FK506-Binding Protein 12.6/1b, a Negative Regulator of [Ca2+], Rescues Memory and Restores Genomic Regulation in the Hippocampus of Aging Rats

Abstract

Hippocampal overexpression of FK506-binding protein 12.6/1b (FKBP1b), a negative regulator of ryanodine receptor Ca²⁺ release, reverses aging-induced memory impairment and neuronal Ca²⁺ dysregulation. Here, we tested the hypothesis that FKBP1b also can protect downstream transcriptional networks from aging-induced dysregulation. We gave hippocampal microinjections of FKBP1b-expressing viral vector to male rats at either 13 months of age (long-term, LT) or 19 months of age (short-term, ST) and tested memory performance in the Morris water maze at 21 months of age. Aged rats treated ST or LT with FKBP1b substantially outperformed age-matched vector controls and performed similarly to each other and young controls (YCs). Transcriptional profiling in the same animals identified 2342 genes with hippocampal expression that was upregulated/downregulated in aged controls (ACs) compared with YCs (the aging effect). Of these aging-dependent genes, 876 (37%) also showed altered expression in aged FKBP1b-treated rats compared with ACs, with FKBP1b restoring expression of essentially all such genes (872/876, 99.5%) in the direction opposite the aging effect and closer to levels in YCs. This inverse relationship between the aging and FKBP1b effects suggests that the aging effects arise from FKBP1b deficiency. Functional category analysis revealed that genes downregulated with aging and restored by FKBP1b were associated predominantly with diverse brain structure categories, including cytoskeleton, membrane channels, and extracellular region. Conversely, genes upregulated with aging but not restored by FKBP1b associated primarily with glial-neuroinflammatory, ribosomal, and lysosomal categories. Immunohistochemistry confirmed aging-induced rarefaction and FKBP1b-mediated restoration of neuronal microtubular structure. Therefore, a previously unrecognized genomic network modulating diverse brain structural processes is dysregulated by aging and restored by FKBP1b overexpression.SIGNIFICANCE STATEMENT Previously, we found that hippocampal overexpression of FK506-binding protein 12.6/1b (FKBP1b), a negative regulator of intracellular Ca²⁺ responses, reverses both aging-related Ca²⁺ dysregulation and cognitive impairment. Here, we tested whether hippocampal FKBP1b overexpression also counteracts aging changes in gene transcriptional networks. In addition to reducing memory deficits in aged rats, FKBP1b selectively counteracted aging-induced expression changes in 37% of aging-dependent genes, with cytoskeletal and extracellular structure categories highly associated with the FKBP1b-rescued genes. Our results indicate that, in parallel with cognitive processes, a novel transcriptional network coordinating brain structural organization is dysregulated with aging and restored by FKBP1b.

Keywords: FKBP12.6; aging; calcium; cytoskeleton; microarray; ryanodine receptor.

Figure 1.

Experimental design. To compare LT with ST exposure to FKBP1b overexpression, aging rats were bilaterally injected in the hippocampus with AAV-FKBP1b at two different ages, one group at 13 months of age (LT) and one group at 19 months of age (ST) (small, vertical arrows). A third group received control vector (AAV-eGFP) at 13 months of age and a YC group received no injections. All animals were tested for spatial learning in the MWM, 3 aged groups at 21 months of age and 1 YC group at 6 months of age (total N = 52). Animals were killed after MWM testing. The mRNA from the dorsal hippocampus of one hemisphere was prepared for qRT-PCR and microarray analyses and the other hemisphere was postfixed for IHC.

Figure 2.

Both LT and ST FKBP1b overexpression countered age-related decline in spatial memory. A, Reference memory probe. FKBP1b treatments countered age-related deficits in reference memory probe performance. B, Reversal memory probe. FKBP1b treatments countered age-related deficits in the reversal memory probe trial. C, Cued testing. With visual cues prominently highlighting location of the escape platform, no group differences were found, indicating that memory test results were not due to differences in locomotor and/or visual abilities. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 significant pairwise contrast versus AC.

Figure 3.

Hippocampal FKBP1b mRNA and protein levels were increased substantially by LT AAV-FKBP1b overexpression. Top, qRT-PCR quantification of hippocampal FKBP1b mRNA expression (FKBP1b/Gapdh) for each treatment group (one-way ANOVA on ranks, p = 0.000050; for pairwise contrast vs AC; *p ≤ 0.05; ***p ≤ 0.001). Bottom, Immunostaining for hippocampal FKBP1b expression. Representative photomicrographs are shown from YC (A), AC (B), aged ST FKBP1b (C), and aged LT FKBP1b (D). Note the substantial increase in FKBP1b expression at both the mRNA and protein levels, particularly in the LT-FKBP1b group. sp, Stratum pyramidale; DG, dentate gyrus. Scale bar, 500 μm.

Figure 4.

Microarray analysis flowchart. Shown are the effects of aging and FKBP1b on hippocampal gene transcription. Left, Total gene probe sets (29,218) were filtered to remove absent (low signal intensity) and incompletely annotated probe sets. The remaining genes (14,828) were tested by ANOVA (p ≤ 0.05) followed by pairwise comparison (Fisher's pLSD, ≤ 0.05 between YC and AC) to define aging-dependent genes. Right, Statistical template algorithm. Aging-dependent genes were categorized based on whether FKBP1b had no effect (templates I and III) or significantly countered aging's effect (templates II and IV). A total of 99.8% of aging-dependent genes were assigned to a template based on criteria described in the text. A Monte Carlo simulation (1000 iterations, see Results) was used to estimate the number of genes expected in each template by chance. The number of genes assigned to each template in the observed data was significantly greater than the number expected by chance (p ≤ 0.0001; binomial test; >11-fold increase for all templates). See also Figure 4-1.

Figure 5.

FKBP1b counters the age-related decrease in hippocampal MAP2 protein expression. Top, Semiquantitative measures of MAP2 IHC staining densities plotted as a function of treatment. Statistical analysis revealed a significant effect (p = 0.0015; one-way ANOVA) with a significant decrease from YC to AC that was countered by ST FKPB1b (**p ≤ 0.01, ***p ≤ 0.001, †n.s. trend p = 0.06; post hoc Fisher's pLSD vs AC). Bottom, Immunostaining for hippocampal MAP2 expression. Representative photomicrographs are shown from YC (rectangle defines region of quantitation; A), AC (B), aged ST FKBP1b (C), and aged LT FKBP1b (D). Scale bar, 500 μm.