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. 2017 Jun 14;37(24):5900-5911.
doi: 10.1523/JNEUROSCI.2343-16.2017. Epub 2017 May 18.

Activation of PERK Elicits Memory Impairment through Inactivation of CREB and Downregulation of PSD95 After Traumatic Brain Injury

Tanusree Sen  1 , Rajaneesh Gupta  2 , Helen Kaiser  2 , Nilkantha Sen  3
Affiliations

Activation of PERK Elicits Memory Impairment through Inactivation of CREB and Downregulation of PSD95 After Traumatic Brain Injury

Tanusree Sen et al. J Neurosci. .

Abstract

The PKR-like ER kinase (PERK), a transmembrane protein, resides in the endoplasmic reticulum (ER). Its activation serves as a key sensor of ER stress, which has been implicated in traumatic brain injury (TBI). The loss of memory is one of the most common symptoms after TBI, but the precise role of PERK activation in memory impairment after TBI has not been well elucidated. Here, we have shown that blocking the activation of PERK using GSK2656157 prevents the loss of dendritic spines and rescues memory deficits after TBI. To elucidate the molecular mechanism, we found that activated PERK phosphorylates CAMP response element binding protein (CREB) and PSD95 directly at the S129 and T19 residues, respectively. Phosphorylation of CREB protein prevents its interaction with a coactivator, CREB-binding protein, and subsequently reduces the BDNF level after TBI. Conversely, phosphorylation of PSD95 leads to its downregulation in pericontusional cortex after TBI in male mice. Treatment with either GSK2656157 or overexpression of a kinase-dead mutant of PERK (PERK-K618A) rescues BDNF and PSD95 levels in the pericontusional cortex by reducing phosphorylation of CREB and PSD95 proteins after TBI. Similarly, administration of either GSK2656157 or overexpression of PERK-K618A in primary neurons rescues the loss of dendritic outgrowth and number of synapses after treatment with a PERK activator, tunicamycin. Therefore, our study suggests that inhibition of PERK phosphorylation could be a potential therapeutic target to restore memory deficits after TBI.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is the leading cause of death and disability around the world and affects 1.7 million Americans each year. Here, we have shown that TBI-activated PKR-like ER kinase (PERK) is responsible for memory deficiency, which is the most common problem in TBI patients. A majority of PERK's biological activities have been attributed to its function as an eIF2α kinase. However, our study suggests that activated PERK mediates its function via increasing phosphorylation of CAMP response element binding protein (CREB) and PSD95 after TBI. Blocking PERK phosphorylation rescues spine loss and memory deficits independently of phosphorylation of eIF2α. Therefore, our study suggests that CREB and PSD95 are novel substrates of PERK, so inhibition of PERK phosphorylation using GSK2656157 would be beneficial against memory impairment after TBI.

Keywords: CREB; GSK3B; PERK; PSD95; TBI.

