Local Inhibition of PERK Enhances Memory and Reverses Age-Related Deterioration of Cognitive and Neuronal Properties.

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of four known kinases that respond to cellular stress by deactivating the eukaryotic initiation factor 2 α (eIF2α) or other signal transduction cascades. Recently, both eIF2α and its kinases were found to play a role in normal and pathological brain function. Here, we show that reduction of either the amount or the activity of PERK, specifically in the CA1 region of the hippocampus in young adult male mice, enhances neuronal excitability and improves cognitive function. In addition, this manipulation rescues the age-dependent cellular phenotype of reduced excitability and memory decline. Specifically, the reduction of PERK expression in the CA1 region of the hippocampus of middle-aged male mice using a viral vector rejuvenates hippocampal function and improves hippocampal-dependent learning. These results delineate a mechanism for behavior and neuronal aging and position PERK as a promising therapeutic target for age-dependent brain malfunction.SIGNIFICANCE STATEMENT We found that local reduced protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) expression or activity in the hippocampus enhances neuronal excitability and cognitive function in young normal mice, that old CA1 pyramidal cells have reduced excitability and increased PERK expression that can be rescued by reducing PERK expression in the hippocampus, and that reducing PERK expression in the hippocampus of middle-aged mice enhances hippocampal-dependent learning and memory and restores it to normal performance levels of young mice. These findings uncover an entirely new biological link among PERK, neuronal intrinsic properties, aging, and cognitive function. Moreover, our findings propose a new way to fight mild cognitive impairment and aging-related cognitive deterioration.

Expression of Quinone Reductase-2 in the Cortex Is a Muscarinic Acetylcholine Receptor-Dependent Memory Consolidation Constraint.

Learning of novel information, including novel taste, requires activation of neuromodulatory transmission mediated, for example, by the muscarinic acetylcholine receptors (mAChRs) in relevant brain structures. In addition, drugs enhancing the function of mAChRs are used to treat memory impairment and decline. However, the mechanisms underlying these effects are poorly understood. Here, using quantitative RT-PCR in Wistar Hola rats, we found quinone reductase 2 (QR2) to be expressed in the cortex in an mAChR-dependent manner. QR2 mRNA expression in the insular cortex is inversely correlated with mAChR activation both endogenously, after novel taste learning, and exogenously, after pharmacological manipulation of the muscarinic transmission. Moreover, reducing QR2 expression levels through lentiviral shRNA vectors or activity via inhibitors is sufficient to enhance long-term memories. We also show here that, in patients with Alzheimer's disease, QR2 is overexpressed in the cortex. It is suggested that QR2 expression in the cortex is a removable limiting factor of memory formation and thus serves as a new target to enhance cognitive function and delay the onset of neurodegenerative diseases. SIGNIFICANCE STATEMENT: We found that: (1) quinone reductase 2 (QR2) expression is a muscarinic-receptor-dependent removable constraint on memory formation in the cortex, (2) reducing QR2 expression or activity in the cortex enhances memory formation, and (3) Alzheimer's disease patients overexpressed QR2. We believe that these results propose a new mechanism by which muscarinic acetylcholine receptors affect cognition and suggest that inhibition of QR2 is a way to enhance cognition in normal and pathological conditions.

PKR Inhibition Rescues Memory Deficit and ATF4 Overexpression in ApoE ε4 Human Replacement Mice.

Sporadic Alzheimer's disease (AD) is an incurable neurodegenerative disease with clear pathological hallmarks, brain dysfunction, and unknown etiology. Here, we tested the hypothesis that there is a link between genetic risk factors for AD, cellular metabolic stress, and transcription/translation regulation. In addition, we aimed at reversing the memory impairment observed in a mouse model of sporadic AD. We have previously demonstrated that the most prevalent genetic risk factor for AD, the ApoE4 allele, is correlated with increased phosphorylation of the translation factor eIF2α. In the present study, we tested the possible involvement of additional members of the eIF2α pathway and identified increased mRNA expression of negative transcription factor ATF4 (aka CREB2) both in human and a mouse model expressing the human ApoE4 allele. Furthermore, injection of a PKR inhibitor rescued memory impairment and attenuated ATF4 mRNA increased expression in the ApoE4 mice. The results propose a new mechanism by which ApoE4 affects brain function and further suggest that inhibition of PKR is a way to restore ATF4 overexpression and memory impairment in early stages of sporadic AD. Significance statement: ATF4 mRNA relative quantities are elevated in ApoE4 allele carriers compared with noncarrier controls. This is true also for the ApoE ε4 human replacement mice. ApoE4 mice injected with PKR inhibitor (PKRi) demonstrate a significant reduction in ATF4 expression levels 3 h after one injection of PKRi. Treatment of ApoE4 human replacement mice with the PKRi before learning rescues the memory impairment of the ApoE4 AD model mice. We think that these results propose a new mechanism by which ApoE4 affects brain function and suggest that inhibition of PKR is a way to restore memory impairment in early stages of sporadic AD.

Genetic or pharmacological reduction of PERK enhances cortical-dependent taste learning.

