Sleep deprivation impairs memory by attenuating mTORC1-dependent protein synthesis

Abstract

Sleep deprivation is a public health epidemic that causes wide-ranging deleterious consequences, including impaired memory and cognition. Protein synthesis in hippocampal neurons promotes memory and cognition. The kinase complex mammalian target of rapamycin complex 1 (mTORC1) stimulates protein synthesis by phosphorylating and inhibiting the eukaryotic translation initiation factor 4E-binding protein 2 (4EBP2). We investigated the involvement of the mTORC1-4EBP2 axis in the molecular mechanisms mediating the cognitive deficits caused by sleep deprivation in mice. Using an in vivo protein translation assay, we found that loss of sleep impaired protein synthesis in the hippocampus. Five hours of sleep loss attenuated both mTORC1-mediated phosphorylation of 4EBP2 and the interaction between eukaryotic initiation factor 4E (eIF4E) and eIF4G in the hippocampi of sleep-deprived mice. Increasing the abundance of 4EBP2 in hippocampal excitatory neurons before sleep deprivation increased the abundance of phosphorylated 4EBP2, restored the amount of eIF4E-eIF4G interaction and hippocampal protein synthesis to that seen in mice that were not sleep-deprived, and prevented the hippocampus-dependent memory deficits associated with sleep loss. These findings collectively demonstrate that 4EBP2-regulated protein synthesis is a critical mediator of the memory deficits caused by sleep deprivation.

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 C57BL6/J mice injected intracerebroventricularly with puromycin. Puromycin signal was normalized to the loading control, GAPDH, 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 SD mice relative to each in those from NSD mice, assessed by qPCR. Expression was normalized to that of housekeeping gene Tuba4a. Data are means ± SEM of 8 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 3 independent assays. a.u., arbitrary units. N, number of mice analyzed, is noted in each bar.

Fig. 2. Schematic of the signaling pathways regulating the initation 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 either through phosphorylation of TSC or Raptor. The binding of mTOR and Raptor is necessary for mTORC1 formation and subsequent phosphorylation of p70 S6 kinase (S6K) or 4EBP2. Phosphorylation (activation) of S6K facilitates translation initiation by its phosphorylation of S6 ribosomal protein (S6). When 4EBP2 is phosphorylated (inhibited), eIF4E is able to form a complex with eukaryotic initiation factor 4G (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 (NSD) mice and sleep-deprived (SD) mice. Data are means ± SEM of 6 mice per condition (* p = 0.032, t test). (B) Representative Western blots and quantitation of hippocampal abundance of p-mTOR (Ser²⁴⁸¹) after immunoprecipitation 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 p-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 NSD and post-5 hours of SD mice. β-tubulin served as a loading control. Data are means ± SEM of 8 mice per condition (*** p < 0.005, t test). (E) Representative Western blots and quantitation of hippocampal abundance of eIF4E after immunoprecipitation with eIF4G from NSD and SD mice. Control input blots for eIF4E and eIF4G are also shown. Data are means ± SEM of 8 mice per condition (* p = 0.047, t test). All blots are representative of at least 3 independent assays. a.u., arbitrary units. N, 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. (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 6 mice in each condition. (** p < 0.005, t test). (C–E) Immunofluorescence for 4EBP2-HA (green), CaMKII (C, red), parvalbumin (D, red) or GFAP (E, red) in the CA3 region of the hippocampus from 4EBP2-HA-injected mice. Arrows, colocalization. Scale bar, 50μm. Images represent 4 mice per condition, 3–5 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 not sleep deprived (NSD) or sleep deprived (SD). Control input blots for eIF4E and eIF4G are also shown. Data are means ± SEM of 6 mice per condition (sleep deprivation effect: F_(1,20) = 9.856, ** p = 0.0052; virus effect: F_(1,20) = 16.53, *** p = 0.0006; interaction effect: F_(1,20) = 6.988, * p = 0.0156; two-way ANOVA). (G) Representative Western blots and quantitation of proteins labeled with puromycin from hippocampi of NSD and SD mice 3 weeks after being injected with eGFP or 4EBP2. Abundance was normalized to GAPDH loading control. Data are means ± SEM of 6 or 8 mice in each condition. (sleep deprivation effect: F_(1,22) = 1.585, p = 0.221; virus effect: F_(1,22) = 0.577, p = 0.456; interaction effect: F_(1,22) = 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 6 or 7 mice in each condition. (eGFP, * p < 0.013; 4EBP2, p = 0.149; Wilcoxon rank sums test). Dotted line indicates chance performance. All blots are representative of at least 3 independent assays. a.u., arbitrary units. N, number of mice analyzed, is noted in each bar.