Volume 1, Issue 6, Pages 676-688 (June 2012) Brain-Specific Disruption of the eIF2α Kinase PERK Decreases ATF4 Expression and Impairs Behavioral Flexibility  Mimi A. Trinh, Hanoch Kaphzan, Ronald C. Wek, Philippe Pierre, Douglas R. Cavener, Eric Klann  Cell Reports  Volume 1, Issue 6, Pages 676-688 (June 2012) DOI: 10.1016/j.celrep.2012.04.010 Copyright © 2012 The Authors Terms and Conditions

Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure 1 Forebrain-Specific Deletion of Perk Suppresses eIF2α Phosphorylation and ATF4 Expression in the PFC (A) Schematic for the conditional allele for Perk. In the top view black triangles represent two loxP sites flanking exons 7–9 (E7–E9) of the Perk gene, and gray boxes represent primers (568F and 568R1) designed to detect recombination. Middle view shows that upon recombination with CaMKIIα-Cre, Perk is deleted in a forebrain-specific manner. Bottom view is PCR identification of alleles of PerkloxP and CaMKIIα-driven Cre. (B) Representative western blot analysis confirming disruption of PERK in hippocampal area CA1. (C) Top view illustrates Nissl-stained coronal sections of PFC. Bottom view shows whole brain from WT and PERK cKO mice. (D) Representative western blot showing CaMKIIα-driven Cre disruption of PERK in other regions of the brain from WT and PERK cKO mice. (E) Representative western blot showing that PERK levels in cKO mice are expressed at similar levels to those in WT mice in tissues outside the central nervous system. (F) Immunohistochemical detection of phosphorylated eIF2α in layer II/III of the mPFC showing decreased expression in PERK cKO (right panel) compared to WT (left panel). Scale bar, 200 μm. (G) Representative western blot showing decreased ATF4 expression in the PFC of PERK cKO mice compared to their WT littermates. Quantification of PERK, eIF2α phosphorylation, and ATF4 expression from the western blot analyses is shown in Figure S1. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure 2 Disruption of PERK-Regulated Translation in the PFC Does Not Alter Protein Synthesis (A) Left view is a western blot image showing newly synthesized proteins labeled with puromycin using the SUnSET technique (see Experimental Procedures). Coronal prefrontal slices from WT and PERK cKO mice were treated with puromycin (5 μg/ml) for 45 min. Right view shows that protein synthesis levels remain unaltered between both genotypes. (cntl, no puromycin control, n = 4; WT, n = 4; cKO, n = 4). (B) Absorbance profile (254 nm) of WT (black trace) and PERK cKO (red trace) prefrontal cortical fractions sedimented through a 20%–50% linear sucrose gradient in the presence of cycloheximide. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure 3 PERK cKO Mice Display Reduced PPI and Enhanced Behavioral Perseveration in Novel Object Recognition and MWM Tasks (A) PERK cKO mice exhibit impaired PPI of the acoustic startle reflex across varying prepulse intensities: 74, 78, 82, 86, and 90 dB. WT, n = 11; cKO, n = 9 (∗p < 0.05, two-way repeated-measure ANOVA followed by Tukey's post hoc test). (B) PERK cKO mice display enhanced perseveration for the familiar object in the novel object recognition task. WT, n = 8; cKO, n = 14 (∗∗∗p < 0.001, two-way repeated-measures ANOVA). (C) Left view illustrates escape latency across 5 days of MWM reference platform task and shows that PERK cKO mice acquired the spatial hidden platform task similarly to WT controls. Right view shows that PERK cKO mice exhibit higher number of previous-day platform position crossing during day 2 reversal phase of task compared to controls. WT, n = 12; cKO, n = 9 (∗p < 0.05, two-way repeated-measures ANOVA, followed by Tukey's post hoc test). See also Figure S3 and Movies S1 and S2. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure 4 PERK cKO Mice Exhibit Cognitive Control Deficits and Behavioral Inflexibility (A) Percent correct arm choice per trial block number (five trials per trial block) during training, test, reversal, and retraining phases of Y maze reversal task. (//) denotes when 12 out of 13 PERK cKO mice had to be retrained because they continued to swim to the originally trained arm choice. WT, n = 10; cKO, n = 13 (∗∗∗p < 0.001, two-way ANOVA). See also Figure S3 and Movies S3 and S4. (B) PERK cKO mice no longer perseverate to originally trained arm choice following same-day immediate reversal training. WT, n = 8; cKO, n = 