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. 2012 Jun 28;1(6):676-88.
doi: 10.1016/j.celrep.2012.04.010. Epub 2012 May 24.

Brain-specific disruption of the eIF2α kinase PERK decreases ATF4 expression and impairs behavioral flexibility

Mimi A Trinh  1 , Hanoch Kaphzan, Ronald C Wek, Philippe Pierre, Douglas R Cavener, Eric Klann
Affiliations

Brain-specific disruption of the eIF2α kinase PERK decreases ATF4 expression and impairs behavioral flexibility

Mimi A Trinh et al. Cell Rep. .

Abstract

Translational control depends on phosphorylation of eIF2α by PKR-like ER kinase (PERK). To examine the role of PERK in cognitive function, we selectively disrupted PERK expression in the adult mouse forebrain. In the prefrontal cortex (PFC) of PERK-deficient mice, eIF2α phosphorylation and ATF4 expression were diminished and were associated with enhanced behavioral perseveration, decreased prepulse inhibition, reduced fear extinction, and impaired behavioral flexibility. Treatment with the glycine transporter inhibitor SSR504734 normalized eIF2α phosphorylation, ATF4 expression, and behavioral flexibility in PERK-deficient mice. Moreover, the expression levels of PERK and ATF4 were reduced in the frontal cortex of human patients with schizophrenia. Together, our findings reveal that PERK plays a critical role in information processing and cognitive function and that modulation of eIF2α phosphorylation and ATF4 expression may represent an effective strategy for treating behavioral inflexibility associated with several neurological disorders such as schizophrenia.

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Figures

Figure 1
Figure 1. Forebrain-specific deletion of Perk suppresses eIF2α phosphorylation and ATF4 expression in the prefrontal cortex
(A) Schematic for the conditional allele for Perk. Top, black triangles represent two loxP sites flanking exons 7–9 (E7–E9) of the Perk gene and grey boxes represent primers (568F and 568R1) designed to detect recombination. Middle, upon recombination with CaMKIIα-Cre, Perk is deleted in a forebrain-specific manner. Bottom, 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, Nissl-stained coronal sections of prefrontal cortex. Bottom, whole brain from wild-type (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 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 medial prefrontal cortex 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 prefrontal cortex 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.
Figure 2
Figure 2. Disruption of PERK-regulated translation in the prefrontal cortex does not alter protein synthesis
(A) Left, Western blot image showing newly synthesized proteins labeled with puromycin using the SUnSET technique (see Experimental Procedures). Coronal prefrontal slices from wild-type and PERK cKO mice were treated with puromycin (5µg/mL) for 45 mins. Right, 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.
Figure 3
Figure 3. PERK cKO mice display reduced prepulse inhibition and enhanced behavioral perseveration in novel object recognition and MWM tasks
(A) PERK cKO mice exhibit impaired prepulse inhibition 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, escape latency across 5 days of MWM reference platform task shows that PERK cKO mice acquired the spatial hidden platform task similarly to wild-type controls. Right, 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.
Figure 4
Figure 4. PERK cKO mice exhibit cognitive control deficits and behavioral inflexibility
(A) Percent correct arm choice per trial block number (5 trials/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 wild-type and PERK cKO mice 30 mins 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, percent of time spent freezing across 2 days of fear extinction training protocol (15 CS presentations/day) indicates that PERK cKO mice exhibit impaired fear extinction learning compared to controls (*p<0.05, two-way ANOVA). Right, 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 values ± SEM.
Figure 5
Figure 5. NMDA-R hypofunction alters eIF2α phosphorylation in the prefrontal cortex
(A) Sample traces of EPSCs recorded from PFC slices of WT (black) and PERK cKO (red) mice. (B) Average NMDA/AMPA EPSCs 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 wild-type 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 (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 values ± SEM.
Figure 6
Figure 6. Treatment with the GlyT1 inhibitor SSR504734 rescues prefrontal cortex-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 wild-type (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 prefrontal cortex of WT and cKO (untreated) compared to chronic treatment with GlyT1 inhibitor. (D) eIF2α phosphorylation is normalized in prefrontal cortex of PERK cKO mice following GlyT1 inhibitor treatment. (E) ATF4 protein expression is enhanced in prefrontal cortex 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 values ± SEM.
Figure 7
Figure 7. PERK and ATF4 expression is reduced in the frontal cortex of schizophrenic, but not bipolar patients
(A) PERK expression levels normalized by GAPDH levels. normal, n=35; schizophrenia (schizo), n=35 (*p<0.05, one-way ANOVA). Scatter plots display the variability and differences in the protein expression leves normalized by each GAPDH expression levels. (B) Gene-specific translation of ATF4 is reduced in the frontal cortex of schizophrenic patients. 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, n.s., one-way ANOVA). (D) ATF4 expression remains unaltered in the frontal cortex of bipolar patients compared to normal controls. normal, n=35; bipolar, n=35 (p>0.05, n.s., one-way ANOVA). A crossbar on each scatter plot represents mean expression levels for each group.
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