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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

Gadd45β expression is increased in the hippocampus following learning. (A) Animals were trained to contextual fear conditioning and area CA1 collected 1 h later (Fear conditioned). A separate group of animals was exposed to the training context for 2 h, followed by the same contextual fear conditioning procedure and area CA1 collected 1 h later (Latent Inhibition). Quantitative RT-PCR revealed an increase in Gadd45β, but not Gadd45α or Gadd45γ, gene expression in area CA1 following fear conditioning (n = 5–6 per group). (B) Western blot analysis confirmed a moderate increase in Gadd45β protein expression at 1 h in the fear conditioned, but not latent inhibition, group (n = 5–6 per group). *p < 0.05 from Naïve. #p < 0.05 from FC.
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Figure 1: Gadd45β expression is increased in the hippocampus following learning. (A) Animals were trained to contextual fear conditioning and area CA1 collected 1 h later (Fear conditioned). A separate group of animals was exposed to the training context for 2 h, followed by the same contextual fear conditioning procedure and area CA1 collected 1 h later (Latent Inhibition). Quantitative RT-PCR revealed an increase in Gadd45β, but not Gadd45α or Gadd45γ, gene expression in area CA1 following fear conditioning (n = 5–6 per group). (B) Western blot analysis confirmed a moderate increase in Gadd45β protein expression at 1 h in the fear conditioned, but not latent inhibition, group (n = 5–6 per group). *p < 0.05 from Naïve. #p < 0.05 from FC.

Mentions: First we tested whether or not learning triggers expression changes of diverse Gadd45 isoforms. For these experiments, animals were trained in a contextual fear conditioning paradigm and after 1 h area CA1 was isolated and we examined changes in Gadd45α, Gadd45β and Gadd45γ gene expression. We chose to assess Gadd45 expression levels at 1 h following fear conditioning, as we have previously found optimal changes in DNA methylation levels in area CA1 of the hippocampus (Lubin et al., 2008). As a control for associative memory, we exposed a separate group of animals to a non-associative latent inhibition learning paradigm procedure, which involves exposure to the fear conditioning chamber only (context) followed by a delayed delivery of the aversive footshock 2 h later, preventing the subject to not associate the unconditioned stimulus (footshock) with the conditioned stimulus (context; Gupta et al., 2010). We found significant increases in Gadd45β (F(2,12) = 4.067, p < 0.05), but not Gadd45α (F(2,12) = 0.674, p = 0.527) or Gadd45γ (F(2,11) = 0.550, p = 0.591) mRNA levels in area CA1 following fear conditioning (Figure 1A). The increase in Gadd45β mRNA levels was not present in the latent inhibition group, confirming that Gadd45β gene expression changes were specific to context-learning along, and occurred with a moderate increase in Gadd45β protein expression (F(2,10) = 3.895, p = 0.056; Figure 1B). Collectively, these results suggest that Gadd45β gene and protein expression are increased in area CA1 as a function of associative learning.


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)

Gadd45β expression is increased in the hippocampus following learning. (A) Animals were trained to contextual fear conditioning and area CA1 collected 1 h later (Fear conditioned). A separate group of animals was exposed to the training context for 2 h, followed by the same contextual fear conditioning procedure and area CA1 collected 1 h later (Latent Inhibition). Quantitative RT-PCR revealed an increase in Gadd45β, but not Gadd45α or Gadd45γ, gene expression in area CA1 following fear conditioning (n = 5–6 per group). (B) Western blot analysis confirmed a moderate increase in Gadd45β protein expression at 1 h in the fear conditioned, but not latent inhibition, group (n = 5–6 per group). *p < 0.05 from Naïve. #p < 0.05 from FC.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4584956&req=5

Figure 1: Gadd45β expression is increased in the hippocampus following learning. (A) Animals were trained to contextual fear conditioning and area CA1 collected 1 h later (Fear conditioned). A separate group of animals was exposed to the training context for 2 h, followed by the same contextual fear conditioning procedure and area CA1 collected 1 h later (Latent Inhibition). Quantitative RT-PCR revealed an increase in Gadd45β, but not Gadd45α or Gadd45γ, gene expression in area CA1 following fear conditioning (n = 5–6 per group). (B) Western blot analysis confirmed a moderate increase in Gadd45β protein expression at 1 h in the fear conditioned, but not latent inhibition, group (n = 5–6 per group). *p < 0.05 from Naïve. #p < 0.05 from FC.
Mentions: First we tested whether or not learning triggers expression changes of diverse Gadd45 isoforms. For these experiments, animals were trained in a contextual fear conditioning paradigm and after 1 h area CA1 was isolated and we examined changes in Gadd45α, Gadd45β and Gadd45γ gene expression. We chose to assess Gadd45 expression levels at 1 h following fear conditioning, as we have previously found optimal changes in DNA methylation levels in area CA1 of the hippocampus (Lubin et al., 2008). As a control for associative memory, we exposed a separate group of animals to a non-associative latent inhibition learning paradigm procedure, which involves exposure to the fear conditioning chamber only (context) followed by a delayed delivery of the aversive footshock 2 h later, preventing the subject to not associate the unconditioned stimulus (footshock) with the conditioned stimulus (context; Gupta et al., 2010). We found significant increases in Gadd45β (F(2,12) = 4.067, p < 0.05), but not Gadd45α (F(2,12) = 0.674, p = 0.527) or Gadd45γ (F(2,11) = 0.550, p = 0.591) mRNA levels in area CA1 following fear conditioning (Figure 1A). The increase in Gadd45β mRNA levels was not present in the latent inhibition group, confirming that Gadd45β gene expression changes were specific to context-learning along, and occurred with a moderate increase in Gadd45β protein expression (F(2,10) = 3.895, p = 0.056; Figure 1B). Collectively, these results suggest that Gadd45β gene and protein expression are increased in area CA1 as a function of associative learning.

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