Elsevier

Molecular Brain Research

Volume 109, Issues 1–2, 30 December 2002, Pages 179-188
Molecular Brain Research

Research report
Characterization of the molecular events following impairment of NF-κB-driven transcription in neurons

https://doi.org/10.1016/S0169-328X(02)00558-2Get rights and content

Abstract

Nuclear factor-κB (NF-κB) is a transcription factor with a pivotal role in neuronal homeostasis. Indeed, NF-κB trans-activates several antiapoptotic genes in neurons and inhibition of NF-κB transcriptional activity triggers neuronal apoptosis. However, the exact mechanisms by which neurons undergo apoptosis in conditions of NF-κB inhibition are poorly understood. To further clarify how NF-κB operates in neurons, and to gather information on the molecular events occurring during NF-κB inhibition-dependent neuronal apoptosis, this study evaluated the effects of recently identified NF-κB inhibitors such as parthenolide, SN50, BAY 11-7082 and helenalin on primary cultures of rat cortical neurons. Data show that NF-κB was constitutively activated in neurons, and demonstrate for the first time that drug-dependent NF-κB inhibition induced rapid mitochondrial release of cytochrome c, caspase-9 and -3 activation, poly(ADP-ribose) polymerase-1 cleavage, membrane blebbing and nuclear fragmentation, without evidence of procaspase-8 and Bid processing. Interestingly, a burst of Akt activation occurred in neurons exposed to NF-κB inhibitors. These events were preceded by selective reduction of mRNAs of NF-κB-dependent, antiapoptotic Bcl-2 family members such as Bcl-xL, Bcl-2 and, in particular, A1/Bfl-1. The present study reports a novel, detailed temporal analysis of the molecular events following impairment of NF-κB-driven transcription in neurons and demonstrates that inhibition of constitutive neuronal NF-κB activity triggers selective activation of the intrinsic apoptotic program.

Introduction

A large body of experimental evidence demonstrates that apoptotic cell death underlies neuronal demise during neurodegenerative disorders [31], [39]. It is also well established that execution of the apoptotic program depends upon the expression of death- and cell cycle-associated effector proteins. Indeed, apoptosis is strictly regulated at the transcriptional level, and aberrant regulation of gene expression is of relevance to human neurological diseases (for reviews see [31], [27], [6]). Hence, a great deal of effort has been directed toward clarifying the transcriptional mechanisms regulating survival and death programs [4], in order to develop apoptosis-based therapeutic agents [38].

Inducible transcription factors are fundamental players in integrating cytoplasmic signaling into gene expression and finely regulate cell fate [4]. One of the most investigated, but not totally understood, transcription factor is nuclear factor-κB (NF-κB), typically present in the cytoplasm as a dimer of different combinations of five proteins such as p65 (RelA), RelB, c-Rel, p50 and p52 [18]. NF-κB plays a pivotal role in cell survival by promoting expression of several anti-apoptotic proteins. These encompass inhibitor-of-apoptosis proteins (IAPs), FLICE inhibitor protein (FLIP), tumor necrosis factor (TNF)-α receptor-associated factor (TRAF)-1 and -2, manganese superoxide dismutase as well as genes of the Bcl-2 family including A1/Bfl-1, Bcl-xL and Bcl-2 itself [18], [57], [32]. Accordingly, extensive data demonstrate that NF-κB activity promotes neuronal survival in vitro and in vivo [32]. In striking contrast with this, however, several lines of evidence emphasize the active role of NF-κB in neuronal cell death. For example, NF-κB trans-activates proapoptotic genes including fas ligand [19] and suppression of NF-κB transcriptional activity provides neuroprotection in vitro [12] and in vivo [45], [49]. In addition, NF-κB promotes glial activation and its detrimental effects on neurons [33], [37]. On this basis, it has been proposed that the ultimate role of NF-κB in survival or death of neurons is stimulus- and activation kinetic-dependent [24], [11], [42]. Yet, the cascade of molecular events triggered by acute suppression of NF-κB-driven transcription and its repercussion on neuronal homeostasis remain in part to be investigated.

