Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Atypon full text link Atypon Free PMC article
Full text links

Actions

Share

.2013 Aug 28;5(200):200ra115.
doi: 10.1126/scitranslmed.3006373.

Molecular mechanism for age-related memory loss: the histone-binding protein RbAp48

Affiliations

Molecular mechanism for age-related memory loss: the histone-binding protein RbAp48

Elias Pavlopoulos et al. Sci Transl Med..

Abstract

To distinguish age-related memory loss more explicitly from Alzheimer's disease (AD), we have explored its molecular underpinning in the dentate gyrus (DG), a subregion of the hippocampal formation thought to be targeted by aging. We carried out a gene expression study in human postmortem tissue harvested from both DG and entorhinal cortex (EC), a neighboring subregion unaffected by aging and known to be the site of onset of AD. Using expression in the EC for normalization, we identified 17 genes that manifested reliable age-related changes in the DG. The most significant change was an age-related decline in RbAp48, a histone-binding protein that modifies histone acetylation. To test whether the RbAp48 decline could be responsible for age-related memory loss, we turned to mice and found that, consistent with humans, RbAp48 was less abundant in the DG of old than in young mice. We next generated a transgenic mouse that expressed a dominant-negative inhibitor of RbAp48 in the adult forebrain. Inhibition of RbAp48 in young mice caused hippocampus-dependent memory deficits similar to those associated with aging, as measured by novel object recognition and Morris water maze tests. Functional magnetic resonance imaging studies showed that within the hippocampal formation, dysfunction was selectively observed in the DG, and this corresponded to a regionally selective decrease in histone acetylation. Up-regulation of RbAp48 in the DG of aged wild-type mice ameliorated age-related hippocampus-based memory loss and age-related abnormalities in histone acetylation. Together, these findings show that the DG is a hippocampal subregion targeted by aging, and identify molecular mechanisms of cognitive aging that could serve as valid targets for therapeutic intervention.

