Ectopic expression of aPKC-mediated phosphorylation in p300 modulates hippocampal neurogenesis, CREB binding and fear memory differently with age

Abstract

Epigenetic modifications have become an emerging interface that links extrinsic signals to alterations of gene expression that determine cell identity and function. However, direct signaling that regulates epigenetic modifications is unknown. Our previous work demonstrated that phosphorylation of CBP at Ser 436 by atypical protein kinase C (aPKC) regulates age-dependent hippocampal neurogenesis and memory. p300, a close family member of CBP, lacks the aPKC-mediated phosphorylation found in CBP. Here, we use a phosphorylation-competent p300 (G442S) knock-in (KI) mouse model that ectopically expresses p300 phosphorylation in a homologous site to CBP Ser436, and assess its roles in modulating hippocampal neurogenesis, CREB binding ability, and fear memory. Young adult (3 months) p300G422S-KI mice exhibit enhanced hippocampal neurogenesis due to increased cell survival of newly-generated neurons, without alterations in CREB binding and contextual fear memory. On the other hand, mature adult (6 months) p300G422S-KI mice display reduced CREB binding, associated with impaired contextual fear memory without alterations in hippocampal neurogenesis. Additionally, we show that repulsive interaction between pS133-CREB and pS422-p300G422S may contribute to the reduced CREB binding to p300G422S. Together, these data suggest that a single phosphorylation change in p300 has the capability to modulate hippocampal neurogenesis, CREB binding, and associative fear memory.

Figure 1

p300G422S-KI increases adult neurogenesis at the age of 3 months by reducing cell death of newborn neurons. (a) Representative images of hippocampal sections from 3-month p300G422S-KI (homologous p300G422S-KI) and their WT, sacrificed 30 days after BrdU injections, and stained for BrdU (green) and NeuN (red). Arrows represent BrdU+/NeuN+ neurons. Scale bar = 20 µm. (b) Quantitative analysis of the total number of BrdU+/NeuN+ newborn neurons in the hippocampi from 3 months WT and p300G422S-KI, as shown in (a). (c) Representative fluorescence images of hippocampal sections from 3 months WT and p300G422S-KI mice, sacrificed one day after BrdU injections, stained for BrdU (green) and Ki67 (red). Arrows denote BrdU+/Ki67+ proliferating cells. Scale bar = 20 μm. (d) Quantitative analysis of the total number of BrdU+/Ki67+ proliferating cells in the hippocampi from 3 months p300G422S-KI and their WT littermates. (e) Representative images of hippocampal sections from 3 months WT and p300G422S-KI mice, sacrificed 12 days after BrdU injections, stained for BrdU (green) and NeuN (red). Arrows represent BrdU+/NeuN+ neurons. Scale bar = 20 µm. (f) Quantitative analysis of the total number of BrdU+/NeuN+ neurons in the hippocampi from WT and p300G422S-KI mice (3-month) as shown in (e). (g) Representative images of hippocampal sections from 3-month WT mice, stained for cleaved caspase 3 (CC3) (green) and DCX (red). Arrows denote CC3+/DCX+ cells. Scale bar = 10 μm. (h) Quantitative analysis of the total number of CC3+/DCX+ cells in the hippocampi from 3 months p300G422S-KI and their WT littermates. (i) Representative images of hippocampal sections from 6 months WT and p300G422S-KI mice, sacrificed 12 days after BrdU injections, stained for BrdU (green) and NeuN (red). Arrows represent BrdU+/NeuN+ neurons. Scale bar = 20 µm. (j,k) Quantitative analysis of total number (j) and the proportion (k) of BrdU+/NeuN+ cells in the hippocampi from WT and p300G422S-KI mice (6 months) as shown in (i). The boxed areas were shown at higher magnification on the right panels in (a,e,i). *p < 0.05; ***p < 0.001, n = 4 animals for each group.

Figure 2

p300G422S-KI shows the impaired CREB binding to p300 at the age of 6 but not 3 months. (a) Co-immunoprecipitation analysis of the interaction between p300 and CREB in the hippocampi of WT and p300G422S-KI mice at 3 and 6 months. Hippocampal lysates were immunoprecipitated with a p300 antibody, washed and blotted with the indicated antibody. Arrow indicates CREB expression band. (b) The graph indicates the fold changes of the relative pulled-down CREB protein over the total p300 amounts, as determined by densitometry and normalized to samples from 3 months WT, (two-way ANOVA: F (1,16) = 4.86, p = 0.04; Tukey’s multiple comparisons, *p < 0.05, n = 5). (c) Co-immunoprecipitation analysis of the interaction between p300 and CREB in C57B6 strain hippocampi at the age of 3 and 6 months as described in (a). The graph data was normalized to one of 3 months old C57B6 samples. (d) Western blot analysis for pS133-CREB and pT410/403-aPKC zeta/iota in hippocampal extracts from 3 and 6 months WT and p300G422S-KI mice. Blots were reprobed for total CREB or aPKC as loading controls. (e) Graphs show relative levels of pS133-CREB and pT410/403-aPKC over total CREB and aPKC, respectively, normalized to one of 3 months WT samples. (f) Western blot analysis for pS133-CREB in hippocampal extracts from 3 and 6 months C57B6 mouse strain. Blots were reprobed for total CREB as a loading control. Graphs show relative levels of pS133-CREB over total CREB, normalized to one of 3 months C57B6 samples. (g) Schematic model showing that both pS133-CREB and pS422-p300 determine the interaction between p300 and CREB. (h) Co-immunoprecipitation analysis of the association of p300 with pS133-CREB and total aPKC in the hippocampi of 6 months WT and p300G422S-KI mice. (i) The graph indicates the fold changes of the relative pulled-down pS133CREB protein and total aPKC over the total p300 amounts, as determined by densitometry and normalized to 6 months WT samples. exp1: individual experiment 1; exp2: individual experiment 2. *p < 0.05; **p < 0.01, n = 3–5 animals for each group. Images derived from different part of the same gel were cropped for layout reasons. Full-length blots/gels are presented in Supplementary Fig. 2.

Figure 3

p300G422S-KI reduces CREB-mediated gene expression at the age of 6 month. 6 months but not 3 months p300G422S-KI hippocampi have reduced expression of a CREB-mediated gene, NR4A1. *p < 0.05. n = 4–6 animals for each group.

Figure 4

p300G422S-KI at the age of 6 months shows reduced hippocampal contextual fear memory. (a) Both 3 and 6 months homozygous p300G422S-KI and their WT littermates, were pre-exposed to the conditioning context at day one, and received an immediate shock (1.0 mA, 2 seconds) within the same context at day two. Percentage of time spent freezing within the first two minutes when the mice were re-placed in the conditioning context at day three without shock. *p < 0.05 (b) Open field test was performed in an open box for 10 minutes. The cumulative time spent within the center and all 4 corners of the box was analyzed. Insets show representative traces of WT and p300G422S -KI mouse during the course of the open field test. (c) Analysis of the total distance travelled during the open field test for WT and p300G422S -KI mice at the age of 3 and 6 months. (d) Analysis of the mean velocity during the open field test at the age of 3 and 6 months. n = 8–13 animals for each group.