The adaptor protein of the anaphase promoting complex Cdh1 is essential in maintaining replicative lifespan and in learning and memory - PubMed (original) (raw)

The adaptor protein of the anaphase promoting complex Cdh1 is essential in maintaining replicative lifespan and in learning and memory

Min Li et al. Nat Cell Biol. 2008 Sep.

Abstract

The anaphase promoting complex (APC) or cyclosome is a multisubunit E3 ubiquitin ligase. Cdc20 (fizzy (fzy)) or p55CDC, and Cdh1 (Hct1, srw1 or fizzy-related 1 (fzr1)) encode two adaptor proteins that bring substrates to the APC. Both APC-Cdc20 and APC-Cdh1 have been implicated in the control of mitosis through mediating ubiquitination of mitotic regulators, such as cyclin B1 and securin. However, the importance of Cdh1 function in vivo and whether its function is redundant with that of Cdc20 are unclear. Here we have analysed mice lacking Cdh1. We show that Cdh1 is essential for placental development and that its deficiency causes early lethality. Cdhl-deficient mouse embryonic fibroblasts (MEFs) entered replicative senescence prematurely because of stabilization of Ets2 and subsequent activation of p6(Ink4a) expression. These results have uncovered an unexpected role of the APC in maintaining replicative lifespan of MEFs. Further, Cdh1 heterozygous mice show defects in late-phase long-term potentiation (L-LTP) in the hippocampus and are deficient in contextual fear-conditioning, suggesting that Cdh1 has a role in learning and memory.

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Figures

Figure 1

Figure 1

Absence of Cdh1 causes early lethality in mice. a and b. Retarded development of Cdh1gt/gt embryos at E8.5 and E9.5 c and d. H&E stained sections of wildtype ( c) and mutant (d) E9.5 whole deciduas. The black outline delineates the boundary between deciduous and placental tissues. e and f. Immunostaining of cyclin B1 on wildtype ( e) and mutant (f) E9.5 placentas. Scale bars in a and b, 40 μm; in c and d, 20 μm; in e and f, 10 μm.

Figure 2

Figure 2

Analyses of Cdh1gt/gt MEFs. a. Growth curve analysis of MEFs at passage 3 (MTT assay). b. BrdU incorporation assays of passage 3 MEFs. c. Immunoblotting of phospho-histone H3 in passage 3 MEFs. d. Growth curve analysis of MEFs at passage 6. e and f. Immunoblotting analysis of various cell cycle regulators in MEFs (e) and E9.0 embryos (f). Results in a, b, and d were from three independent experiments. Error bars are standard deviations.

Figure 3

Figure 3

Cdh1 regulates the expression of p16 through modulating the stability of Ets2. a. Western blot analysis of p16, p53 and Ets2 in MEFs. b. Quantitation of Ets2 levels in a. c. RT-PCR analysis of gene expression in MEFs. d. Quantitation of p16 levels in c. e. Analysis of Ets2 stability in wildtype and Cdh1 mutant MEFs treated with cyclohexamide (CHX). f. Quantitation of Ets2 levels in e. g. The effect of Cdh1 overexpression on the stability of Ets2 in wildtype MEFs. Full scans of the gels in a, e, and g are presented in supplemental figure 5.

Figure 4

Figure 4

Ets2 is potential substrate of APC-Cdh1. a. Immunoblotting analysis of ETS2 (wildtype and destruction box mutated) in 293T cells transfected with control or _Cdh1_-expressiong plasmids. b. Analysis of wildtype and destruction box-mutated Ets2 expressed in HeLa cells treated with cyclohexamide (CHX). c. Quantitation of the results in b. c. Ets2 interacts with Cdh1. Myc-Cdh1 and Flag-Ets2 were transiently expressed in 293T cells. Flag-Ets2 was immunoprecipitated and blotted for the presence of Myc-Cdh1. e. Analysis of the effect of Est2 expression on the proliferative potential of wildtype MEFs. f. Quantitation of cells positive for senescence-associated β-galactosidase. Results in e and f were from three independent experiments. Error bars indicate standard deviation. Full scans of the gels in a, b, and d are presented in supplemental figure 5.

Figure 5

Figure 5

Learning and memory defects in Cdh1 heterozygous mice. a. Early-phase LTP induced by a single 100 Hz stimulus (1s). 5 slices per mouse from a total of 5 mice of each genotype were used. Representative fEPSP recordings from time points A and B are shown for each condition. Calibration: 1 mV, 5 ms. b. Late-phase LTP elicited by four 100 Hz trains (1s) with 5 min intertrain interval. 10 slices per mouse from a total of 8 mice of each genotype were used. Representative fEPSP recordings from time points A and B are shown for each condition. Calibration: 1 mV, 5 ms. c. Analysis of contextual fear conditioning. d. Analysis of cued fear conditioning. 10 measurements were made in c and d and error bars indicate standard deviation.

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