PubMed Disclaimer

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. RbAp48 deficiency in the aging human hippocampal formation
(A)RbAp48 gene expression over life span in (a) the DG showing a decline (β = −0.77,P = 0.026) and (b) the EC (β = 0.07,P = 0.87) showing no decline. (c) DG/EC ratio of RbAp48 declines with age (β = −0.94,P = 0.0005) (n = 8). (B) RbAp48 protein in separate group of brains (n = 10). RbAp48 protein levels decline over the life span (a) in the DG (β = −0.72,P = 0.02), but not (b) in the EC (β = 0.13,P = 0.71). (c) DG protein expression normalized to EC protein expression. In (A) and (B), each data point corresponds to one human subject and microarray experiment. (C) Human subjects used in the studies. (D) Representative Western blot showing RbAp48 levels in a young and an old sample. SUB, subiculum; ACT, actin (control; data from DG are shown).
Fig. 2
Fig. 2. Generation and characterization of DT mice expressing RbAp48-DN in the forebrain
(A) (Left) Radioactive in situ hybridization ofRbAp48 mRNA on a sagittal brain slice from adult wild-type (WT) mice (3.5 months). (Right) High magnification of left image showingRbAp48 mRNA in the hippocampus (hp). See table S2 for hybridization probe sequence (oligo1). (B) Representative Western blot (different mouse in each lane) and averaged values below. RbAp48 values were normalized to tubulin values from the same mouse, and data from all mice were averaged and expressed as fold difference change in aged mice (15 months) compared to young mice (3.5 months) ± SEM. RbAp48 is reduced in the DG of aged mice compared to young mice (*P = 3.2 × 10−8).n = 24 per age (six mice per age; four independent experiments). (C) (a) Schematic representation of doxycycline (dox)–regulated expression of Flag–RbAp48-DN in the forebrain. In DT mice that carried thetetOpromoter–RbAp48-DN and theCaMKIIαpromoter-tTA transgenes, the heterologous transactivator tTA bound to thetetO promoter and activatedRbAp48-DN expression only in the forebrain where theCaMKIIα promoter is active. Maintaining the DT mice on doxycycline-containing food inhibitsRbAp48-DN transcription because doxycycline prevents the binding of the tTA to thetetO promoter. (Right)RbAp48-DN mRNA in sagittal brain sections from adult (day P95) DT animals with no doxycycline treatment after day P40 (transgene ON; on/off dox diet) or with doxycycline treatment after day P80 to inhibit the transgene expression (transgene OFF; on/off/on dox diet). (b) Diet protocols for DT mice and control littermates used in all studies. (c) Western blot analysis of RbAp48-DN (anti-Flag) and endogenous RbAp48 in the hippocampus of 95-day-old mice. DT on dox, DT mouse treated with doxycycline (RbAp48-DN OFF); DT, mouse not fed with doxycycline in adulthood (RbAp48-DN ON); Control,tetO–RbAp48-DN single transgenic mouse. RbAp48-DN is detected only in DT. Four independent experiments gave similar results. (D) Confocal hippocampal images from adult (3.5 months) DT mouse andtetO–RbAp48-DN (tetO) single transgenic animal (control). (Insets) High-magnification DG images. (E) Binding of RbAp48 to histone 4 (H4). (a) Representative Western blots showing RbAp48 level in adult (3.5 months) hippocampal lysates before (input; left) and after (right) coimmunoprecipitation (IP) with anti-H4 antibody. Immunoprecipitated H4 is also shown (right). DT, RbAp48-DN–expressing mouse; ctl, control littermates. (b) Representative Western blots of immunoprecipitated H4 and coimmunoprecipitated RbAp48 from adult (3.5 months) hippocampal lysates from DT mice kept on doxycycline-containing food (RbAp48-DN OFF) and control littermates kept on or off dox in adulthood. IgG and beads: controls for the specificity of the anti-H4 immunoprecipitations. (Graphs) Averaged data (mean ± SEM). (a) DT:n = 4, ctl:n = 6 (tetO = 2, tTA = 2, WT = 2); (b) DT on dox:n = 3; ctl on dox:n = 3 (tetO = 1, tTA = 1, WT = 1). One experiment per mouse. The values of RbAp48 level after the anti-H4 immunoprecipitation were normalized to the H4 values from the same mouse, and data from all mice were averaged and expressed as fold difference change in DT compared to controls in (a) and fold difference change in DT on dox and controls on dox compared to controls off dox in (b). Significant difference was found between DT and controls in (a) [*P = 2 × 10−6, analysis of variance (ANOVA)].
Fig. 3
Fig. 3. Effect of RbAp48-DN in the adult forebrain on object recognition memory