Signaling pathways activated by glutamate [12], [16], oxygen radicals, interleukin-1β, TNF-α [32], [33], neurotrophic cytokines [34] and nerve growth factor [28] lead to NF-κB activation in neurons. Most of these signaling pathways typically trigger IκB kinase complex (IKK) activation that in turn induces degradation of the inhibitory proteins IκBs and nuclear translocation of NF-κB [17], [21]. In addition to this canonical pathway, recent evidence suggests that other effectors, including Akt (also known as protein kinase B), regulate in a phosphorylation-dependent manner the activity of NF-κB once inside the nucleus [21], [50]. Various strategies and tools have been used to block NF-κB-driven transcription. These include genetic deletion of NF-κB subunits, degradation-resistant IκBα (the so-called IκBα super-repressor), proteasome inhibitors as well as κB-decoy DNA [18], [32]. NF-κB inhibition has also been obtained with the cell-permeable synthetic peptide SN50. This compound bears the nuclear localization sequence (amino acids 360–369) of NF-κB p50 and inhibits nuclear translocation of NF-κB active complex. Amino acid substitution at Lys363 and Arg364 as in mutated SN50 (SN50M) abolishes inhibition of NF-κB nuclear translocation [23]. In addition, sesquiterpene lactones are recently identified alkaloids able to inhibit NF-κB activity at different levels. For instance, parthenolide (PAR) inhibits IKKβ [15], whereas helenalin directly inactivates NF-κB p65 [25] in immune cells.

In order to investigate the molecular mechanisms by which NF-κB regulates neuronal homeostasis, the present study evaluated the effects of SN50, SN50M, parthenolide, helenalin and BAY 11-7082 (an IKK inhibitor [43]) on pure cultures of rat cortical neurons. It is shown here for the first time a detailed temporal analysis of the selective changes in gene expression, mitochondrial dysfunction and activation of the intrinsic cell death program that follow inhibition of NF-κB in neurons.

Section snippets

Primary cultures of cortical neurons

Primary cultures of cortical neurons were prepared from E17 rat embryos. Cortices were isolated in cold phosphate buffered saline (PBS) and then incubated 10 min at 37 °C in phosphate buffered saline containing 0.25% trypsin and 0.05% DNase. After blocking enzymatic digestion with the addition of 10% heat-inactivated fetal bovine serum, cortices were mechanically disrupted, washed and the cells resuspended in neurobasal medium plus B27 (Gibco, Life Technologies, Rockville, MD, USA) and plated at

Effects of PAR and SN50 on the DNA binding activity of NF-κB in cortical neurons

The appearance of a retarded band in the gel shift assay (Fig. 1Aa) revealed constitutive binding activity to the κB oligoprobe in extracts of neurons under resting conditions. Exposure of neurons to TNFα (10 ng/ml), a well-known inducer of NF-κB in neurons [32], increased the binding activity to the κB oligoprobe, whereas the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC, 50 μM) reduced the effect of TNFα. The κB-binding activity induced by TNFα was reduced by the addition of an antibody

Discussion

Lack of specificity is always of concern when using pharmacological tools such as PAR or SN50 [52], [2]. However, (i) amino acid substitution in the sequence that specifically prevents NF-κB nuclear translocation (as in SN50M) abrogated the effects of SN50 on both DNA binding of NF-κB and neuronal survival; (ii) neurotoxicity of PAR was prevented by β-mercaptoethanol; (iii) two selective NF-κB inhibitors structurally unrelated to PAR and SN50 such as helenalin and BAY 11-7082 also induced

Acknowledgements

The invaluable support of J. V. Walsh is gratefully acknowledged. This work was supported in part by the Project for Young Investigator of the University of Florence. The author wishes to thank E. Rapizzi for advice and L. Scorrano for the anti-Bid antibody.

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