9. (C) Western blot analysis of eIF2α phosphorylation in PFC of WT and PERK cKO mice 30 min following training and reversal learning. WT, n = 6; cKO, n = 6. (∗p < 0.05, one-way ANOVA; n.s., not significant). (D) Left and middle views show percentage of time spent freezing across 2 days of fear extinction training protocol (15 CS presentations/day) and indicate that PERK cKO mice exhibit impaired fear extinction learning compared to controls (∗p < 0.05, two-way ANOVA). Right view illustrates percent freezing time during long-term memory extinction test on day 3. WT, n = 8; cKO, n = 10 (∗p < 0.05, Student's t test). All data represent mean ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure 5 NMDAR Hypofunction Alters eIF2α Phosphorylation in the PFC (A) Sample traces of EPSCs recorded from PFC slices of WT (black) and PERK cKO (red) mice. (B) Average NMDA/AMPA EPSC ratios from pyramidal cells in layer II of the mPFC show no significant difference between either genotype. WT, n = 16; cKO, n = 17. (C) eIF2α phosphorylation is increased in the frontal cortex of WT mice upon single-dose (acute) treatment with MK-801 (0.2 mg/kg, i.p.) compared to saline. saline, n = 3; MK-801, n = 3 (∗p < 0.05, Student's t test). (D) Western blot analysis showing a decrease of eIF2α phosphorylation following 15 days (chronic) MK-801 (0.1 mg/kg, i.p.) treatment. saline, n = 4; MK-801, n = 5 (∗p < 0.05, Student's t test). All data represent mean ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure 6 Treatment with the GlyT1 Inhibitor SSR504734 Rescues PFC-Dependent Molecular and Cognitive Deficits in PERK cKO Mice (A) Y maze reversal task performance following 21 days of treatment with either saline (Veh) or glycine transporter-1 inhibitor (GlyT1 inh) SSR504734 (20 mg/kg, i.p.). (B) Upon chronic GlyT1 inhibitor treatment, PERK cKO mice behave like WT controls and require similar number of trials to meet criterion during reversal phase of the Y maze task. See also Movies S5 and S6. (C) Western blot analysis of basal PERK expression in PFC of WT and cKO (untreated) compared to chronic treatment with GlyT1 inhibitor. (D) eIF2α phosphorylation is normalized in PFC of PERK cKO mice following GlyT1 inhibitor treatment. (E) ATF4 protein expression is enhanced in PFC of PERK cKO mice following GlyT1 inhibitor treatment compared to untreated controls. ∗p < 0.05, ∗∗∗p < 0.001, two-way ANOVA. All data represent mean ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure 7 PERK and ATF4 Expression Are Reduced in the Frontal Cortex of Patients with Schizophrenia but Not Patients with Bipolar Disorder (A) PERK expression levels normalized by GAPDH levels. normal, n = 35; schizophrenia (schizo), n = 35 (∗p < 0.05, one-way ANOVA). Scatterplots display the variability and differences in the protein expression levels normalized by each GAPDH expression level. (B) Gene-specific translation of ATF4 is reduced in the frontal cortex of patients with schizophrenia. normal, n = 35; schizo, n = 33 (∗∗∗p < 0.001, one-way ANOVA). (C) PERK expression levels do not differ between control and bipolar patient brain samples. PERK expression normalized by tubulin levels. normal, n = 35; bipolar, n = 35 (p > 0.05, not significant, one-way ANOVA). (D) ATF4 expression remains unaltered in the frontal cortex of patients with bipolar disorder compared to normal controls. normal, n = 35; bipolar, n = 35 (p > 0.05, not significant, one-way ANOVA). A crossbar on each scatterplot represents mean expression levels for each group. See also Tables S1 and S2. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure S1 Quantification of PERK, eIF2α Phosphorylation, and ATF4 Expression in Brain Regions and Tissue Examined, Related to Figure 1 (A) PERK expression is quantified as the percentage of wild-type (WT) expression for the brain regions and tissue examined. Lysates were prepared from tissues extracted from WT and cKO mice; 40 μg/lane loaded for CNS tissue, 100 μg/lane loaded for non-CNS tissue. PERK cKO expression: hippocampus (Hip), 63%; area CA1, 14%; area CA3, 35%; dentate gyrus (DG), 99%; prefrontal cortex (PFC), 11%; amygdala (Amg), 26%; striatum (Stm), 98%; cerebellum (Cer), 99%, liver (Liv), 101.1%; heart (Hrt), 99%; pancreas (Pan), 100.3%; lung (Lng), 99.5%. (B) eIF2α phosphorylation is quantified as a ratio to total-eIF2α levels and normalized to WT values. PERK cKO expression: PFC, 40%; Amg, 