Data from novel object recognition task (mean ± SEM; one experiment). (A) Mice kept off doxycycline during the task. (a) Fifteen-minute training [DT:n = 11; control:n = 22 (tetO = 6, tTA = 8, WT = 8)]. DT (RbAp48-DN ON) mice performed worse than did controls during the 48-hour memory test (genotype × test effect:P = 0.0077, repeated-measures ANOVA;P = 0.0001,t test). (b) Ten-minute training [different groups of mice; DT:n = 12; control:n = 12 (tetO = 5, tTA = 4, WT = 3)]. DT mice performed worse than did controls in the 24-hour memory test (genotype × test effect:P = 0.0158, repeated-measures ANOVA;P = 0.0023,t test). (B) Animals kept on doxycycline during the task (mean ± SEM; one experiment). (a) Fifteen-minute training [DT: n = 10; control: n = 17 (tetO = 5, tTA = 5, WT = 7)]. (b) Ten-minute training [different groups; DT:n = 12; controls:n = 21 (tetO = 7, tTA = 7, WT = 7)]. DT on dox (RbAp48-DN OFF) and Control on dox displayed similar performance (P > 0.2, repeated-measures ANOVA). (C) Confocal images (30 μm) showing immunostaining against doublecortin (DCX) and Ki67 in the DG of 4-month-old RbAp48-DN–expressing (DT) and control mice. Graphs: Averaged data (±SEM). The numbers of DCX-and Ki67-expressing cells in DT and controls were similar (P > 0.5, ANOVA). DT:n = 24 (six mice; four slices per mouse) and controls:n = 24 [6 mice (tetO = 2, tTA = 2, WT = 2); four slices per mouse]. NeuN, marker of mature neurons. (D) Data from novel object recognition (mean ± SEM; one experiment). Young (3.5 months) and aged (15 months) WT mice. (a) Fifteen-minute training (n = 8 mice per age). Aged mice showed lower performance than did young mice in the 48-hour memory test (age × test effect:P = 0.0052, repeated-measures ANOVA;P = 0.0002,t test). (b) Ten-minute training (different group;n = 10 per age). Aged mice did not form 24-hour memory (age × test effect:P = 0.0021, repeated-measures ANOVA;P = 0.01,t test). *P = 0.01, **P = 0.0023, ***P < 0.0003 (A, B, and D). See table S3 for detailed statistics.
Fig. 4
Fig. 4. Effect of RbAp48-DN in the adult forebrain on spatial memory
Mice tested in the Morris water maze. (Left) Path lengths (mean ± SEM) for mice to reach the platform over the days of training. (Right) Number of platform crossings (mean ± SEM) during probe trials 1 day after the end of training. In the probe trial, the platform was removed from the pool and the mice swam for 1 min. The number of times that the mice cross the platform location in the training quadrant (TQ; the quadrant of the pool where the platform was during training) indicates the strength of their spatial memory. (a) Visible and hidden platform versions of the task. (b) Transfer phase of the task (the mice are trained to learn a new hidden platform location). The green schematics depict the quadrants of the pool, and the small circle in them depicts the platform locations. (A) DT and control mice kept off doxycycline in adulthood [same mice as in Fig. 3A (a); DT:n = 11 and control:n = 22 (tetO = 6, tTA = 8, WT = 8); one experiment]. No differences were observed between DT and controls [P > 0.137, repeated-measures ANOVA for visible (a), hidden/acquisition (a), and hidden/transfer (b)]. In the probe trials (right), DT displayed significantly lower performance than did controls [repeated-measures ANOVA, quadrant × genotype effect:P = 0.04 (a) andP = 0.03 (b);t test in training quadrant:P = 0.017 (a) andP = 0.035 (b)]. (B) DT and control groups treated with doxycycline in adulthood [same mice as in Fig. 3B (a); DT on dox:n = 10 and control:n = 17 (tetO = 5, tTA = 5, WT = 7); one experiment]. No differences were observed (repeated-measures ANOVA; visible/hidden-acquisition/hidden-transfer:P > 0.16; probe trials:P > 0.34). (C) Young adult (3.5 months) and aged (15 months) WT mice (n = 14 per age; one experiment). Groups showed similar path lengths during training [P > 0.1, repeated-measures ANOVA for (a) and (b)]. During the probe trials, aged mice crossed the platform location significantly less often than did young animals [repeated-measures ANOVA, age × quadrant effect:P = 0.0003 (a) andP = 0.0002 (b);t test for training quadrant:P = 0.0015 (a) andP = 0.002 (b)]. *P < 0.036; **P < 0.0021. See table S3 for detailed analysis.
Fig. 5
Fig. 5. RbAp48-DN in the DG
(A) (Left) Individual examples of CBV maps of the hippocampal formation [EC, DG (circle), CA3-CA1, and subiculum (SUB)], generated with MRI, in DT mice on or off dox in adulthood and respective control siblings. The CBV maps are color-coded such that cooler colors reflect less basal metabolism. (Right) Group data analysis (mean ± SEM) of relative CBV (rCBV) (DT off dox:n = 9, controls off dox:n = 19, DT on dox:n = 12, controls on dox:n = 15; one measure per subregion per mouse). Selective decrease in rCBV of DG in DT off dox (F = 6.3,P = 0.019). (B) Immunohistochemistry (top) and quantification (mean ± SEM) (bottom) of AcH2B(Lys20) and AcH4(Lys12). Their levels were significantly reduced in the DG of DT off dox (comparisons with controls off dox and controls and DT on dox;P < 0.003, ANOVA). AcH2B(Lys20): DT off dox:n = 27 slices (five mice), DT on dox:n = 15 slices (three mice), controls off dox:n = 45 slices (seven mice), controls on dox:n = 15 slices (four mice). AcH4(Lys12): DT off dox:n = 44 slices (five mice), DT on dox:n = 15 slices (three mice), controls off dox:n = 52 slices (seven mice), controls on dox:n = 11 slices (four mice). (C) Immunohistochemistry (left) and quantification (mean ± SEM) (right) of AcH3(Lys9). No differences were observed (P > 0.42, ANOVA). DT off dox:n = 16 slices (five mice), controls off dox:n = 28 slices (seven mice). Mice on dox were not analyzed because no difference was detected in mice off dox. (Insets) High-magnification DG images. (B and C) See tables S1 and S3 for detailed analysis. *P < 0.05; **P < 0.01.
