NR4A nuclear receptors support memory enhancement by histone deacetylase inhibitors

Abstract

The formation of a long-lasting memory requires a transcription-dependent consolidation period that converts a short-term memory into a long-term memory. Nuclear receptors compose a class of transcription factors that regulate diverse biological processes, and several nuclear receptors have been implicated in memory formation. Here, we examined the potential contribution of nuclear receptors to memory consolidation by measuring the expression of all 49 murine nuclear receptors after learning. We identified 13 nuclear receptors with increased expression after learning, including all 3 members of the Nr4a subfamily. These CREB-regulated Nr4a genes encode ligand-independent "orphan" nuclear receptors. We found that blocking NR4A activity in memory-supporting brain regions impaired long-term memory but did not impact short-term memory in mice. Further, expression of Nr4a genes increased following the memory-enhancing effects of histone deacetylase (HDAC) inhibitors. Blocking NR4A signaling interfered with the ability of HDAC inhibitors to enhance memory. These results demonstrate that the Nr4a gene family contributes to memory formation and is a promising target for improving cognitive function.

Figure 1. The formation of contextual fear memories induces expression of NR genes in the hippocampus.

(A) Contextual fear conditioning produces a long-lasting memory for the training context and the association of this context with a mild foot-shock. RNA was collected from whole hippocampi at multiple time points after training to survey the impact of training on NR gene expression using a standard ΔΔCT approach. (B) High-throughput qPCR data are illustrated for the 13 NR genes with statistically significant changes in gene expression during the first 2 hours after training, the window in which the majority of changes were observed. (C) The data from this screen indicate that 13 NRs have increased expression in the hippocampus within the first 2 hours after training (red), whereas 13 NRs are not appreciably expressed in the hippocampus (black). The remaining 23 NRs (blue) show no evidence of altered hippocampal expression in the first 2 hours after training. Expression changes are illustrated within clusters defined by anatomical expression profiling (IA, IB, IC, IIA, IIB, IIC) in a diagram modified with permission from Cell; ref. 17. (D) Nr4a1 expression is potently induced in the first hour after learning (P < 0.001). (E) Nr4a2 expression increases after fear conditioning (P = 0.033). (F) Nr4a3 expression increases after fear conditioning (P = 0.004). HC, home cage. Error bars represent SEM. *P < 0.05. See also Supplemental Figure 1.

Figure 2. Blocking function of NR4A family NRs in the hippocampus impairs long-term memory formation.

(A) To impede NR4A signaling in forebrain neurons, a tTA transgene expressed selectively in the forebrain was used to activate a dominant-negative Nr4a transgene (NR4ADN) under control of the tetO. (B) An antibody to the YFP tag on the transgenic NR4ADN protein coimmunoprecipitates endogenous NR4A2 protein from hippocampal protein extracts, confirming the ability of the dominant-negative transgenic protein to heterodimerize with NR4A protein. (C) In the top row, immunolabeling for the NR4ADN hemagglutinin (HA) tag (brown) with cresyl violet counterstain (purple) shows expression in the hippocampus as well as in cortex and striatum (original magnification, ×100). Fluorescent immunolabeling for the YFP tag (middle row) and propidium iodide counterstaining (bottom row) illustrates transgene expression in hippocampal subregions CA1 (original magnification, ×250) and the dentate gyrus (DG) (original magnification, ×250) but not the amygdala (original magnification, ×62.5). (D) NR4ADN mice have selective deficits in long-term contextual fear memory, whereas neither short-term contextual nor long-term cued fear conditioning are impaired. (E) No difference in 24-hour contextual fear memory performance was detected between wild-type and NR4ADN mice after 4 weeks of doxycycline (dox) treatment (P = 0.87, n = 12 mice/group). All error bars denote SEM. *P < 0.05. See also Supplemental Figure 2.

Figure 3. The ability of an HDAC inhibitor to enhance memory is blocked by the Nr4a dominant-negative transgene.

(A) Intrahippocampal injection of the HDAC inhibitor TSA (T) enhances acetylation of histone H3 (AcH3), as illustrated at 1 hour after TSA injection after training. Veh/V, vehicle. (B and C) Intrahippocampal TSA also increases expression of the (B) NR4A1 and (C) NR4A3 protein at the same time point. (D) Intrahippocampal TSA increases mRNA levels for each of the 3 Nr4a genes at 1 hour after injection. (E) Injection of TSA into hippocampi after training enhances 24-hour contextual fear memory of wild-type mice but fails to enhance memory of NR4ADN littermates. Error bars represent SEM. *P < 0.05. See also Supplemental Figure 3. Lanes were run on the same gel but were noncontiguous (white lines).

Figure 4. Common gene targets are impaired by NR4ADN expression and increased by TSA treatment.

(A) NR4ADN transgenic mice have reduced expression of several putative Nr4a target genes, including Bdnf, Fosl2, and Pak6. (B) Intrahippocampal TSA treatment increases expression of several genes, including Fosl2 and Bdnf. Error bars represent SEM. *P < 0.05. See also Supplemental Figure 4.

Figure 5. NR4A signaling contributes to memory formation and enhancement by HDAC inhibitors.

HDAC inhibitors increase Nr4a gene expression, and blocking NR4A signaling prevents memory enhancement by HDAC inhibitors, suggesting a model in which NR4A target genes contribute to memory enhancement by HDAC inhibition. In this figure, arrows represent processes that stimulate gene expression and enhance memory formation. In contrast, blunt ends signify pathways that repress gene expression and limit memory formation. Nucleosomes are indicated by the green barrels that are encircled by the gray ribbon, which illustrates promoter DNA. Acetylation (ac) of the histone proteins that constitute the nucleosome (N) is dictated by a dynamic equilibrium between HDAC and HAT activity. Impairing HAT activity would be predicted to reduce Nr4a gene expression and impair memory formation. As illustrated in this study, blocking HDAC activity increases Nr4a gene expression and enhances memory formation. Also, inhibiting the function of NR4A proteins using a dominant-negative protein blocks memory enhancement by HDAC inhibition and impedes expression of several putative NR4A target genes. The increase in Nr4a gene expression observed after TSA injection after training is accompanied by increased expression of the putative NR4A target genes, Bdnf and Fosl2, two memory-associated genes that may contribute to the molecular mechanism of memory enhancement by HDAC inhibitors.