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NF-κB mediates Gadd45β expression and DNA demethylation in the hippocampus during fear memory formation.

Jarome TJ, Butler AA, Nichols JN, Pacheco NL, Lubin FD - Front Mol Neurosci (2015)

Bottom Line: Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity.Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression.Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama at Birmingham Birmingham, AL, USA.

ABSTRACT
Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus sequences within the Gadd45β gene promoter. Whether NF-κB activity regulates Gadd45 expression and associated DNA demethylation in neurons during memory formation is unknown. Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity. Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression. The learning-induced increases in Gadd45β mRNA levels, Gadd45β binding at the BDNF gene and BDNF DNA demethylation were blocked in area CA1 of the c-rel knockout mice. Additionally, local siRNA-mediated knockdown of c-rel in area CA1 prevented fear conditioning-induced increases in Gadd45β expression and BDNF DNA demethylation, suggesting that c-Rel containing NF-κB transcription factor complex is responsible for Gadd45β regulation during memory formation. Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

No MeSH data available.


Related in: MedlinePlus

Local knockdown of c-rel in the hippocampus prevents fear conditioning-induced changes in Gadd45β expression and BDNF DNA methylation. (A) Rats were infused Accell siRNAs against c-rel or a negative control (Scr-siRNA) 5 days prior to fear conditioning and area CA1 collected 1 h later. (B) Fear conditioning increased c-Rel protein expression in the hippocampus which was blocked by the c-rel siRNA, confirming effective gene knockdown by the siRNA infusion (left). Fear conditioning-induced increases in c-Rel protein expression in the retrosplenial cortex (RSC) were not altered by the c-rel siRNA when infused in the hippocampus, confirming that the siRNA did not diffuse up the injection tract (right). (C) Knockdown of c-rel prevented the fear conditioning induced increases in Gadd45β gene (n = 3–4 per group) and protein expression (n = 5 per group) in the hippocampus. (D) Knockdown of c-rel prevented the fear conditioning induced decreases in rat BDNF Promoter 4 DNA methylation in the hippocampus (n = 3 per group). *p < 0.05 Scr-siRNA Naïve. #p < 0.05 from Scr-siRNA FC.
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Figure 5: Local knockdown of c-rel in the hippocampus prevents fear conditioning-induced changes in Gadd45β expression and BDNF DNA methylation. (A) Rats were infused Accell siRNAs against c-rel or a negative control (Scr-siRNA) 5 days prior to fear conditioning and area CA1 collected 1 h later. (B) Fear conditioning increased c-Rel protein expression in the hippocampus which was blocked by the c-rel siRNA, confirming effective gene knockdown by the siRNA infusion (left). Fear conditioning-induced increases in c-Rel protein expression in the retrosplenial cortex (RSC) were not altered by the c-rel siRNA when infused in the hippocampus, confirming that the siRNA did not diffuse up the injection tract (right). (C) Knockdown of c-rel prevented the fear conditioning induced increases in Gadd45β gene (n = 3–4 per group) and protein expression (n = 5 per group) in the hippocampus. (D) Knockdown of c-rel prevented the fear conditioning induced decreases in rat BDNF Promoter 4 DNA methylation in the hippocampus (n = 3 per group). *p < 0.05 Scr-siRNA Naïve. #p < 0.05 from Scr-siRNA FC.

Mentions: An alternative explanation for the effects described above is that the alterations in Gadd45β expression and BDNF DNA demethylation in area CA1 of c-rel−/− mice are due to the loss of c-rel in multiple brain regions simultaneously, which could result in wide-scale epigenetic changes across the neural circuit. To test this possibility, we locally knocked-down c-rel in area CA1 of the hippocampus in adult animals using siRNA technology and examined changes in Gadd45β expression and BDNF Promoter 4 DNA demethylation following learning (Figure 5A). First, we confirmed the effectiveness of our siRNA by examining c-Rel protein expression in area CA1 and the surrounding cortical region following fear conditioning (Figure 5B). In area CA1, we found that fear conditioning increased c-Rel expression, which was attenuated in c-rel siRNA infused animals (F(2,10) = 4.150, p < 0.05). However, in the RSC, which requires de novo protein synthesis for the consolidation of contextual fear memories (Kwapis et al., 2015), we found a learning-induced increase in c-Rel expression that was unaffected by infusion of the c-rel siRNA into the hippocampus (F(2,10) = 3.838, p = 0.058). These results suggest that our siRNA effectively targeted c-rel in the hippocampus and supports previous studies that found increased expression of NF-κB proteins during enhanced synaptic activity (Meberg et al., 1996). Next, we examined what effect c-rel knockdown had on Gadd45β expression and DNA demethylation following fear conditioning. We found that siRNA-mediated knockdown of c-rel in adulthood completely abolished the learning-induced increases in Gadd45β gene (F(2,7) = 8.760, p < 0.05) and largely reduced the increases in protein expression (F(2,12) = 3.762, p = 0.053) in area CA1 (Figure 5C), confirming what we observed in c-rel−/− mice. Additionally, c-rel siRNA knockdown abolished the learning-induced decreases in BDNF Promoter 4 DNA methylation (F(2,6) = 6.786, p < 0.05; Figure 5D). In combination with our c-rel−/− mice data, these results strongly suggest that c-Rel regulates Gadd45β expression and BDNF Promoter 4 DNA demethylation in the hippocampus during memory consolidation.


