The hypothalamic–pituitary–adrenal (HPA) axis activity is one of several proposed mechanisms involved in brain aging. While there are considerable individual differences in circadian cortisol levels with age, excessive circulating levels of glucocorticoids associated with exogenous administration, Cushing’s syndrome, or HPA axis hyperactivity in response to chronic stress can cause cognitive impairment and reversible hippocampal atrophy (Brown et al. 2004; Miller and O'Callaghan 2005). In a longitudinal study of elderly people, those with elevated basal cortisol levels were more likely to have deficits in hippocampus-mediated declarative memory performance, as well as reduced hippocampal volumes (Lupien et al. 2005). One factor that may account for cognitive decline in subpopulations of the elderly is individual differences in the stress-response system, specifically genomic glucocorticoid signaling in the HPA axis (Goosens and Sapolsky 2007; Oitzl et al. 2009).

In the brain, the glucocorticoid receptor (GR) is ubiquitous and particularly enriched in the hippocampus, prefrontal cortex, hypothalamus, and amygdala in correspondence with their role in modulating HPA axis activity (Lupien et al. 2005). Results from a cellular system revealed an intracellular cascade involved in the mediation of GR in genomic glucocorticoid signaling in mammalian cells (Grad and Picard 2007; Wochnik et al. 2005). In the absence of ligand binding, the GR heterocomplex residing in the cytoplasm is assembled with molecular chaperones, mainly heat shock protein (Hsp) 90, p23, and immunophilin FK506 binding protein (FKBP) 51. Upon GR binding to its hormone ligands in the cytoplasm, FKBP51 (known to inhibit nuclear translocation of the GR heterocomplex by disrupting the attachment of the GR heterocomplex to the transport protein dynein) is competitively replaced by FKBP52 (Davies et al. 2002; Wochnik et al. 2005). By interacting with Hsp90, FKBP52, and dynein, GR is guided along cytoskeletal tracts to the nucleus to exert influences on specific gene transcription (Binder 2009). The activity of GR is therefore dependent on the relative levels of FKBP51 and FKBP52 in the cytoplasm (Grad and Picard 2007).

Examination of functional, anatomical, cellular, and neurochemical differences between aged animals that exhibit intact cognitive performance and those that are impaired has been pursued across rodents and non-human primates (Buccafusco 2006). In aged rats, elevated basal corticosterone levels predicted spatial memory impairment (Issa et al. 1990). In adult rodents, chronic stress or chronic exogenous administration of corticosterone caused reversible changes in the brain, such as dendritic retraction of pyramidal neurons in the prefrontal cortex and hippocampal CA3, decreased neurogenesis in the dentate gyrus, and hippocampal volume loss that was not associated with reduced neuron numbers (Lupien et al. 2009). It is not clear, however, whether downstream intracellular regulators of glucocorticoid signaling also play a role in cognitive decline.

We found in our present study that GR and FKBP52 expression were increased in aged mice irrespective of their object recognition performance compared to young mice, while FKBP51 expression was decreased only in aged mice with recognition memory impairment. In mammalian cells, the activity of GR is dependent on the relative levels of FKBP51 and FKBP52 in the cytoplasm where FKBP51 acts as an inhibitor and FKBP52 as a facilitator for nuclear translocation of GR upon hormone binding (Grad and Picard 2007; Wochnik et al. 2005). Our data further showed that the FKBP51/FKBP52 ratio correlated directly with DR in aged mice. These findings suggest that GR-mediated glucocorticoid signaling in the forebrain is increased with age by up-regulation of GR and FKBP52, and aging-related impairment in short-term recognition memory is associated with the concomitant FKBP51 down-regulation that further promotes glucocorticoid signaling. In agreement with our findings, a study using transgenic mice suggested that GR over-expression in forebrain was associated with accelerated aging-related deficiencies in feedback inhibition of the HPA axis activity, as well as mild dysfunction in long-term spatial memory, which was observed after reversal learning training in the Morris water maze test (Wei et al. 2007).

The single-trial object recognition test for rodents is a novelty-preference paradigm aimed at assessing non-spatial recognition memory and requiring innate motivation to explore novel stimuli (Bevins and Besheer 2006; Dere et al. 2007). This behavioral test measures the ability to recognize a familiar object and discriminate it from a novel object (Dere et al. 2007). Because aged mice spent significantly more time exploring both objects than young mice did during the retention phase, we tested whether there was any relationship between the FKBP51 expression and the total object exploration time. It appeared that the findings of FKBP51 expression were relatively selective to the object memory performance, as this neurochemical marker was not significantly correlated with the total object exploration time during either the training or retention phase in aged mice. Moreover, it does not appear that the direct correlation between the FKBP51 expression and DR in aged mice (Fig. 5) can be explained by differences in exploration or motivation. The total object exploration time during the retention phase did not predict DR in aged mice, nor did it correlate with the FKBP51 expression, suggesting this exploration time measure is orthogonal to DR. It is possible that aged animals showed reduced habituation to the objects compared to young animals. Dysregulation in GR signaling in the forebrain may be one of several potential mechanisms involved in aging-related impairment in short-term recognition memory. Any genomic GR signaling is not likely to occur during the behavioral task of relatively short period in the single-trial object recognition test. The combination of decreased FKBP51 expression and increased expression of GR and FKBP52 in forebrain in our present study may represent the molecular substrate for exaggerated genomic glucocorticoid signaling that adversely affects the functional condition of mice prior to the learning task and consequently predisposes them to develop short-term recognition memory impairment.

