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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

Conditional mutation of p65/relA does not alter Gadd45β expression in the hippocampus following learning. (A) Floxed p65/relA mice (relAflox/+) were injected with Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) adenoviruses 2 weeks prior to fear conditioning to induce mutation in the p65/relA gene. The day after training, mice were tested to the auditory cue followed by the context 2 h later. (B) Immunohistochemistry showing GFP expression in the hippocampus. DAPI was used to visualization the different hippocampus sub-regions. (C) While there were no differences between group during training or testing to the auditory cue, relAflox/+ mice receiving Cre injections had impaired contextual fear memory relative to controls (n = 5–7 per group). (D)relAflox/+ were injected with Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) adenoviruses 2 weeks prior to fear conditioning and area CA1 collected 1 h later. (E) Western blot analysis confirmed knockdown of p65 and IκBα expression in the Cre-infused mice (n = 4–5 per group). (F) There were no differences in Gadd45β gene or protein expression following Cre-infusion relative to GFP-infused controls (n = 4–5 per group). *p < 0.05 from GFP. #p = 0.054 from GFP.
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Figure 3: Conditional mutation of p65/relA does not alter Gadd45β expression in the hippocampus following learning. (A) Floxed p65/relA mice (relAflox/+) were injected with Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) adenoviruses 2 weeks prior to fear conditioning to induce mutation in the p65/relA gene. The day after training, mice were tested to the auditory cue followed by the context 2 h later. (B) Immunohistochemistry showing GFP expression in the hippocampus. DAPI was used to visualization the different hippocampus sub-regions. (C) While there were no differences between group during training or testing to the auditory cue, relAflox/+ mice receiving Cre injections had impaired contextual fear memory relative to controls (n = 5–7 per group). (D)relAflox/+ were injected with Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) adenoviruses 2 weeks prior to fear conditioning and area CA1 collected 1 h later. (E) Western blot analysis confirmed knockdown of p65 and IκBα expression in the Cre-infused mice (n = 4–5 per group). (F) There were no differences in Gadd45β gene or protein expression following Cre-infusion relative to GFP-infused controls (n = 4–5 per group). *p < 0.05 from GFP. #p = 0.054 from GFP.

Mentions: While our pharmacological manipulation with DDTC suggests a role for NF-κB activity in regulation Gadd45β expression and BDNF DNA demethylation in the hippocampus during memory formation, our studies do not yet distinguish between the contributions of different NF-κB subunits that may have been involved. The p65/RelA and p50 heterodimer is critical for nuclear translocation and activation of the NF-κB complex, thus we tested if manipulation of p65/RelA would mimic the effects of inhibiting NF-κB signaling activity with DDTC on Gadd45β expression following learning. We conditionally mutated p65/relA using a Cre-loxP insert spanning exons 7–10 containing the Rel homology domain and nuclear translocation site (Algul et al., 2007). To induce the relAΔ mutation in area CA1 of the adult hippocampus, we infused Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) viral vectors 2 weeks prior to fear conditioning (Figures 3A,B). Consistent with a role for NF-κB signaling in hippocampus-dependent memory formation, we found that while relAflox/+ mice successfully acquired the fear memory during training (t(10) = 0.9807, p = 0.349), when tested 24 h later, relAflox/+ mice had significant impairments in memory retention for contextual (t(10) = 2.182, p = 0.054) but not hippocampus-independent auditory (t(9) = 1.184, p = 0.266), fear memory (Figure 3C). This is the first evidence that local knockdown of p65/relA in the adult hippocampus impairs long-term memory formation. Next, we tested if mutation of p65/relA altered Gadd45β expression following learning (Figure 3D). Interestingly, while we confirmed that relAflox/+ mice had reduced expression of p65 (t(6) = 3.583, p < 0.05) and the NF-κB associated protein IκBα (t(7) = 2.624, p < 0.05) relative to controls (Figure 3E), we found no effect of the p65/relA mutation on Gadd45β mRNA (t(6) = 0.214, p = 0.837) or protein (t(7) = 0.427, p = 0.681) levels following learning (Figure 3F). Thus far, our results suggest that while NF-κB signaling and p65/RelA activity in the hippocampus are critical for memory formation, p65/RelA is not responsible for the NF-κB-dependent regulation of Gadd45β expression during the memory consolidation process.


