Survey of the Arc Epigenetic Landscape in Normal Cognitive Aging.

Aging is accompanied by aberrant gene expression that ultimately affects brain plasticity and the capacity to form long-term memories. Immediate-early genes (IEGs) play an active role in these processes. Using a rat model of normal cognitive aging, we found that the expression of Egr1 and c-Fos was associated with chronological age, whereas Arc was more tightly linked to cognitive outcomes in aging. More specifically, constitutive Arc expression was significantly elevated in aged rats with memory impairment compared to cognitively intact aged rats and young adult animals. Since alterations in the neuroepigenetic mechanisms that gate hippocampal gene expression are also associated with cognitive outcome in aging, we narrowed our focus on examining potential epigenetic mechanisms that may lead to aberrant Arc expression. Employing a multilevel analytical approach using bisulfite sequencing, chromatin immunoprecipitations, and micrococcal nuclease digestion, we identified CpG sites in the Arc promoter that were coupled to poor cognitive outcomes in aging, histone marks that were similarly coupled to spatial memory deficits, and nucleosome positioning that also varied depending on cognitive status. Together, these findings paint a diverse and complex picture of the Arc epigenetic landscape in cognitive aging and bolster a body of work, indicating that dysfunctional epigenetic regulation is associated with memory impairment in the aged brain.

Experience Modulates the Effects of Histone Deacetylase Inhibitors on Gene and Protein Expression in the Hippocampus: Impaired Plasticity in Aging.

The therapeutic potential of histone deacetylase inhibitor (HDACi) treatment has attracted considerable attention in the emerging area of cognitive neuroepigenetics. The possibility that ongoing cognitive experience importantly regulates the cell biological effects of HDACi administration, however, has not been systematically examined. In an initial experiment addressing this issue, we tested whether water maze training influences the gene expression response to acute systemic HDACi administration in the young adult rat hippocampus. Training powerfully modulated the response to HDACi treatment, increasing the total number of genes regulated to nearly 3000, including many not typically linked to neural plasticity, compared with <300 following HDACi administration alone. Although water maze training itself also regulated nearly 1800 genes, the specific mRNAs, gene networks, and biological pathways involved were largely distinct when the same experience was provided together with HDACi administration. Next, we tested whether the synaptic protein response to HDACi treatment is similarly dependent on recent cognitive experience, and whether this plasticity is altered in aged rats with memory impairment. Whereas synaptic protein labeling in the young hippocampus was selectively increased when HDACi administration was provided in conjunction with water maze training, combined treatment had no effect on synaptic proteins in the aged hippocampus. Our findings indicate that ongoing experience potently regulates the molecular consequences of HDACi treatment and that the interaction of recent cognitive experience with histone acetylation dynamics is disrupted in the aged hippocampus. SIGNIFICANCE STATEMENT: The possibility that interventions targeting epigenetic regulation could be effective in treating a range of neurodegenerative disorders has attracted considerable interest. Here we demonstrate in the rat hippocampus that ongoing experience powerfully modifies the molecular response to one such intervention, histone deacetylase inhibitor (HDACi) administration. A single learning episode dramatically shifts the gene expression profile induced by acute HDACi treatment, yielding a qualitatively distinct hippocampal transcriptome compared with the influence of behavioral training alone. The downstream synaptic protein response to HDACi administration is similarly experience-dependent, and we report that this plasticity is disrupted in the aged hippocampus. The findings highlight that accommodating the modulatory influence of ongoing experience represents a challenge for therapeutic development in the area of cognitive neuroepigenetics.

A fine balance: Regulation of hippocampal Arc/Arg3.1 transcription, translation and degradation in a rat model of normal cognitive aging.

Memory decline is a common feature of aging. Expression of the immediate-early gene Arc is necessary for normal long-term memory, and although experience dependent Arc transcription is reportedly reduced in the aged rat hippocampus, it has not been clear whether this effect is an invariant consequence of growing older, or a finding linked specifically to age-related memory impairment. Here we show that experience dependent Arc mRNA expression in the hippocampus fails selectively among aged rats with spatial memory deficits. While these findings are consistent with the possibility that blunted Arc transcription contributes to cognitive aging, we also found increased basal ARC protein levels in the CA1 field of the hippocampus in aged rats with memory impairment, together with a loss of the experience dependent increase observed in young and unimpaired aged rats. Follow-up analysis revealed that increased basal translation and blunted ubiquitin mediated degradation may contribute to increased basal ARC protein levels noted in memory impaired aged rats. These findings indicate that Arc expression is regulated at multiple levels, and that several of these mechanisms are altered in cognitively impaired aged rats. Defining the influence of these alterations on the spatial and temporal fidelity of synapse specific, memory-related plasticity in the aged hippocampus is an important challenge.

Reassessing the effects of histone deacetylase inhibitors on hippocampal memory and cognitive aging.

Converging results link histone acetylation dynamics to hippocampus-dependent memory, including evidence that histone deacetylase inhibitor (HDACi) administration enhances long-term memory. Previously, we demonstrated that aging disrupts the coordinated epigenetic response to recent experience observed in the young adult hippocampus. Here, we extended that work to test the cognitive effects of a novel, brain-penetrant HDACi (EVX001688; EVX) that we confirmed yields robust, relatively long lasting dose-dependent increases in histone acetylation in the hippocampus. In young rats, acute systemic EVX administration, scheduled to yield elevated histone acetylation levels during training in a contextual fear conditioning (CFC) task, had no effect on memory retention at 24 h at any dose examined (10, 30, or 60 mg/kg). Pretraining injection of another HDACi, sodium butyrate, also failed to affect fear memory, and CFC training itself had no influence on hippocampal histone acetylation at 1 hour in mice or two strains of rats. EVX administration before water maze training in young rats yielded a modest effect such that the middle dose produced marginally better 24-h retention than either the low or high dose, but only a small numerical benefit relative to vehicle. Guided by those findings, a final experiment tested the influence of pretraining EVX treatment on age-related spatial memory impairment. The results, revealing no effect on performance, are consistent with the idea that effective procognitive HDACi treatments in aging may require intervention aimed at restoring coordinated epigenetic regulation rather than bulk increases in hippocampal histone acetylation.

