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. 2016 Sep 14;11(9):e0162766.
doi: 10.1371/journal.pone.0162766. eCollection 2016.

PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility

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Free PMC article

PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility

Siying Zhu et al. PLoS One. .
Free PMC article

Abstract

PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK's role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. BrPerk KO mice are impaired in spontaneous alternation Y-maze task.
(A) Western blot analysis showing substantial knockdown of PERK in the prefrontal cortex (PFC) of BrPerk KO mice. PERK was also significantly knocked down in the hippocampus (HIP) but not in the cerebellum (CER). (BKO: BrPerk KO; *** p<0.001, * p<0.05, n.s. not significant, two-tailed student’s t-Test; n = 5 for each genotype). (B) Spontaneous alternation Y-maze task. BrPerk KO mice exhibited a lower alternation rate compared to the wild-type littermates, indicating their poor spatial working memory (WT n = 15, BrPerk KO n = 22; ** p<0.01, two-tailed student’s t-Test). No difference was observed in total number of arms entered (n.s. not significant, two-tailed student’s t-Test), indicating similar motor ability and curiosity for the environment between two genotypes. Impaired spontaneous alternation was observed in BrPerk KO group in both genders. The figures represent pooled results of both genders.
Fig 2
Fig 2. BrPerk KO mice are impaired in the delay match-to place working memory task.
(A) Spatial reference memory test in radial-arm water maze. BrPerk KO mice exhibited normal reference memory in a 4 day reference platform task in radial-arm water maze (WT n = 5, BrPerk KO n = 6). Each mouse was given 8 trials per day during the 4 day task. A trial block represents the average number of errors of 4 trials. Only male mice were used in the experiment. (B) The delay match-to-place task in radial-arm water maze. BrPerk KO mice made more errors and required longer escape latency than their wild-type littermates to locate the hidden platform in the match trial (WT n = 16, BrPerk KO n = 20; *** p<0.001, ** p<0.01, two-tailed student’s t-Test). Impaired spatial working memory was observed in BrPerk KO group in both genders. The figures represent pooled results of both genders.
Fig 3
Fig 3. WT mice with short-term PERK inhibition are impaired in Y-maze spontaneous alternation.
(A) Western blot analysis showing PERK inhibitor (PI) pretreatment prevented thapsigargin (TG)-induced phosphorylation of PERK and its substrate eIF2α in primary cortical neurons (one-way ANOVA followed by Bonferroni’s post-hoc test, * p<0.05, ** p<0.01). Cells were pretreated with 500 nM PI or DMSO for 15 min followed by co-treatment with or without 100 nM TG for 30 min. The phosphorylation level of PERK is shown by p-PERK blot, and the shifted band in PERK blot due to the dimerization and auto-phosphorylation of PERK after its activation. 3 replicates were included in the experiment and the quantification was performed on pooled results. (B) Schematic of PERK inhibitor treatment in spontaneous alternation Y-maze task. (C) Spontaneous alternation Y-maze task. Short-term PERK-inhibited mice showed a lower alteration rate compared to vehicle group (Vehicle n = 17, PERKi n = 18; ***p<0.001, two-tailed student’s t-Test). No difference was observed in the total number of arms entered (n.s. not significant, two-tailed student’s t-Test). Only male mice were used in the experiment.
Fig 4
Fig 4. WT mice with short-term PERK inhibitor gavage administration are impaired in fear extinction.
(A) Fear conditioning on day 0. No difference was observed between treatments. (B) Fear extinction over 3 days (3 CS presentations/block; Vehicle, n = 6; PERKi, n = 5; * p<0.05, two-tailed student’s t-Test). PERK-inhibited mice exhibited higher freezing rate over 3 days’ extinction session (p<0.001, non-parametric paired sign test). Only male mice were used in the experiment. (C) Average freezing rate over 3 days’ extinction session. Short-term PERK-inhibited mice exhibited higher average freezing rate on the 2nd and 3rd day of extinction session, indicating their impairment in fear extinction (* p<0.05, two-tailed student’s t-Test).
Fig 5
Fig 5. WT mice with long-term PERK inhibitor gavage administration are impaired in fear extinction.
(A) Fear conditioning on day 0. No difference was observed between treatments. (B) Fear extinction over 2 days (3 CS presentations/block; Vehicle n = 10, PERKi n = 8, * p<0.05, *** p<0.001, two-tailed student’s t-Test). Long-term PERK-inhibited mice exhibited delayed fear extinction over the 2 days (p<0.01, non-parametric paired sign test). Only male mice were used in the experiment. (C) Average freezing rate over 2 days’ extinction session. PERK-inhibited mice exhibited higher average freezing rate over 2 days’ extinction session, indicating their impairment in fear extinction (* p<0.05, two-tailed student’s t-Test).

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