and p50 protein in the nuclear extracts was also increased, as compared with the control group. However, the expression of p65 and p50 protein from cytosolic extracts did not show any significant change. Dexamethasone down-regulated not only NF-κB DNA-binding activity but also the expression of p65 protein in the nuclear extracts. From these data, we have concluded that NF-κB activation and nuclear translocation of NF-κB play a key role in the molecular mechanism of brain tissue injury in endotoxic shock. Dexamethasone may alleviate brain injury by inhibiting NF-κB activation.
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To better understand the role of inflammatory responses in temporal lobe epilepsy, we characterized Interleukin1-β (IL1-β), Nuclear Factor-κB (NF-κB), and Cyclooxygenase-2 (COX-2) expression together with neurodegeneration in the rat lithium–pilocarpine model. The immunohistochemical expression of IL1-β, NF-κB, and COX-2 started by 12 h post-injection, persisted for 24 h (status epilepticus period), and returned to basal levels by 3 and 6 days (latent period). The regional distribution of IL1-β, NF-κB, and COX-2 occurred mainly in structures prone to develop neuronal damage. Using double-staining protocols, we detected IL1-β expression in glial cells, COX-2 expression in neurons, and NF-κB in both cell types. The presence of Fluoro-Jade-B-positive degenerating neurons was associated with IL1-β, NF-κB, and COX-2 proteins expression during status epilepticus but not during the latent period while neurons were still degenerating. These data suggest that seizure-related IL1-β, NF-κB, and COX-2 expression may contribute to the pathophysiology of epilepsy by inducing neuronal death and astrocytic activation.
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Nuclear factor-κB in rat brain: Enhanced DNA-binding activity following convulsant-induced seizures

Abstract

Biochem. Biophys. Acta

Cell

Cell

Eur. J. Pharmacol.

Neurosci. Lett.

Neurosci. Lett.

Curr. Opin. Cell Biol.

Cell

Trends Pharmacol. Sci.

Neuron

Cell

Neuron

IκB: A specific inhibitor of the NF-κB transcription factor

Science

A 65-kD subunit of active NF-κB is required for inhibition of NF-κB by IκB

Genes Dev.