Restoring synaptic plasticity and memory in mouse models of Alzheimer’s disease by PKR inhibition

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with deficits in cognition and synaptic plasticity. While accumulation of amyloid β (Aβ) and hyper-phosphorylation of tau are parts of the etiology, AD can be caused by a large number of different genetic mutations and other unknown factors. Considering such a heterogeneous nature of AD, it would be desirable to develop treatment strategies that can improve memory irrespective of the individual causes. Reducing the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) was shown to enhance long-term memory and synaptic plasticity in naïve mice. Moreover, hyper-phosphorylation of eIF2α is observed in the brains of postmortem AD patients. Therefore, regulating eIF2α phosphorylation can be a plausible candidate for restoring memory in AD by targeting memory-enhancing mechanism. In this study, we examined whether PKR inhibition can rescue synaptic and learning deficits in two different AD mouse models; 5XFAD transgenic and Aβ1–42-injected mice. We found that the acute treatment of PKR inhibitor (PKRi) can restore the deficits in long-term memory and long-term potentiation (LTP) in both mouse models without affecting the Aβ load in the hippocampus. Our results prove the principle that targeting memory enhancing mechanisms can be a valid candidate for developing AD treatment. Electronic supplementary material The online version of this article (10.1186/s13041-017-0338-3) contains supplementary material, which is available to authorized users.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive deficits and synaptic dysfunction, for which there is currently no effective treatment available. Genetic studies have shown that mutations in specific set of genes such as APP, PSEN1, and PSEN2 are associated with early-onset of familial AD (FAD) [1][2][3]. APP encodes amyloid β (Aβ) precursor protein, while PSEN1 and PSEN2 encodes presenilin-1 and presenilin-2, respectively. These proteins are involved in Aβ processing pathway and consequently support a hypothesis that Aβ accumulation in the brain is critical for the onset of AD [4]. In addition to Aβ accumulation, hyperphosphorylation of tau is another well-known hallmark for AD [5]. Interestingly, both Aβ accumulation and tau hyper-phosphorylation are regulated by eukaryotic translation initiation factor 2α (eIF2α) [6,7]. Hyperphosphorylation of eIF2α at Ser 51 is observed in the brains of postmortem AD patients as well as in several AD mouse models [8][9][10][11]. In addition, Aβ treatment was shown to induce the phosphorylation of eIF2α in cultured neurons [12]. Whereas the phosphorylation of eIF2α inhibits general mRNA translation, eIF2α phosphorylation enhances translation of the specific group of mRNAs such as β-site APP cleaving enzyme 1 (BACE1) and activating transcriptional factor 4 (ATF4), a suppressor of memory formation by inhibiting cAMP responsive element binding protein (CREB)-dependent transcription [12][13][14]. Since CREB is essential for long-term memory formation and long-term synaptic plasticity [15][16][17][18], reducing eIF2α phosphorylation enhanced long-term potentiation (LTP) and long-term memory by reducing ATF4 translation in mice [19]. In addition to eIF2α, the double-stranded RNA-activated protein kinase (PKR), one of eIF2α kinases, is highly phosphorylated in AD brains [7,11,20]. PKR becomes active through the auto-phosphorylation when it binds to ATP and dsRNA [21]. Previous studies revealed that either genetic or pharmacological blockage of PKR enhances LTP and memory in mice [22,23].
Recent studies have suggested that reducing the phosphorylation level of eIF2α could be one of treatment strategies for AD [9,10,13,24]. Genetic reduction of PERK and GCN2, which are other kinases of eIF2α, ameliorated AD-related phenotypes in synaptic plasticity and behavior in AD mouse models such as APP/PS1 mice and 5XFAD mice [9,10] (but also see [8]). However, most of the previous studies focused on eIF2α signaling pathway in mainly relation to the production of Aβ [8][9][10]13].
We hypothesized that PKR inhibition may enhance synaptic plasticity and subsequently rescue memory deficits in AD mouse models even at late stage of the disease. We used Aβ 1-42 -injected wild-type mice and 5XFAD transgenic mice as acutely induced and genetic model of AD, respectively [25,26]. Our data showed that PKR inhibitor (PKRi) restored LTP deficit in both AD mouse models. Moreover, we found that PKRi treatment rescued the hippocampus-dependent memory deficits in both mouse models. In addition, acute PKR inhibition did not cause any change in Aβ load in the hippocampus of 5XFAD mice. Taken together, this study suggests that enhancing synaptic plasticity by targeting PKR-eIF2α signaling pathway can be a potential therapeutic target for AD.