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Figures

Figure 1.
Figure 1.
Experimental design for TBI mice after with or without treatment with GSK2656157. A, B, mice were treated with GSK2656157 for 24 d after TBI and then biochemical analysis was done on day 25. In other experiments, the mice were trained for the MWM test for 6 d after completion of GSK2656157 treatment and the probe trial was done on day 31 after TBI. The same sets of mice were used to monitor the synaptic density of TBI or sham mice on day 31 after TBI. C, Either PERK or PERK-K618A was overexpressed in the brain 7 d before TBI. Biochemical analysis was performed on day 25 after TBI. The procedure for TBI and overexpression of PERK/PERK-K618A were described in the Materials and Methods.
Figure 2.
Figure 2.
Treatment with GSK2656157 reduces PERK phosphorylation. A, Western blot analysis of the expression of PERK and eIF2α phosphorylation in pericontusional cortex after TBI. *p < 0.05, n = 9, one-way ANOVA, mean ± SEM. B, Western blot analysis to measure uncleaved and cleaved ATF6 after sham or TBI. C, Western blot analysis to measure phosphorylation level of IRE1α after TBI. D, Western blot analysis of phosphorylation of both PERK and eIF2α after treatment with GSK2656157 at 10, 20, and 50 mg/kg after TBI. *p < 0.05, n = 5, one-way ANOVA, mean ± SEM. E, Western blot analysis of eIF2α phosphorylation after overexpression of either PERK or PERK-K618A in the brain before TBI. The overexpression of PERK was monitored by Western blot hybridization. F, Confocal microscopic analysis to measure PERK phosphorylation with or without GSK2656157 treatment after TBI. G, Primary neurons were treated with tunicamycin (3 μg/ml) with or without GSK2656157 (15 μg/ml) treatment for 6 h and phosphorylation of PERK and eIF2α were monitored by Western blot hybridization. *p < 0.05, n = 9, one-way ANOVA, mean ± SEM.
Figure 3.
Figure 3.
Effect of GSK2656157 on spine density and memory functions after TBI. A–D, Dendritic spines/10 μm (A, B), spine length (A, C), and spine surface area (A, D) were measured after treatment with GSK2656157 (50 mg/kg) after TBI. *p < 0.05, n = 5, one-way ANOVA, mean ± SEM. E, F, Latency to find the platform during training period of 6 d at the end of 2 trials in a day (E). *p < 0.05, n = 7, two-way ANOVA between TBI and TBI + GSK2656157 groups for all days during training (mean ± SEM) and the day of the probe trial (F). G, Percentage of time in each quadrant was measured for the sham-, sham + GSK2656157-, TBI-, and TBI + GSK2656157-treated groups. The quadrant with the platform was designated as TQ and the quadrant from which the mice started their swimming was designated as OP for “opposite.” The quadrant on the left side of OP was designated as AL for “adjacent left” and the quadrant on the right side of OP was designated as AR for “adjacent right.” *p < 0.05, n = 5–6, two-way ANOVA, mean ± SEM. H, Mouse speed was monitored in the sham-, sham + GSK2656157-, TBI-, and TBI + GSK2656157-treated groups. I, Percentage of time for thigmotaxis was monitored in the sham-, sham + GSK2656157-, TBI-, and TBI + GSK2656157-treated groups. *p < 0.05, n = 10–12, one-way ANOVA, mean ± SEM.
Figure 4.
Figure 4.
Effect of GSK2656157 on CREB phosphorylation and dendritic outgrowth. A, Western blot analysis of CREB phosphorylation at S129 and S133 in pericontusional cortex after TBI. *p < 0.05, n = 5, one-way ANOVA, mean ± SEM. B, Confocal microscopic analysis of CREB phosphorylation (S129). C, Coimmunoprecipitation assay to monitor the interaction between either CREB or phospho-CREB (S129) and CBP after TBI. D, Western blot analysis of BDNF protein level. E, Western blot analysis of the protein level of CREB phosphorylation (S129) and BDNF after GSK2656157 treatment after TBI. F, Confocal microscopic analysis of Myc in cortex after overexpression of either Myc-PERK or Myc-PERK-K618A. Western blot analysis to monitor CREB phosphorylation at S129, BDNF, Actin, and Myc protein levels after TBI. *p < 0.05, n = 8–10, one-way ANOVA, mean ± SEM. G, Western blot analysis to monitor CREB phosphorylation at S129 after treatment with lithium (5 mg/kg) with or without the addition of GSK2656157. *p < 0.05, n = 5, one-way ANOVA, mean ± SEM. H, In vitro kinase assay for CREB phosphorylation by PERK. Western blot analysis to monitor CREB phosphorylation at S129 residue. I, J, Primary neurons were overexpressed with GFP and treated with isoflurane. Neurons were imaged by confocal microscopy to monitor dendritic morphology (I). Total dendritic length was measured after GSK2656157 treatment and cells overexpressed with either PERK or PERK-K618A before treatment with tunicamycin (J). *p < 0.05, n = 8–10, one-way ANOVA, mean ± SEM.
Figure 5.
Figure 5.
Effect of GSK2656157 on PSD95 phosphorylation and puncta numbers. A, Western blot analysis of PSD95 phosphorylation at T19 after TBI. *p < 0.05, n = 9, one-way ANOVA, mean ± SEM. B, Confocal microscopic analysis of PSD95 phosphorylation at T19 after TBI. C, Western blot analysis of PSD95 phosphorylation (T19) after treatment with GSK2656157. *p < 0.05, n = 5, one-way ANOVA, mean ± SEM. D, Western blot analysis and quantitative measurement to monitor PSD95 phosphorylation and protein level of PSD95 after overexpression of either PERK wild-type or PERK-K618A in the cortex. *p < 0.05, n = 5, one-way ANOVA, mean ± SEM. E, Western blot analysis to monitor PSD95 phosphorylation at T19 after treatment with lithium (1 mm) with or without the addition of GSK2656157. F, In vitro kinase assay for PSD95 phosphorylation by PERK. Western blot analysis to monitor PSD95 phosphorylation at the T19 residue. G–I, Confocal microscopic analysis of the number (G, H) and size (G, I) of PSD95-positive puncta after tunicamycin treatment with or without GSK2656157. *p < 0.05, n = 9, one-way ANOVA, mean ± SEM. J–L, Confocal microscopic analysis of the number (J, K) and size (J, L) of PSD95-positive puncta in cells overexpressed with either PERK or PERK-K618A mutant after TBI. *p < 0.05, n = 8–10, one-way ANOVA, mean ± SEM. M, Schematic representation showing that TBI leads to an increase in PERK phosphorylation that will subsequently phosphorylate CREB and PSD95 at the S129 and T19 residues, respectively. The increase in CREB phosphorylation causes a downregulation of BDNF level and PSD95 phosphorylation causes its downregulation after TBI. These events lead to spine loss and memory impairment after TBI.
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