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)

Conditional mutation of p65/relA does not alter Gadd45β expression in the hippocampus following learning. (A) Floxed p65/relA mice (relAflox/+) were injected with Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) adenoviruses 2 weeks prior to fear conditioning to induce mutation in the p65/relA gene. The day after training, mice were tested to the auditory cue followed by the context 2 h later. (B) Immunohistochemistry showing GFP expression in the hippocampus. DAPI was used to visualization the different hippocampus sub-regions. (C) While there were no differences between group during training or testing to the auditory cue, relAflox/+ mice receiving Cre injections had impaired contextual fear memory relative to controls (n = 5–7 per group). (D)relAflox/+ were injected with Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) adenoviruses 2 weeks prior to fear conditioning and area CA1 collected 1 h later. (E) Western blot analysis confirmed knockdown of p65 and IκBα expression in the Cre-infused mice (n = 4–5 per group). (F) There were no differences in Gadd45β gene or protein expression following Cre-infusion relative to GFP-infused controls (n = 4–5 per group). *p < 0.05 from GFP. #p = 0.054 from GFP.
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Figure 3: Conditional mutation of p65/relA does not alter Gadd45β expression in the hippocampus following learning. (A) Floxed p65/relA mice (relAflox/+) were injected with Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) adenoviruses 2 weeks prior to fear conditioning to induce mutation in the p65/relA gene. The day after training, mice were tested to the auditory cue followed by the context 2 h later. (B) Immunohistochemistry showing GFP expression in the hippocampus. DAPI was used to visualization the different hippocampus sub-regions. (C) While there were no differences between group during training or testing to the auditory cue, relAflox/+ mice receiving Cre injections had impaired contextual fear memory relative to controls (n = 5–7 per group). (D)relAflox/+ were injected with Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) adenoviruses 2 weeks prior to fear conditioning and area CA1 collected 1 h later. (E) Western blot analysis confirmed knockdown of p65 and IκBα expression in the Cre-infused mice (n = 4–5 per group). (F) There were no differences in Gadd45β gene or protein expression following Cre-infusion relative to GFP-infused controls (n = 4–5 per group). *p < 0.05 from GFP. #p = 0.054 from GFP.
Mentions: While our pharmacological manipulation with DDTC suggests a role for NF-κB activity in regulation Gadd45β expression and BDNF DNA demethylation in the hippocampus during memory formation, our studies do not yet distinguish between the contributions of different NF-κB subunits that may have been involved. The p65/RelA and p50 heterodimer is critical for nuclear translocation and activation of the NF-κB complex, thus we tested if manipulation of p65/RelA would mimic the effects of inhibiting NF-κB signaling activity with DDTC on Gadd45β expression following learning. We conditionally mutated p65/relA using a Cre-loxP insert spanning exons 7–10 containing the Rel homology domain and nuclear translocation site (Algul et al., 2007). To induce the relAΔ mutation in area CA1 of the adult hippocampus, we infused Cre-containing (AAV-CMV-Cre-GFP) or empty (AAV-CMV-GFP) viral vectors 2 weeks prior to fear conditioning (Figures 3A,B). Consistent with a role for NF-κB signaling in hippocampus-dependent memory formation, we found that while relAflox/+ mice successfully acquired the fear memory during training (t(10) = 0.9807, p = 0.349), when tested 24 h later, relAflox/+ mice had significant impairments in memory retention for contextual (t(10) = 2.182, p = 0.054) but not hippocampus-independent auditory (t(9) = 1.184, p = 0.266), fear memory (Figure 3C). This is the first evidence that local knockdown of p65/relA in the adult hippocampus impairs long-term memory formation. Next, we tested if mutation of p65/relA altered Gadd45β expression following learning (Figure 3D). Interestingly, while we confirmed that relAflox/+ mice had reduced expression of p65 (t(6) = 3.583, p < 0.05) and the NF-κB associated protein IκBα (t(7) = 2.624, p < 0.05) relative to controls (Figure 3E), we found no effect of the p65/relA mutation on Gadd45β mRNA (t(6) = 0.214, p = 0.837) or protein (t(7) = 0.427, p = 0.681) levels following learning (Figure 3F). Thus far, our results suggest that while NF-κB signaling and p65/RelA activity in the hippocampus are critical for memory formation, p65/RelA is not responsible for the NF-κB-dependent regulation of Gadd45β expression during the memory consolidation process.

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