Fig. 6
Fig. 6. Effect of RbAp48 up-regulation in the DG on histone acetylation and age-related memory loss
(A) (a) Schematic representation and confocal images of the hippocampi from WT aged mice (15 months) expressing DG RbAp48-HA (hemagglutinin) or green fluorescent protein (GFP) (control) via lentiviral injections. (Insets) High-magnification DG images. (b) Western blot showing DG-specific expression of RbAp48-HA and GFP. RB1 to RB3 and GFP1 to GFP3 indicate the numbers of RbAp48-HA– and GFP-injected mice. (c) Western blot and averaged data (±SEM) of total RbAp48.n = 9 measures per virus (three mice per virus; three independent blots). Significant increase of RbAp48 in the DG of RbAp48-HA mice compared to GFP controls (F1,16 = 22.584; *P = 0.0002, ANOVA). RbAp48 level in CA3-CA1 was similar between the two groups (F1,16 = 0.397;P = 0.7357, ANOVA). (B) Data from novel object recognition (a) and the Morris water maze (b) (mean ± SEM; one experiment for each task). (a) RbAp48-HA–injected aged mice (15 months;n = 12) performed better than GFP-injected age-matched littermates (n = 10) during the 24-hour memory test (injection × session effect:P = 0.0192, repeated-measures ANOVA;P = 0.0275,t test). (b) (Top) Both groups performed similarly during training in all phases of the water maze (repeated-measures ANOVA; visible:P = 0.36; acquisition:P = 0.0703; transfer:P = 0.6996). (Bottom) RbAp48-HA mice performed better than GFP controls during probe trials 1 day after at the end of training (repeated-measures ANOVA; injection × quadrant effect; acquisition:P = 0.0094,t test for training quadrant:P = 0.0133; transfer:P = 0.0443,t test for training quadrant:P = 0.016). *P < 0.03. (C) Immunohistochemistry and quantification (mean ± SEM) of acetylated histones. AcH2BK20 and AcH4K12 levels were significantly increased in the DG of RbAp48-HA–injected aged mice compared to GFP-injected siblings (*P < 0.0015, ANOVA). AcH3K9 remained unaltered in DG and CA3-CA1 (P > 0.095, ANOVA).n = 12 slices per virus (three mice per virus; four slices per mouse). See tables S1 and S3 for detailed analysis.
Fig. 7
Fig. 7. Effect of expression of RbAp48-DN in the forebrain on CBP HAT activity
Averaged HAT activity (±SEM) of CBP immunoprecipitated from DG and CA3-CA1 lysates expressed as fold difference from IgG control immunoprecipitations. (Insets) Raw data acquired from HAT assays [3H counts per minute (CPM)]. (A) Assays in DT mice (3.5 months) and control littermates kept off doxycycline or doxycycline in adulthood. DT, DT off dox; Control, control off dox.n = 9 measurements per genotype per treatment (three measurements per immunoprecipitation per mouse; three mice per genotype per treatment). Significantly reduced CBP HAT activity in the DG of DT compared to all other groups (P < 0.0005, ANOVA). (B) Young (3.5 months) and aged (15 months) WT mice.n = 12 measurements per age (three measurements per immunoprecipitation per mouse; four mice per age). Significantly reduced CBP HAT activity in the DG of aged mice (P = 0.0006, ANOVA). (C) WT aged mice (15 months) virally expressing in DG RbAp48-HA (RbAp48 up-regulation in DG) or GFP (control).n = 9 measurements per virus (three measurements per immunoprecipitation per mouse; three mice per virus). CBP HAT activity was significantly increased in the DG of RbAp48-HA mice compared to GFP controls (P = 0.0001, ANOVA). *P < 0.0006. For detailed analysis, see table S3.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Small SA, Schobel SA, Buxton RB, Witter MP, Barnes CA. A pathophysiological framework of hippocampal dysfunction in ageing and disease. Nat Rev Neurosci. 2011;12:585–601. - PMC - PubMed
    1. Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Del Tredici K. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol. 2006;112:389–404. - PMC - PubMed
    1. Gómez-Isla T, Price JL, McKeel DW, Jr, Morris JC, Growdon JH, Hyman BT. Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer’s disease. J Neurosci. 1996;16:4491–4500. - PMC - PubMed
    1. Thal DR, Rüb U, Orantes M, Braak H. Phases of Aβ-deposition in the human brain and its relevance for the development of AD. Neurology. 2002;58:1791–1800. - PubMed
    1. West MJ. Regionally specific loss of neurons in the aging human hippocampus. Neurobiol Aging. 1993;14:287–293. - PubMed

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full text links
Atypon full text link Atypon Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp