REDD1 is essential for stress-induced synaptic loss and depressive behavior

Abstract

Major depressive disorder (MDD) affects up to 17% of the population, causing profound personal suffering and economic loss. Clinical and preclinical studies have revealed that prolonged stress and MDD are associated with neuronal atrophy of cortical and limbic brain regions, but the molecular mechanisms underlying these morphological alterations have not yet been identified. Here, we show that stress increases levels of REDD1 (regulated in development and DNA damage responses-1), an inhibitor of mTORC1 (mammalian target of rapamycin complex-1; ref. 10), in rat prefrontal cortex (PFC). This is concurrent with a decrease in phosphorylation of signaling targets of mTORC1, which is implicated in protein synthesis-dependent synaptic plasticity. We also found that REDD1 levels are increased in the postmortem PFC of human subjects with MDD relative to matched controls. Mutant mice with a deletion of the gene encoding REDD1 are resilient to the behavioral, synaptic and mTORC1 signaling deficits caused by chronic unpredictable stress, whereas viral-mediated overexpression of REDD1 in rat PFC is sufficient to cause anxiety- and depressive-like behaviors and neuronal atrophy. Taken together, these postmortem and preclinical findings identify REDD1 as a critical mediator of the atrophy of neurons and depressive behavior caused by chronic stress exposure.

Figure 1. Chronic unpredictable stress increases REDD1 and decreases mTORC1 signaling in rat PFC

(a) Schematic showing how increased REDD1 could result in decreased mTORC1 signaling via Rheb and the TSC1/TSC2 complex (10), and subsequent decreased translation and synaptogenesis. (b) Rats received 21 d of CUS or regular handling (control) then sacrificed 4 h following the final stressor. Results are mean ± SEM fold change of PFC REDD1 mRNA [CUS n=8; Control n=8] and protein [CUS n=10; Control n=12] relative to control. (c) Rats received 1 d of mild stress or handling (control) then sacrificed 4 h following the final stressor. Results are mean ± SEM fold change PFC REDD1 mRNA [1 d mild stress n=6; control n=6] and protein [1 d mild stress n=6; control n=6] relative to control. For (b) and (c), protein levels were normalized to GAPDH, and representative blots are displayed to the right. (d) Rats received 21 d of CUS (n=10) or regular handling (control) (n=12), then sacrificed 4 h following the final stressor. (e) Rats received 1 d mild stress (n=6) or handling (control) (n=6), then sacrificed 4 h following the final stressor. For (d) and (e), phospho-protein levels in PFC were normalized to total protein levels; results are shown as mean (± SEM) fold change. Representative blots are displayed to the right. (*) p < 0.05 (#) p < 0.10 relative to control. Abbreviations: mTORC1, mammalian/mechanistic target of rapamycin complex 1; ERK, extracellular signal-regulated kinase; Akt, serine threonine kinase or protein kinase B; S6K, P70 ribosomal protein S6 kinase; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; TSC, tuberous sclerosis complex.

Figure 2. REDD1 mRNA is increased in the dlPFC of MDD patients

(a) Results are the mean ± SEM fold change relative to control of REDD1 mRNA in dlPFC of MDD subjects or psychiatrically-healthy controls (Control n=36; MDD n=36). (b) Results are the mean ± SEM fold change relative to control of mTOR mRNA in dlPFC of MDD subjects or psychiatrically-healthy controls (Control n=35; MDD n=37). White data points indicate subjects from cohort 1; green data points indicate subjects from cohort 2. Target mRNA levels have been normalized to GAPDH mRNA. (*) p < 0.05 control vs. MDD.

Figure 3. REDD1 knock out mice are resilient to CUS-induced alterations in the PFC

(a) REDD1 KO mice and their wild-type littermates received 21 d of CUS or regular handling (Ctrl), then were behaviorally tested. After testing, mice continued to receive CUS or regular handling for 7 d, then sacrificed 4 h after the final stressor. (b) Ratio of sucrose to water consumption (± SEM) over a 1 h test is depicted for WT Ctrl (n=13), WT CUS (n=14), KO Ctrl (n=16), and KO CUS (n=17) groups. ANOVA revealed a significant effect of genotype [F(,56) = 4.47; p < 0.05], stress [F(,56) = 5.809; p < 0.03], and genotype x stress interaction [F(,56) = 4.157; p < 0.05]. (c) Phospho-protein levels in PFC were analyzed in WT Ctrl (n=13), WT CUS (n=14), KO Ctrl (n=16), and KO CUS (n=17) groups. Results are the mean ± SEM fold change. Phospho-protein levels were normalized to total protein levels and representative blots are displayed. ANOVA for phospho-S6K revealed a significant genotype x stress interaction [F(, 56) = 7.632, p < 0.01] and a main effect of genotype [F(, 56) = 12.08, p < 0.01], but no main effect of stress. ANOVA for phospho-4EBP1 showed a significant genotype x stress interaction [F(, 56) = 4.939, p < 0.05] and a main effect of genotype [F(, 56) = 4.077, p < 0.05], but no main effect of stress. (d) Left. Sample whole-cell voltage-clamp traces of serotonin (5-HT) and hypocretin (Hcrt)-induced EPSCs in layer V pyramidal cells. Scale is depicted on the lower right. Right. Mean ± SEM frequency of 5-HT and Hcrt-induced EPSCs from WT Ctrl (5-HT n=16; Hcrt n=13), WT CUS (5-HT n=16; Hcrt n=16), KO Ctrl (5-HT n=16; Hcrt n=15), and KO CUS (5-HT n=16; Hcrt n=16) groups is depicted. ANOVA for 5-HT-induced EPSCs in layer V pyramidal neurons of the mPFC showed a significant main effect of stress [F(,60) = 6.131, p < 0.05] and a trend for a main effect of genotype [F(,60) = 3.124, p < 0.10], but no stress x genotype interaction. ANOVA of Hcrt-induced EPSCs revealed a significant main effect of stress [F(,56)=7.809, p < 0.01] and a significant stress x genotype interaction [F(,56)=4.046, p < 0.05], but no main effect of genotype. (e) Left. Representative images are shown of high magnification Z-stack projections of segments of the layer V pyramidal cell apical tuft dendrites (scale: 5 μm). Right. Mean ± SEM of spine density from WT Ctrl (n=30), WT CUS (n=41), KO Ctrl (n=27), and KO CUS (n=23) groups is depicted. ANOVA for spine density in the recorded cells revealed a significant main effect of stress [F(,117) = 4.487, p < 0.05] and genotype [F(,117) = 4.183, p < 0.05], but no stress x genotype interaction (*) p < 0.05 relative to WT Ctrl mice. (#) p < 0.10 relative to WT Ctrl mice.

Figure 4. REDD1 over-expression in the rat mPFC increases depressive and anxiety-related behaviors and decreases mTORC1 signaling

(a) Top. Human REDD1 was subcloned into an AAV2 plasmid under transcriptional regulation of the CMV promoter (AAV2-REDD1-IRES-eYFP). AAV2-IRES-eYFP plasmid without the human REDD1 gene was used as the control. Bottom. Schematic of experiment. Representative photomicrographs of injection sites in the mPFC are shown to the right. (b) Left. Mean sucrose preference (± SEM) for control (n=7) and AAV-REDD1 infused animals (n=7). Center. Mean immobility time (± SEM) for control (n=6) and AAV-REDD1 injected (n=7) animals. Right. Mean latency to feed (± SEM) for control (n=6) and AAV-REDD1 injected (n=7) animals. (c) Left. Mean entries to the center in the open field (+/− SEM) for control (n=7) and AAV-REDD1 injected (n=7) animals. Right. Mean center duration (± SEM) in the open field for control (n=7) and AAV-REDD1 injected (n=7) animals. (d) Left. Mean number (± SEM) of open arm entries in the elevated plus maze for control (n=6) and AAV-REDD1 injected (n=7) animals. Center. Mean open arm duration (± SEM) in the elevated plus maze for control (n=6) and AAV-REDD1 injected (n=7) animals. Right. Mean number (± SEM) of closed arm entries in the elevated plus maze for control (n=6) and AAV-REDD1 injected (n=7) animals. (e) Mean (± SEM) percent co-localization of eYFP and phospho-S6 Ribosomal protein in control (n=5) and REDD1 virus-injected (n=6) rats. Right. Representative co-localization images. (f) Mean (± SEM) fold change of protein from mPFC microdissections from control (n=10) or AAV-REDD1 injected (n=10) rats. Representative blots are shown to the right. (g) Left Top. Representative images of a co-localized mPFC layer V pyramidal neurons positive for GFP and DiI. Left Bottom. Representative images of high magnification Z-stack projections of segments of layer V pyramidal cell primary apical dendrites. Right. Mean ± SEM of spine density from control (n=12) and AAV-REDD1 (n=12) groups. (h) Mean ± SEM of spine density of Filopodia (F), Long-Thin (LT), and Mushroom/Stubby (M/S) spines from control and AAV-REDD1 groups is depicted (F Ctrl n=11; F REDD1 n=12; LT Ctrl n=11; LT REDD1 n=11; M/S Ctrl n=12; M/S REDD1 n=11). (*) p < 0.05 relative to control.