53%; Stm, 99%; Cer, 101.5%. (C) ATF4 expression is significantly reduced in PFC of PERK cKO mice compared to WT. WT, n = 5; cKO, n = 5. (∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA). All data represent mean values ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure S2 PERK cKO Mice Show Enhanced Vertical Activity and Normal Anxiety, Grooming, and Marble-Burying Behavior, Related to Figure 3 (A) PERK cKO mice have increased vertical activity compared to WT littermates in the open field arena. WT, n = 10; cKO, n = 13. (∗p < 0.05, ∗∗p < 0.01, Student's t test). (B) PERK cKO mice demonstrate no difference for total distance traveled in the open field task when compared to WT littermates. WT, n = 10; cKO, n = 13. (C) PERK cKO mice spend similar amount of time in center zone of the open field arena compared to their WT littermates indicating normal anxiety. WT, n = 10; cKO, n = 14. (D) No differences were observed between either group for time spent in the closed arms versus the aversive open arms of the elevated plus maze. WT, n = 10; cKO, n = 14. (p > 0.05, n.s., two-way ANOVA). (E) PERK cKO mice display normal grooming behavior compared to WT littermates. WT, n = 9; cKO, n = 8. (F) PERK cKO mice bury similar number of marbles compared to controls. WT, n = 23; cKO, n = 13. All data represent mean values ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure S3 PERK cKO Mice Display Impaired Spatial Reversal Learning in MWM Task and Enhanced Perseveration for Previously Trained Arm Choice in Y-Maze Reversal Task, Related to Figures 3 and 4 (A) PERK cKO mice and their wild-type (WT) littermates showed similar preferences for the training quadrant during a probe trial given on day 5. Percent of time spent in the initial training quadrant (TQ), the opposite quadrant (OQ), and the average of the two adjacent quadrants (AQ) is shown. WT, n = 12; cKO, n = 9. (p > 0.05, n.s., one-way ANOVA). (B) PERK cKO mice spend more time in quadrant where hidden platform was previously placed during reversal phase on day 2 of MWM task. WT, n = 12; cKO, n = 9. (∗∗p < 0.01, Student's t test). (C) PERK cKO mice display normal escape latency during visible platform task. WT, n = 12; cKO, n = 9. (p > 0.05, n.s., two-way repeated-measures ANOVA). (D) (left side of graph) The number of correct arm choices during training phase (20 trials) of Y-maze reversal task. (right side of graph) The number of trials needed to meet criterion (9/10 correct) during reversal phase of Y-maze reversal task. WT, n = 10; cKO, n = 13. (∗∗∗p < 0.001, two-way ANOVA). All data represent mean values ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure S4 PERK cKO Mice Exhibit Normal Motor Function during Rotarod Test, Related to Figure 4 Motor coordination and learning was assessed using the accelerating rotarod test. Latency to fall from the rotating rod was measured over two days (4 trials/day). PERK cKO mice displayed normal motor coordination and motor memory compared to their WT littermates. WT, n = 10; cKO, n = 14. (p > 0.05, n.s., two-way repeated-measures ANOVA). All data represent mean values ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure S5 GCN2 KO Mice Display Normal Reversal Learning in Y-Maze Reversal Task, Related to Figure 4A Reversal learning was assessed in mice lacking the eIF2α kinase GCN2 during the Y-maze reversal task. Percent correct arm choice per trial block number (5 trials/trial block) is plotted for training, test, and reversal phases of task. WT, n = 6; cKO, n = 6. (p > 0.05, n.s., ANOVA). All data represent mean values ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions

Figure S6 Chronic SSR504734 Administration Does Not Correct Either the Increase in Vertical Activity or the Impairment in Sensorimotor Gating Exhibited by the PERK cKO Mice, Related to Figure 6 (A) Increased vertical activity persists in the PERK cKO mice despite chronic treatment with the glycine transporter-1 inhibitor (GlyT1 inh.) SSR504734 (20 mg/kg, i.p.) WT, n = 10; cKO, n = 10. (∗∗p < 0.01, Student's t test). (B) Chronic GlyT1 inhibitor treatment does not correct sensorimotor gating impairment in the PERK cKO mice. WT, n = 8; cKO, n = 8. (∗p < 0.05, two-way repeated-measures ANOVA, followed by Tukey's post hoc test). All data represent mean values ± SEM. Cell Reports 2012 1, 676-688DOI: (10.1016/j.celrep.2012.04.010) Copyright © 2012 The Authors Terms and Conditions