Research ArticleNeuroscience

Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis

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Science Signaling  26 Apr 2016:
Vol. 9, Issue 425, pp. ra41
DOI: 10.1126/scisignal.aad4949
  • Fig. 1 Sleep deprivation impairs hippocampal protein synthesis in vivo.

    (A) Western blotting analysis for puromycin, as a proxy for protein synthesis, in the hippocampus from non–sleep-deprived (NSD) or sleep-deprived (SD) male C57BL/6J mice injected intracerebroventricularly with puromycin. Puromycin signal was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control and quantified (right panel). Data are means ± SEM of 13 mice in each condition (*P = 0.047, t test). (B) Gene expression of Arc and Hspa5/BiP in hippocampal extracts from sleep-deprived mice relative to each in those from non–sleep-deprived mice, assessed by quantitative polymerase chain reaction (qPCR). Expression was normalized to that of housekeeping gene Tuba4a. Data are means ± SEM of eight mice in each condition (***P < 0.001, t test). (C) Representative Western blots and quantitation of Arc and Hspa5/BiP abundance in hippocampal extracts. Abundance was normalized to the β-tubulin loading control. Data are means ± SEM of 16 or 17 mice in each condition. All blots are representative of at least three independent assays. a.u., arbitrary units. The number of mice analyzed is noted in each bar.

  • Fig. 2 Schematic of the signaling pathways regulating the initiation of protein synthesis.

    The insulin signaling pathway, which includes AMPK and mTOR, is one pathway that regulates protein synthesis initiation. When AMPK is activated, it inhibits mTORC1 activity through phosphorylation of either TSC or Raptor. The binding of mTOR and Raptor is necessary for mTORC1 formation and subsequent phosphorylation of p70 S6K or 4EBP2. Phosphorylation (activation) of S6K facilitates translation initiation by its phosphorylation of S6 ribosomal protein (S6). When 4EBP2 is phosphorylated (inhibited), eIF4E can form a complex with eIF4G to start cap-dependent translation.

  • Fig. 3 Sleep deprivation affects AMPK-mTORC1-4EBP2 signaling pathway in the hippocampus.

    (A) Representative Western blots and quantitation of the ratios of phosphorylated to total AMPKα in hippocampal extracts from non–sleep-deprived mice and sleep-deprived mice. Data are means ± SEM of six mice per condition (*P = 0.032, t test). (B) Representative Western blots and quantitation of hippocampal abundance of phosphorylated mTOR (p-mTOR) (Ser2481) after immunoprecipitation (IP) with Raptor (left) or Rictor (right). Negative control blots for Rictor in the Raptor IP (left, below Raptor blot) and Raptor in the Rictor IP (right, below Rictor blot) are shown. Control input blots for phosphorylated mTOR (Ser2481), Raptor, and Rictor are also shown. Data are means ± SEM of 14 mice per condition (*P = 0.042, t test). (C and D) Representative Western blots and quantitation of phosphorylated 4EBP2 (p-4EBP2) (C) or phosphorylated S6K1 (p-S6K1) and phosphorylated S6 ribosomal protein (p-S6) (D) relative to their respective total protein abundance from hippocampus of non–sleep-deprived and sleep-deprived (after 5 hours) mice. β-Tubulin served as a loading control. Data are means ± SEM of eight mice per condition (***P < 0.005, t test). (E) Representative Western blots and quantitation of hippocampal abundance of eIF4E after immunoprecipitation with eIF4G from non–sleep-deprived and sleep-deprived mice. Control input blots for eIF4E and eIF4G are also shown. Data are means ± SEM of eight mice per condition (*P = 0.047, t test). All blots are representative of at least three independent assays. The number of mice analyzed is noted in each bar.

  • Fig. 4 Viral expression of 4EBP2 in the hippocampus prevents deficits in protein synthesis and memory impairment caused by sleep deprivation.