Age-related memory impairment is associated with disrupted multivariate epigenetic coordination in the hippocampus.

Mounting evidence linking epigenetic regulation to memory-related synaptic plasticity raises the possibility that altered chromatin modification dynamics might contribute to age-dependent cognitive decline. Here we show that the coordinated orchestration of both baseline and experience-dependent epigenetic regulation seen in the young adult hippocampus is lost in association with cognitive aging. Using a well-characterized rat model that reliably distinguishes aged individuals with significant memory impairment from others with normal memory, no single epigenetic mark or experience-dependent modification in the hippocampus uniquely predicted differences in the cognitive outcome of aging. The results instead point to a multivariate pattern in which modification-specific, bidirectional chromatin regulation is dependent on recent behavioral experience, chronological age, cognitive status, and hippocampal region. Whereas many epigenetic signatures were coupled with memory capacity among young adults and aged rats with preserved cognitive function, such associations were absent among aged rats with deficits in hippocampal memory. By comparison with the emphasis in current preclinical translational research on promoting chromatin modifications permissive for gene expression, our findings suggest that optimally successful hippocampal aging may hinge instead on enabling coordinated control across the epigenetic landscape.

Age-related spatial learning impairment is unrelated to spinophilin immunoreactive spine number and protein levels in rat hippocampus.

Age-related impairments in hippocampus-dependent learning and memory tasks are not associated with a loss of hippocampal neurons, but may be related to alterations in synaptic integrity. Here we used stereological techniques to estimate spine number in hippocampal subfields using immunostaining for the spine-associated protein, spinophilin, as a marker. Quantification of the immunoreactive profiles was performed using the optical disector/fractionator technique. Aging was associated with a modest increase in spine number in the molecular layer of the dentate gyrus and CA1 stratum lacunosum-moleculare. By comparison, spinophilin protein levels in the hippocampus, measured by Western blot analysis, failed to differ as a function of age. Neither the morphological nor the protein level data were correlated with spatial learning ability across individual aged rats. The results extend current evidence on synaptic integrity in the aged brain, indicating that a substantial loss of dendritic spines and spinophilin protein in the hippocampus are unlikely to contribute to age-related impairment in spatial learning.

Selective cholinergic depletion of the hippocampus spares both behaviorally induced Arc transcription and spatial learning and memory.

We demonstrated previously that when hippocampal-dependent learning and plasticity are compromised by fornix lesions, behaviorally induced expression of the immediate early gene, Arc, is correspondingly low. The medial septum and the vertical diagonal band are major sources of subcortical afferents that innervate the hippocampus via the fornix. Here we assessed the specific contribution of cholinergic afferents from these regions to the impairments in spatial learning and behavioral induction of Arc transcription produced by fornix lesions. The immunotoxin, 192 IgG-saporin, was used to produce selective lesions of cholinergic cell bodies in the medial septum and vertical diagonal band. Rats were then trained on both cued and spatial delayed match-to-place tasks in a radial arm water maze. Animals with 192 IgG-saporin lesions learned both cue and place discrimination tasks in the water maze normally, and showed only a mild and transient impairment when switching from the cued to the spatial version of the task. Following behavioral testing, rats explored two novel environments sequentially in a setting known to induce Arc expression in hippocampal pyramidal neurons. In marked contrast to the effects of complete fornix transection, quantitative in situ autoradiography revealed no differences in Arc mRNA expression between sham and lesion animals in CA1, CA3 or stratum radiatum. The conclusion from these data is that cholinergic deafferentation alone cannot account for the spatial learning deficits or impaired behavioral induction of Arc transcription produced by fornix lesions.

Fornix lesions decouple the induction of hippocampal arc transcription from behavior but not plasticity.

The immediate-early gene (IEG) Arc is transcribed after behavioral and physiological treatments that induce synaptic plasticity and is implicated in memory consolidation. The relative contributions of neuronal activity and learning-related plasticity to the behavioral induction of Arc remain to be defined. To differentiate the contributions of each, we assessed the induction of Arc transcription in rats with fornix lesions that impair hippocampal learning yet leave cortical connectivity and neuronal firing essentially intact. Arc expression was assessed after exploration of novel environments and performance of a novel water maze task during which normal rats learned the spatial location of an escape platform. During the same task, rats with fornix lesions learned to approach a visible platform but did not learn its spatial location. Rats with fornix lesions had normal baseline levels of hippocampal Arc mRNA, but unlike normal rats, expression was not increased in response to water maze training. The integrity of signaling pathways controlling Arc expression was demonstrated by stimulation of the medial perforant path, which induced normal synaptic potentiation and Arc in rats with fornix lesions. Together, the results demonstrate that Arc induction can be decoupled from behavior and is more likely to indicate the engagement of synaptic plasticity mechanisms than synaptic or neuronal activity per se. The results further imply that fornix lesions may impair memory in part by decoupling neuronal activity from signaling pathways required for long-lasting hippocampal synaptic plasticity.