The GR-mediated effects of glucocorticoids are implicated in terminating ongoing stress reaction, facilitating recovery, and promoting memory storage in preparation for future events (de Kloet et al. 2005). Upon GR activation, increased influx of calcium through voltage-gated calcium channels helps to slowly reverse temporarily raised electrical activity in adaptive response to stress (de Kloet et al. 2005; Joëls 2001). Aging-related up-regulation of GR and FKBP52 found in our present study may form the molecular basis for excessive GR-mediated glucocorticoid signaling during stress response. In this maladaptive reaction, calcium influx may be enhanced beyond control, predisposing neurons in vulnerable brain regions to injury (de Kloet et al. 2005; Joëls 2001). Moreover, the concomitant down-regulation of FKBP51 observed in our impaired aged mice might represent the molecular substrate for exaggerated GR-mediated glucocorticoid signaling during stress reaction. The potential neurobiological correlates of aging-related cognitive impairments include dendritic atrophy, synaptic degeneration, and deficiency in adult hippocampal neurogenesis (McEwen and Magarinos 2001; Miller and O'Callaghan 2005). Experimental studies in mice showed that overall levels of adult neurogenesis drastically declined in old age, although voluntary physical exercise and long-term environmental enrichment could induce neurogenesis (Kempermann et al. 2002; McEwen and Magarinos 2001; van Praag et al. 1999). In the subgranular layer of the dentate gyrus, GR expression in neural precursor cells involved in adult neurogenesis was shown to be increased in aged mice compared to young mice, which might enhance their vulnerability to adverse glucocorticoid-mediated influences (Garcia et al. 2004).

There are individual differences in basal and stress-response levels of circulating glucocorticoids, depending on genetic processes or epigenetic modifications associated with early-life psychosocial experiences affecting the HPA axis function (Goosens and Sapolsky 2007). Aging-related changes in basal corticosterone levels in rodents might be strain dependent (Herbert et al. 2006). In the rat, elevated corticosterone levels were not a necessary consequence of aging, but were more prevalent in those exhibiting spatial memory deficits than in cognitively intact animals (Herbert et al. 2006). By using the Morris water maze test to assess hippocampus-dependent spatial learning, Bizon et al. (2001) found no differences in basal or peak-stress-induced levels of circulating corticosterone between young or aged rats, irrespective of cognitive status. However, plasma corticosterone levels in cognitively impaired aged rats were slower to return to baseline after acute restraint stress compared to young or unimpaired aged rats. By in situ hybridization, GR mRNA levels were lower in the ventral hippocampal CA1 and CA3, dorsal and ventral dentate granule cell layer, and medial prefrontal cortex of impaired aged rats compared to young rats; no quantitative GR protein data shown in this report (Bizon et al. 2001). In a study using transgenic mice with forebrain-specific GR over-expression, Wei et al. (2007) reported that both young and aged transgenic mice in response to strong acute restraint stress displayed a delayed termination of circulating corticosterone elevation, compared to the respective wild-type mice. Specifically, following strong acute stress young transgenic mice exhibited a corticosterone recovery pattern similar to that seen in aged wild-type mice. This finding suggested that forebrain-dependent feedback inhibition of the HPA axis activity was deficient in GR-overexpressed mice, which might be related to decreased gene expression in the glutamate receptor signaling system shown in the RNA microarray analysis of the hippocampus (Wei et al. 2007). In our present study, circulating levels of corticosterone were not measured in relation to object recognition performance or stress response, nor were GR mRNA expression levels in the forebrain.

In addition to the role in GR signaling, FKBP52 with its ability to bind Hsp90 is involved in the intracellular signaling of progesterone, estrogen, and androgen receptors (Davies and Sánchez 2005). Most of FKBP immunophilin family members (e.g., FKBP12, FKBP51, and FKBP52) exhibit peptidyl-prolyl cis-trans isomerase activity, which is involved in the slow protein-folding process (Kang et al. 2008). Unlike FKBP12, FKBP52 when bound to the immunosuppressive drug FK506 does not inhibit calcineurin involved in T cell activation (Davies and Sánchez 2005). FKBP38 can protect cells from apoptosis by localizing anti-apoptotic proteins Bcl-2 and Bcl-X_L to the mitochondrial membrane (Kang et al. 2008). Immunophilin ligands (e.g., FK506, cyclosporin A, and rapamycin) have been reported to have neuroprotective effects via unclear mechanisms (Kang et al. 2008). Non-immunosuppressive rapamycin analogs, WYE-592 and ILS-920, were shown to protect neurons from calcium-induced cell death by modulating calcium channels and promote neuronal survival and neurite outgrowth through FKBP52 binding (Ruan et al. 2008).

In summary, our present study correlated the combination of decreased FKBP51 expression and increased expression of GR and FKBP52 in forebrain with short-term recognition memory impairment in aged mice. This intracellular neurochemical dysregulation may represent the molecular substrate for exaggerated GR-mediated glucocorticoid signaling during stress response. Extensive evidence has suggested that chronic stress and prolonged elevation of circulating glucocorticoids of various etiologies have deleterious effects on cognitive function (Brown et al. 2004; Goosens and Sapolsky 2007; Miller and O'Callaghan 2005). Glucocorticoids, while their circulating levels are high, need to first bind to GR in the cytoplasm in order to initiate a cascade of nuclear translocation of the GR heterocomplex to eventually exert their genomic influences on target cells either in adaptive or maladaptive direction (Grad and Picard 2007; Wochnik et al. 2005). These GR-dependent effects may be complemented by faster non-genomic effects mediated by the membrane mineralocorticoid receptor (MR) displaying a 10-fold lower affinity for corticosterone than nuclear MR (de Kloet et al. 2008). Even in the absence of chronic stress or prolonged excessive levels of circulating glucocorticoids, the combination of decreased FKBP51 expression and increased expression of GR and FKBP52 in the brain may lead to abnormal glucocorticoid signaling particularly in response to sporadic acute stress.