NF-κB mediates Gadd45β expression and DNA demethylation in the hippocampus during fear memory formation.

Jarome TJ, Butler AA, Nichols JN, Pacheco NL, Lubin FD - Front Mol Neurosci (2015)

Local knockdown of c-rel in the hippocampus prevents fear conditioning-induced changes in Gadd45β expression and BDNF DNA methylation. (A) Rats were infused Accell siRNAs against c-rel or a negative control (Scr-siRNA) 5 days prior to fear conditioning and area CA1 collected 1 h later. (B) Fear conditioning increased c-Rel protein expression in the hippocampus which was blocked by the c-rel siRNA, confirming effective gene knockdown by the siRNA infusion (left). Fear conditioning-induced increases in c-Rel protein expression in the retrosplenial cortex (RSC) were not altered by the c-rel siRNA when infused in the hippocampus, confirming that the siRNA did not diffuse up the injection tract (right). (C) Knockdown of c-rel prevented the fear conditioning induced increases in Gadd45β gene (n = 3–4 per group) and protein expression (n = 5 per group) in the hippocampus. (D) Knockdown of c-rel prevented the fear conditioning induced decreases in rat BDNF Promoter 4 DNA methylation in the hippocampus (n = 3 per group). *p < 0.05 Scr-siRNA Naïve. #p < 0.05 from Scr-siRNA FC.
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Figure 5: Local knockdown of c-rel in the hippocampus prevents fear conditioning-induced changes in Gadd45β expression and BDNF DNA methylation. (A) Rats were infused Accell siRNAs against c-rel or a negative control (Scr-siRNA) 5 days prior to fear conditioning and area CA1 collected 1 h later. (B) Fear conditioning increased c-Rel protein expression in the hippocampus which was blocked by the c-rel siRNA, confirming effective gene knockdown by the siRNA infusion (left). Fear conditioning-induced increases in c-Rel protein expression in the retrosplenial cortex (RSC) were not altered by the c-rel siRNA when infused in the hippocampus, confirming that the siRNA did not diffuse up the injection tract (right). (C) Knockdown of c-rel prevented the fear conditioning induced increases in Gadd45β gene (n = 3–4 per group) and protein expression (n = 5 per group) in the hippocampus. (D) Knockdown of c-rel prevented the fear conditioning induced decreases in rat BDNF Promoter 4 DNA methylation in the hippocampus (n = 3 per group). *p < 0.05 Scr-siRNA Naïve. #p < 0.05 from Scr-siRNA FC.
Mentions: An alternative explanation for the effects described above is that the alterations in Gadd45β expression and BDNF DNA demethylation in area CA1 of c-rel−/− mice are due to the loss of c-rel in multiple brain regions simultaneously, which could result in wide-scale epigenetic changes across the neural circuit. To test this possibility, we locally knocked-down c-rel in area CA1 of the hippocampus in adult animals using siRNA technology and examined changes in Gadd45β expression and BDNF Promoter 4 DNA demethylation following learning (Figure 5A). First, we confirmed the effectiveness of our siRNA by examining c-Rel protein expression in area CA1 and the surrounding cortical region following fear conditioning (Figure 5B). In area CA1, we found that fear conditioning increased c-Rel expression, which was attenuated in c-rel siRNA infused animals (F(2,10) = 4.150, p < 0.05). However, in the RSC, which requires de novo protein synthesis for the consolidation of contextual fear memories (Kwapis et al., 2015), we found a learning-induced increase in c-Rel expression that was unaffected by infusion of the c-rel siRNA into the hippocampus (F(2,10) = 3.838, p = 0.058). These results suggest that our siRNA effectively targeted c-rel in the hippocampus and supports previous studies that found increased expression of NF-κB proteins during enhanced synaptic activity (Meberg et al., 1996). Next, we examined what effect c-rel knockdown had on Gadd45β expression and DNA demethylation following fear conditioning. We found that siRNA-mediated knockdown of c-rel in adulthood completely abolished the learning-induced increases in Gadd45β gene (F(2,7) = 8.760, p < 0.05) and largely reduced the increases in protein expression (F(2,12) = 3.762, p = 0.053) in area CA1 (Figure 5C), confirming what we observed in c-rel−/− mice. Additionally, c-rel siRNA knockdown abolished the learning-induced decreases in BDNF Promoter 4 DNA methylation (F(2,6) = 6.786, p < 0.05; Figure 5D). In combination with our c-rel−/− mice data, these results strongly suggest that c-Rel regulates Gadd45β expression and BDNF Promoter 4 DNA demethylation in the hippocampus during memory consolidation.

Bottom Line: Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity.Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression.Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama at Birmingham Birmingham, AL, USA.

ABSTRACT
Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus sequences within the Gadd45β gene promoter. Whether NF-κB activity regulates Gadd45 expression and associated DNA demethylation in neurons during memory formation is unknown. Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity. Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression. The learning-induced increases in Gadd45β mRNA levels, Gadd45β binding at the BDNF gene and BDNF DNA demethylation were blocked in area CA1 of the c-rel knockout mice. Additionally, local siRNA-mediated knockdown of c-rel in area CA1 prevented fear conditioning-induced increases in Gadd45β expression and BDNF DNA demethylation, suggesting that c-Rel containing NF-κB transcription factor complex is responsible for Gadd45β regulation during memory formation. Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

No MeSH data available.


Related in: MedlinePlus