Neuroadaptations in human chronic alcoholics: dysregulation of the NF-kappaB system

Abstract

Background: Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-kappaB) family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics.

Methods and findings: Analysis of DNA-binding of NF-kappaB (p65/p50 heterodimer) and the p50 homodimer as well as NF-kappaB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant kappaB binding factor in analyzed tissues. NF-kappaB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA) mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-kappaB target DNA sites, kappaB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with kappaB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex.

Conclusions: We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-kappaB, when repeated over years downregulate RELA expression and NF-kappaB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of kappaB regulated genes. Alterations in expression of p50 homodimer/NF-kappaB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.

Figure 1. WB images of p65, p50 and IKKβ proteins in the PFC.

Representative blots of three control subjects (c) and three alcoholics (a).

Figure 2. Stoichiometry of p65/p50 and IKKβ/p65 proteins (A), and RELA/NFKB1 and IKKβ/RELA mRNAs (B).

Results are presented for the PFC of alcoholics (n = 15) and control subjects (n = 15) as mean±SD. Significance of differences between groups was evaluated by Mann Whitney U-test (A, p65/p50), covariance by multiple regression analysis (A, IKKβ/p65, covariate age; age×group interaction), or Student's t-test (B). * P<0.05.

Figure 3. κB–binding factors in the PFC identified by EMSA.

(A) The constitutively active κB-DNA binding activity was determined by incubation of tissue extracts with labeled κB-oligonucleotide followed by treatment with 0.03% DOC solution (DOC-) that did not affect DNA-protein complex formation and was used as a control. Latent factors were activated for binding to DNA by treatment with 0.6% DOC (DOC+) that dissociated IκB inhibitory protein from complexes with NF-κB, thus allowing to measure the total DNA-binding. Total NF-κB activity consisted of the constitutively active and DOC-activated DNA binding activities. The specificity of DNA-protein complexes was assessed by competition with wild type-κB (wt) or mutant-κB (m) oligonucleotides. Upper sequence-specific complexes I and II consisted of NF-κB and p50 homodimer, respectively, as demonstrated previously , . The lower complex III showed no DNA-binding sequence specificity and was probably formed by Ku protein , . For the upper image, film was exposured with a gel for longer time than for the lower one. (B) Representative picture of the constitutively active (DOC-) and total (DOC+) κB–binding activities in the PFC of control subjects and alcoholics. Lanes 3 (subject 13C) and 15 (subject 3A), only weak or no complexes were formed (see Table 1 for the description of subjects).

Figure 4. Correlations between DNA-binding activity, protein and mRNA levels.

(A) Constitutively active NF-κB DNA binding correlates with IKKβ protein levels in the PFC of alcoholics (n = 13; P = 0.002) and control subjects (n = 11; P = 0.007). No significant difference was found between slopes in the alcohol and control group. (B) p65 and p50 protein levels (15 control subjects: P = 0.01; 15 alcoholics: P = 0.06) and (C) RELA and NFKB1 mRNA levels (15 control subjects: P<0.0001; 15 alcoholics: P<0.0002) correlate in the PFC.

Figure 5. κB elements in genes differentially expressed in alcoholics.

Frequencies of occurrence of the “NF-κB”- and “p50 homodimer”-subtypes of κB binding sites in 270 and 209 genes downregulated and upregulated in alcoholics, respectively, and 1164 control genes (Table S1). (A,B) Matrices from the JASPAR database (http://mordor.cgb.ki.se/cgi-bin/jaspar2005/jaspar_db.pl) used to identify “NF-κB” (A) and “p50 homodimer” (B) subtypes of κB binding sites. Both NF-κB and p50 homodimer bind to the “NF-κB” subtype of κB elements with high affinity and specificity whereas p50 homodimer has higher affinity for more symmetric “p50 homodimer”-subtype of κB binding sites , . The matrix logos provide visual representation profiles of nucleotide conservation in κB elements. The maximal conservation amounts are 2 bits for each position. Higher conservation indicates increased biological importance for a base. (C, D). Frequencies of occurrence of the “NF-κB”- (C) and “p50 homodimer”- (D) subtypes of κB binding sites in 270 and 209 genes downregulated and upregulated in alcoholics, respectively, and in 1164 control genes (Table S1). Significance of differences between upregulated, downregulated and control genes in the frequency of occurrence of genes with κB elements were evaluated with Chi-square test (df = 2). Pairwise comparison of the up- with downregulated genes, the upregulated with control genes, and the downregulated with control genes was performed with Chi Square test (df = 1). ** P<0.01, * P<0.05.

Figure 6. Model for the molecular adaptations in the NF-κB system in the PFC in alcoholics.

(A) Chronic alcohol intake inhibits RELA gene transcription and induces alterations in stoichiometry of RELA/NFKB1 mRNAs and p65/p50 proteins, resulting in downregulation of NF-κB DNA-binding. In cells with dominant NF-κB this adaptation results in inhibition of NF-κB-dependent transcription. (B) Alcohol acts upstream of an unidentified protein kinase that modulates DNA-binding of the p50 homodimer. This results in hypophosphorylation of p50 and downregulation of p50 homodimer DNA-binding. In control individuals the p50 homodimer inhibits gene transcription acting through κB binding sites in cells in which it prevails over NF-κB. In alcoholics, downregulation of the p50 homodimer eliminates transcriptional repression leading to upregulation of κB-mediated transcription. Dysregulation of the NF-κB/p50 homodimer-dependent gene transcription contributes to cellular neuroplastic adaptations and neurodegeneration.