Discussion
AD is a highly heterogeneous disease caused by multiple known and unknown factors. Therefore, it would be extremely difficult to develop treatments by targeting specific causes for individual cases. Based on a hypothesis that manipulating memory-enhancing mechanisms may be beneficial to AD animal models irrespective of their individual etiology [18,31], we examined whether suppressing PKR/eIF2α signaling can restore synaptic plasticity and behaviors in AD mouse models. It has been previously shown that inhibiting eIF2α phosphorylation can enhance synaptic plasticity and memory in mice [19,22,23,37]. Our results in the electrophysiological recording show that impaired synaptic plasticity can be rescued by PKRi in two different AD mouse models. We assume that these changes in synaptic plasticity consequently contributed to restoring the memory deficit in AD mouse models. Suppressing eIF2α phosphorylation was shown to enhance CREB activity as well as LTP by reducing the translation of ATF4 [19,38]. It is also known that elevated CREB activity increases the density and complexity of dendritic spines and enhances presynaptic neurotransmitter release [39,40]. A previous study showed that overexpression of CREB in CA1 rescued spatial memory deficit and altered structure of dendritic spines in 5XFAD mice, which had lower level of CREB phosphorylation [40]. However, we found that p-CREB level was slightly, but not statistically significantly altered by either Aβ  or PKRi treatment in the hippocampus under our experimental condition. Although further experiments are required, we speculate that our sample preparation time (2 days after Aβ 1-42 -injection, 30 min after PKRi injection) might not be optimal to observe the impact of Aβ 1-42 -injection and PKR inhibition on CREB phosphorylation.
Previous studies have reported that genetic disruption of PERK in AD mouse models such as APP/PS1 and 5XFAD mice can rescue AD-associated phenotypes, suggesting that inhibiting the upstream kinase of eIF2α may be beneficial to AD [9,10,13]. In contrast, there is an inconsistency in the effect of genetic disruption of GCN2 on AD mouse models [8,9]. Ma and colleagues found that the conditional knockout of GCN2 rescued the deficits in LTP and spatial memory in APP/PS1 mice [9], whereas Devi and Ohno showed that GCN2 deletion could not rescue AD-related phenotypes in 5XFAD mice [8]. Moreover, crossing 5XFAD to eIF2α S51A knock-in line failed to rescue memory deficits in 5XFAD mice [41]. These findings suggest that manipulation of different eIF2α kinases may have distinct impact on cognitive c PKRi treatment showed a trend to decrease eIF2α phosphorylation in Aβ 1-42 -treated mice, but the effect was not statistically significant (normalized p-eIF2α, vehicle, 1.00 ± 0.04, 17 hippocampi from 11 mice; Aβ 1-42 , 1.28 ± 0.11, 19 hippocampi from 13 mice; Aβ 1-42 + PKRi, 1.14 ± 0.10, 18 hippocampi from 12 mice; unpaired t-test, vehicle vs Aβ 1-42 , *p < 0.05; Aβ 1-42 vs Aβ 1-42 + PKRi, p = 0.354). (D) CREB phosphorylation was slightly reduced by Aβ  and was rescued by PKRi treatment although it was not statistically significant (normalized p-CREB, vehicle, 1.00 ± 0.07, 15 hippocampi from 9 mice; Aβ 1-42 , 0.93 ± 0.05, 15 hippocampi from 9 mice; Aβ 1-42 + PKRi, 1.01 ± 0.07, 16 hippocampi from 10 mice; unpaired t-test, vehicle vs Aβ 1-42 , p = 0.426; Aβ 1-42 vs Aβ 1-42 + PKRi, p = 0.390). Bars represent as mean ± SEM functions in AD mouse models. A recent study showed that PKRi treatment rescued memory deficits in an AD mouse model expressing the human APOE4 allele, which is consistent with our results [24]. However, to our knowledge, our data is the first showing the beneficial effect of PKRi on synaptic plasticity as well as memory in two independent AD mouse models, which might support the possibility that PKRi could be a potential broad-spectrum drug for AD treatment.
We found that PKR inhibition also reversed the deficits in basal synaptic transmission and short-term plasticity assessed by PPR in 5XFAD mice. Zhu and colleagues recently showed that PKRi treatment in naïve mice decreased GABAergic output of inhibitory networks, resulting the hyperactivity of excitatory neuronal networks [22]. A previous study showed that 5XFAD mice had lower activity of excitatory neural networks compared to their WT littermates [42], which may contribute to the deficits in basal synaptic transmission in 5XFAD mice. We also found that 5XFAD mice showed LTP deficit only when LTP was induced by TBS protocol which is more sensitive to changes in inhibition, but not by high frequency stimulation (100 Hz) protocol (Additional file 1: Figure S5) [43], suggesting an imbalance between excitation and inhibition in 5XFAD mice. It is plausible to speculate that PKRi might have rescued the deficits in basal synaptic transmission and long-term synaptic plasticity by restoring the activity of excitatory networks although it remains to be further investigated. Furthermore, it is worthy to note that changes in inflammation processes involving interferon gamma may underlie the beneficial effect of PKRi on AD mouse models since it has been reported that genetic deletion or inhibition of PKR upregulates the level of interferon gamma, which in turn increases neural excitability and enhances cognitive functions [22,44].
In contrast to previous studies [19,22], PKRi treatment did not enhance LTP or learning in our study. Although the reason for the difference is not clear, different experimental conditions such as genetic background of the mice (ICR or B6SJL in our study vs. C57Bl/6J in [19]) may contribute to the difference. Also, Segev and colleagues did not see the memory enhancement in control ApoE3 mice [24].
It is worthy to note that we could rescue the deficits in 12-month-old 5XFAD mice by acute PKRi treatment, suggesting that PKRi might be effective in late stage of AD in spite of the substantial accumulation of amyloid β in the brain. Indeed, we showed that acute PKRi treatment can reverse deficits in LTP and memory in 5XFAD mice without affecting Aβ load in the hippocampus. Considering recent reports on failures in AD clinical trials by targeting amyloid β [45,46] (but also see [47]), pharmacological interventions enhancing plasticity such as suppressing PKR may provide a promising alternative strategy for developing AD treatment.

