HDAC3-Mediated Repression of the Nr4a Family Contributes to Age-Related Impairments in Long-Term Memory

Abstract

Aging is accompanied by cognitive deficits, including impairments in long-term memory formation. Understanding the molecular mechanisms that support preserved cognitive function in aged animals is a critical step toward identifying novel therapeutic targets that could improve memory in aging individuals. One potential mechanism is the Nr4a family of genes, a group of CREB-dependent nuclear orphan receptors that have previously been shown to be important for hippocampal memory formation. Here, using a cross-species approach, we tested the role of Nr4a1 and Nr4a2 in age-related memory impairments. Using a rat model designed to identify individual differences in age-related memory impairments, we first identified Nr4a2 as a key gene that fails to be induced by learning in cognitively impaired male aged rats. Next, using a mouse model that allows for genetic manipulations, we determined that histone deacetylase 3 (HDAC3) negatively regulates Nr4a2 in the aged male and female hippocampus. Finally, we show that overexpression of Nr4a1, Nr4a2, or both transcripts in the male mouse dorsal hippocampus can ameliorate age-related impairments in object location memory. Together, our results suggest that Nr4a2 may be a key mechanism that promotes preserved cognitive function in old age, with HDAC3-mediated repression of Nr4a2 contributing to age-related cognitive decline. More broadly, these results indicate that therapeutic strategies to promote Nr4a gene expression or function may be an effective strategy to improve cognitive function in old age.SIGNIFICANCE STATEMENT Aging is accompanied by memory impairments, although there is a great deal of variability in the severity of these impairments. Identifying molecular mechanisms that promote preserved memory or participate in cognitive reserve in old age is important to develop strategies that promote healthy cognitive aging. Here, we show that learning-induced expression of the CREB-regulated nuclear receptor gene Nr4a2 is selectively impaired in aged rats with memory impairments. Further, we show that Nr4a2 is regulated by histone deacetylase HDAC3 in the aged mouse hippocampus. Finally, we demonstrate that hippocampal overexpression of either Nr4a2 or its family member, Nr4a1, can ameliorate age-related memory impairments. This suggests that promoting Nr4a expression may be a novel strategy to improve memory in aging individuals.

Keywords: HDAC3; Nr4a1; Nr4a2; aging; epigenetics; memory.

Figure 1.

Behavioral characterization of young, AU, and AI rats. A, LI scores from young, AU, and AI rats. Aged rats show significantly higher LI scores and a larger distribution of scores compared with young rats. B, OLM experimental design. C, Young and AU rats show significantly better memory for OLM than AI rats. D, No group differences were observed in total object exploration time. E, LI scores in the water maze negatively correlate with DI scores in OLM. All data are shown as mean ± SEM. *p < 0.05, **p < 0.01.

Figure 2.

Learning-induced hippocampal Nr4a2 is impaired in cognitively impaired aged rats. A, Experimental design. Young, AU, or AI rats were killed 1 h after OLM or were killed from the HC. B, Nr4a1 mRNA is upregulated in the dorsal hippocampus following OLM in young, but not AU or AI rats. C, Nr4a2 mRNA is upregulated by OLM in the dorsal hippocampus of young and AU rats but fails to increase following learning in AI rats. D, Hippocampal cFos expression is significantly increased by OLM in young, AU, and AI rats. All data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

HDAC3 negatively regulates both Nr4a2 mRNA and acetylation at the Nr4a2 promoter in the aged mouse hippocampus. (A) Experimental design for B–C. Young and old mice were sacrificed 1h after OLM training or were sacrificed from HC. (B) Nr4a1 mRNA was significantly increased in the dorsal hippocampus of both young and old mice following OLM. (C) Nr4a2 mRNA was upregulated in the dorsal hippocampus of young mice after OLM but failed to increase in old mice. (D) Experimental design for E–H. 18-month-old HDAC3^+/+ and HDAC3^flox/flox mice injected with AAV-CaMKII-Cre into the dorsal hippocampus were sacrificed 1h after OLM training (OLM) or were sacrificed from HC. (E) Nr4a1 mRNA was significantly increased in the dorsal hippocampus of both aged HDAC3^+/+ and aged HDAC3^flox/flox mice following OLM. (F) Nr4a2 mRNA failed to increase after OLM in aged HDAC3^+/+ mice, but is significantly upregulated by OLM in the absence of HDAC3 in aged HDAC3^flox/flox mice. (G) Occupancy of H4K8Ac at the Nr4a1 promoter was not affected by OLM training in either HDAC3^+/+ or HDAC3^flox/flox mice. (H) Occupancy of H4K8Ac at the Nr4a2 promoter was not altered by learning in HDAC3^+/+ mice, but H4K8Ac was enriched at the Nr4a2 promoter following OLM training in aged HDAC3^flox/flox mice. All data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Hippocampal overexpression of Nr4a1, Nr4a2, or both transcripts enhances OLM. A, Experimental design. Following injection of AAV-EV, AAV-HA-Nr4a1, AAV-V5-Nr4a2, or both AAV-HA-Nr4a1 and AAV-V5-Nr4a2, mice were trained and tested in OLM. A subset of these animals was then trained in ORM. B, Representative immunofluorescence images showing expression of the V5 epitope tag (green) in the dorsal hippocampus following injection of EV, AAV-HA-Nr4a1, AAV-V5-Nr4a2, or both viruses. V5 labeling was observed in animals injected with AAV-V5-Nr4a2 or both viruses. Neurons are counterstained with NeuroTrace (red), a fluorescent Nissl stain. Immunolabeling of the HA epitope tag on AAV-HA-Nr4a1 was unsuccessful and is not shown. C–F, RT-qPCR verification of viral expression. C, The HA-Nr4a1 transcript was expressed in the groups infused with AAV-HA-Nr4a1 and both viruses. D, V5-Nr4a2 was expressed in groups infused with AAV-v5-Nr4a2 or both viruses. E, Endogenous Nr4a1 mRNA was expressed at significantly higher levels in groups injected with either AAV-HA-Nr4a1 or both viruses. F, Endogenous Nr4a2 mRNA was expressed at significantly higher levels in group injected with either AAV-v5-Nr4a2 or both viruses. G, Mice injected with either AAV-HA-Nr4a1, AAV-v5-Nr4a2, or both viruses showed significantly better memory for OLM than AAV-EV controls. H, All groups showed similar levels of total object exploration during the OLM test. I, Hippocampal infusion of the viruses did not improve memory for ORM. J, All groups showed similar levels of total object exploration during the ORM test. All data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.