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| Status |
Public on Dec 19, 2017 |
| Title |
FFK506-Binding Protein 12.6/1b, a negative regulator of [Ca2+], rescues memory and restores genomic regulation in the hippocampus of aging rats |
| Organism |
Rattus norvegicus |
| Experiment type |
Expression profiling by array
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| Summary |
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 test 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) or 19-months-of-age (short-term) and tested memory performance in the Morris water maze at 21-months-of-age. Aged rats treated short- or long-term with FKBP1b substantially outperformed age-matched vector controls and performed similarly to each other and young controls. Transcriptional profiling in the same animals identified 2342 genes whose hippocampal expression was up-/down-regulated in aged controls vs. young controls (the aging effect). Of these aging-dependent genes, 876 (37%) also showed altered expression in aged FKBP1b-treated rats compared to aged controls, 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 young controls. 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 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. Thus, a previously-unrecognized genomic network modulating diverse brain structural processes is dysregulated by aging and restored by FKBP1b overexpression.
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| Overall design |
All experiments and procedures were performed in accordance with the University of Kentucky guidelines and were approved by the Animal Care and Use Committee. Hippocampal overexpression of FKBP1b was induced using methods and doses similar to those we described and validated previously (Gant et al., 2015). Briefly, bilateral injection of adeno-associated virus (AAV) vector harboring the transgene for FKBP1b under control of the CAMKII promoter (AAV2/9.CAMKII 0.4.ratFkbp1b.RGB; or AAV-FKBP1b) into the CA1 region of the hippocampus. A control vector harboring the transgene for enhanced green fluorescent protein (AAV2/9.CAMKII 0.4.eGFP.RGB, or, AAV-eGFP) was administered with the same procedure to a vector control group of aged rats. The AAV vectors were constructed at the University of Pennsylvania vector core (Philadelphia, PA). A total of 23 male F344 rats that completed behavioral training were used for microarray measurement, divided into 4 treatment groups: 1) young controls receiving no injections (YC) (n = 6, 4-5 months of age at receipt) 2) Long-term aged vector control (AC) (n = 5, bilateral injections of AAV-eGFP 1.86e13 GC/mL, 2 µl per side; at 13 months-of-age; 3) Short-term FKBP1b (ST) (n = 6, bilateral injections of AAV.FKBP1b 1.99e12 GC/mL, 2 µl per side, at 19 months-of-age); and 4) Long-term FKBP1b (LT) (n = 6, bilateral injections of AAV-FKBP1b 1.99e12 GC/mL, 2 µl per side; at 13 months-of-age). All aged animals used in this study arrived together and were housed in our animal care facility for the same duration. All animals underwent Morris Water Maze (MWM) training similar to prior work (e.g., Rowe et al., 2007; Latimer et al., 2014) and brain dorsal hippocampi were harvested for gene chip as in prior work (Kadish et al., 2009; Searcy et al., 2012; Latimer et al., 2014; Gant et al., 2015). Tissue was placed in RNase-free sample tubes and stored at -80° C until further use. Dorsal hippocampal RNA was extracted according to standard protocols, and evaluated using Agilent Bioanalyzer. All samples were of sufficient quality and did not differ significantly among treatment groups (RNA Integrity Number [RIN]: 9.4 ± 0.1 for all groups; p = 0.16, ANOVA across YC, AC, ST and LT groups). Microarray procedures were similar to our prior microarray studies on hippocampal aging in rats (Blalock et al., 2003; Rowe et al., 2007; Kadish et al., 2009). Briefly, RNA extracted from dorsal hippocampus was labeled and hybridized to Affymetrix Rat Gene 1.0 ST arrays (one array per animal). Signal intensities were calculated using the Robust Multi-array Average (RMA) algorithm (Bolstad et al., 2003) at the transcript level and data were associated with vendor-provided annotation information. Total probe sets were filtered prior to statistical testing to retain only unique, annotated probe sets with adequate signal intensity (defined as unlogged signal intensity ≥ 40 on ≥ 4 arrays in the study), and outlier values (> 2 SD of the group mean) were treated as missing values. Filtered signal intensities were analyzed by one-way ANOVA to identify significant differences, and the False Discovery Rate (FDR) procedure (Hochberg and Benjamini, 1990) was used to estimate the error of multiple testing. Significant genes were assigned to one of four expression templates based on post hoc pairwise comparisons (Fisher’s protected Least Significant Difference- please see Results).
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| Contributor(s) |
Blalock EM, Gant JC, Chen K, Kadish I, Thibault O, Porter NM, Landfield PW |
| Citation(s) |
29255009 |
| Submission date |
Jul 31, 2017 |
| Last update date |
Jul 25, 2021 |
| Contact name |
Eric M Blalock |
| E-mail(s) |
eric.blalock@uky.edu
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| Phone |
859-323-8033
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| Organization name |
University of Kentucky
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| Department |
Molecular and Biomedical Pharmacology
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| Lab |
Blalock
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| Street address |
800 Rose St.
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| City |
Lexington |
| State/province |
KY |
| ZIP/Postal code |
40475 |
| Country |
USA |
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| Platforms (1) |
| GPL6247 |
[RaGene-1_0-st] Affymetrix Rat Gene 1.0 ST Array [transcript (gene) version] |
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| Samples (23)
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| Relations |
| BioProject |
PRJNA396498 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE102054_RAW.tar |
94.9 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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