Methods
Animals 4-6-week-old male ICR mice were purchased from Orient Bio Inc. and B6SJL-Tg (APPSwFlLon, PSEN1*M146 L*L286V)6799Vas/Mmjax mice (5XFAD) were generous gifts from Dr. Woo Keun Song (Gwangju Institute of Science and Technology, Korea) and Dr. Inhee Mook-Jung (Seoul National University College of Medicine, Korea). Both male and female 5XFAD mice were used. Mice were maintained on a 12 h light-dark cycle and food and water were provided ad libitum in vivarium at Seoul National University College of Medicine and Chung-Ang University. Mice were assigned in a group of 4 to 6 per cage and acclimated to the vivarium at least one week before experiments. Prior to experiments, mice were individually handled for 5 min in the testing room each day for 4 days.

PKRi treatment
PKRi (C-16, Cal-biochem, # 527450) stock solution was dissolved in DMSO (670 μg/ml). For behavioral test, PKRi were further diluted in distilled water to a final DMSO concertation of 10% immediate before i.p. injection (0.335 mg/kg body weight). For control group, 10% DMSO in distilled water was used as vehicle. For electrophysiology, PKRi was diluted in ACSF to 1 μM.

Novel object recognition (NOR) task
Prior to Aβ 1-42 injection, mice were habituated to a test arena (33 cm × 33 cm × 33 cm) without an object for 15 min per day for 2 days. Training was performed 2 days after the stereotaxic surgery. During the training session, mice were placed in the test arena containing two identical objects and allowed to explore the objects for 15 min. Twenty-four hours later, mice were placed again in the same test arena but one of the objects was replaced with a novel object. Behavior was recorded by a video camera. The exploration time to each object was scored manually. The test box was cleaned with 70% ethanol between each trial. The experimenter was blinded to the treatments for all the behavioral tests.

Fear conditioning
Prior to fear conditioning training, mice were acclimated to the testing room for 1 h. Mice were placed in the fear conditioning chamber (Coulbourn Instruments) for 2 min and received two pairs of a tone (2800 Hz, 85 dB, 30 s) and a co-terminating electric foot-shock (0.7 mA, 2 s) with 30 s intervals. One day after the training, mice were placed again in the chamber to test contextual fear memory for 3 min. The freezing behavior was automatically measured by Freeze Frame software (ActiMetrics, IL, USA). Data from one mouse that had freezing rate of deviation more than 2 standard deviations were excluded from the analysis.

Statistics
Effects of PKRi treatment on different groups were analyzed by using two-way ANOVA followed by appropriate post-hoc tests. Some behavioral, electrophysiological and western blotting data were analyzed by using unpaired two-tailed t-test as indicated in the results section. Data are presented as mean ± standard error of the mean (SEM).

Additional files
Additional file 1: Figure S1. Confirmation of Aβ 1-42 oligomerization. Figure S2. Inhibition of PKR restores basal synaptic dysregulation in 5XFAD mice. Figure S3. ICR mice showed the low standard of the freezing behavior in contextual fear conditioning. Figure S4. Neither Aβ 1-42 nor PKRi affected basal synaptic transmission and short-term synaptic plasticity. Figure S5