    (A) Schematics of the pAAV9-CaMKIIα0.4-eGFP or pAAV9-CaMKIIα0.4-eIF4EBP2-HA vectors used to express eGFP or 4EBP2 in hippocampal excitatory neurons in mice. ITR, inverted terminal repeat. (B) Representative Western blots and quantitation of the ratio between phosphorylated 4EBP2 and total 4EBP2 in the hippocampus of mice injected with either the eGFP or the 4EBP2 vector. Abundance was normalized to GAPDH loading control. Data are means ± SD from six mice in each condition (**P < 0.005, t test). (C to E) Immunofluorescence for 4EBP2-HA (green), CaMKII (red), parvalbumin (Pvalb; red), or GFAP (red) in the CA3 region of the hippocampus from 4EBP2-HA–injected mice. Arrows indicate colocalization. Scale bar, 50 μm. Images represent four mice per condition (three to five images per mouse). (F) Representative Western blots and quantitation of hippocampal abundance of eIF4E after immunoprecipitation with eIF4G from eGFP- and 4EBP2-HA–injected mice that were either non–sleep-deprived or sleep-deprived. Control input blots for eIF4E and eIF4G are also shown. Data are means ± SEM of six mice per condition [sleep deprivation effect: F1,20 = 9.856, **P = 0.0052; virus effect: F1,20 = 16.53, ***P = 0.0006; interaction effect: F1,20 = 6.988, *P = 0.0156; two-way analysis of variance (ANOVA)]. (G) Representative Western blots and quantitation of proteins labeled with puromycin from hippocampi of non–sleep-deprived and sleep-deprived mice 3 weeks after being injected with eGFP or 4EBP2. Abundance was normalized to GAPDH loading control. Data are means ± SEM of six or eight mice in each condition [sleep deprivation effect: F1,22 = 1.585, P = 0.221; virus effect: F1,22 = 0.577, P = 0.456; interaction effect: F1,22 = 5.201, *P = 0.033; two-way ANOVA]. (H) Performance of mice expressing eGFP or 4EBP2 in a hippocampus-dependent object-place recognition task (schematic, top) when either sleep-deprived for 5 hours immediately after training or left undisturbed. Data are means ± SEM of six or seven mice in each condition. (eGFP, *P < 0.013; 4EBP2, P = 0.149; Wilcoxon rank-sum test). Dotted line indicates chance performance. All blots are representative of at least three independent assays. The number of mice analyzed is noted in each bar.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/425/ra41/DC1

    Fig. S1. Schematic showing the mechanism of attenuated translation in the hippocampus caused by sleep deprivation.

    Fig. S2. Control immunoblots of hippocampal extracts treated with rabbit IgG.

    Fig. S3. The abundance of phosphorylated TSC2 does not change after 5 hours of sleep deprivation.

    Fig. S4. Five hours of sleep deprivation reduces the abundance of phosphorylated 4EBP2, which rebounds after 2.5 hours of recovery sleep.

    Fig. S5. Abundance of phosphorylated eIF2α does not change after 5 hours of sleep deprivation.

  • Supplementary Materials for:

    Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis

    Jennifer C. Tudor, Emily J. Davis, Lucia Peixoto, Mathieu E. Wimmer, Erik van Tilborg, Alan J. Park, Shane G. Poplawski, Caroline W. Chung, Robbert Havekes, Jiayan Huang, Evelina Gatti, Philippe Pierre, Ted Abel*

    *Corresponding author. Email: abele{at}sas.upenn.edu

    This PDF file includes:

    • Fig. S1. Schematic showing the mechanism of attenuated translation in the hippocampus caused by sleep deprivation.
    • Fig. S2. Control immunoblots of hippocampal extracts treated with rabbit IgG.
    • Fig. S3. The abundance of phosphorylated TSC2 does not change after 5 hours of sleep deprivation.
    • Fig. S4. Five hours of sleep deprivation reduces the abundance of phosphorylated 4EBP2, which rebounds after 2.5 hours of recovery sleep.
    • Fig. S5. Abundance of phosphorylated eIF2α does not change after 5 hours of sleep deprivation.

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    Citation: J. C. Tudor, E. J. Davis, L. Peixoto, M. E. Wimmer, E. van Tilborg, A. J. Park, S. G. Poplawski, C. W. Chung, R. Havekes, J. Huang, E. Gatti, P. Pierre, T. Abel, Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis. Sci. Signal. 9, ra41 (2016).

    © 2016 American Association for the Advancement of Science

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