Protein translation initiation is controlled by levels of eIF2α phosphorylation (p-eIF2α) on Ser51. In addition, increased p-eIF2α levels impair long-term synaptic plasticity and memory consolidation, whereas decreased levels enhance them. Levels of p-eIF2α are determined by four kinases, of which protein kinase RNA-activated (PKR), PKR-like endoplastic reticulum kinase (PERK), and general control nonderepressible 2 are extensively expressed in the mammalian mature brain. Following identification of PERK as the major kinase to determine basal levels of p-eIF2α in primary neuronal cultures, we tested its function as a physiological constraint of memory consolidation in the cortex, the brain structure suggested to store, at least in part, long-term memories in the mammalian brain. To that aim, insular cortex (IC)-dependent positive and negative forms of taste learning were used. Genetic reduction of PERK expression was accomplished by local microinfusion of a lentivirus harboring PERK Short hairpin RNA, and pharmacological inhibition was achieved by local microinfusion of a PERK-specific inhibitor (GSK2606414) to the rat IC. Both genetic reduction of PERK expression and pharmacological inhibition of its activity reduced p-eIF2α levels and enhanced novel taste learning and conditioned taste aversion, but not memory retrieval. Moreover, enhanced extinction was observed together with enhanced associative memory, suggesting increased cortical-dependent behavioral plasticity. The results suggest that, by phosphorylating eIF2α, PERK functions in the cortex as a physiological constraint of memory consolidation, and its downregulation serves as cognitive enhancement.

Bridging the Accessibility Gap of Cannabinoid Medicine and Arabic Culture.

Arabs are a large minority group in the Israeli society. With the increasing use of medical cannabis throughout Israel due to changing governmental policies, the interactions of the Arab society with medical cannabis becomes of scientific and medical relevance. Recreational cannabis use is considered haram (forbidden) in Islam. However, most religious scholars agree that medical cannabis usage might be justified as zarurat (emergency and life-saving, therefore allowed) use. Obstacles to medical cannabis use within the Arabic population may relate to language barrier and/or cultural barriers. There are few Arabic-speaking web-based medical-cannabis support groups, and little official information about it is available in the Arabic language. In order for the full benefits of medical cannabis to reach the entire Israeli population, a government-sponsored web-based educational program is necessary in Hebrew and Arabic, both of which are among the nation's official languages, thereby contributing to the equalization of health resource accessibility.

Trace Fear Conditioning: Procedure for Assessing Complex Hippocampal Function in Mice.

The trace fear conditioning protocol is designed to measure hippocampal function in mice. The protocol includes a neutral conditioned stimulus (tone) and an aversive unconditioned stimulus (shock), separated in time by a trace interval. The trace interval between the tone and the shock critically involves the hippocampus and could be used to evaluate hippocampal-dependent learning and memory. In this protocol, we presented mice with five pairings of tone and shock separated by a 20 sec trace interval. Freezing was measured 24 h after conditioning to evaluate contextual memory by placing mice in the conditioned chamber. In addition, 48 h after conditioning, freezing was measured in a dark chamber, which served as a different context. This method enables precise detection of hippocampal-dependent learning and memory following pharmacological and genetic manipulations that impair or enhance hippocampal function.

A dual role for interleukin-1 in hippocampal-dependent memory processes.

Ample research demonstrates that pathophysiological levels of the pro-inflammatory cytokine interleukin-1 (IL-1) produces detrimental effects on memory functioning. However, recent evidence suggests that IL-1 may be required for the normal physiological regulation of hippocampal-dependent memory. To substantiate the physiological role of IL-1 in learning and memory we examined the induction of IL-1 gene expression following a learning experience, and the effects of IL-1 signaling blockade, by either genetic or pharmacological manipulations, on memory functioning. We show that IL-1 gene expression is induced in the hippocampus 24h following fear-conditioning in wild type mice, but not in two mouse strains with impaired IL-1 signaling. Moreover, we report that mice with transgenic over-expression of IL-1 receptor antagonist restricted to the CNS (IL-1raTG) display impaired hippocampal-dependent and intact hippocampal-independent memory in the water maze and fear-conditioning paradigms. We further demonstrate that continuous administration of IL-1ra via osmotic minipumps during prenatal development disrupt memory performance in adult mice, suggesting that IL-1 plays a critical role not only in the formation of hippocampal-dependent memory but also in normal hippocampal development. Finally, we tested the dual role of IL-1 in memory by intracerebroventricular (ICV) administration of different doses of IL-1beta and IL-1ra following learning, providing the first systematic evidence that the involvement of IL-1 in hippocampal-dependent memory follows an inverted U-shaped pattern, i.e., a slight increase in brain IL-1 levels can improve memory, whereas any deviation from the physiological range, either by excess elevation in IL-1 levels or by blockade of IL-1 signaling, results in impaired memory.

Transcriptional regulation of the murine Presenilin-2 gene reveals similarities and differences to its human orthologue.

Inherited Presenilin-2 mutations cause familial Alzheimer's disease, and its regulation may play a role in sporadic cases. The human Presenilin-2 (PSEN2) regulatory region includes two separate promoters modulated by Egr-1, a transcription factor involved in learning and memory. To enable in-vivo analysis of Presenilin-2 regulation, we characterized the murine Presenilin-2 (Psen2) promoter. We identified novel Psen2 Transcription start sites (TSSs) 10 kb upstream of previously reported sites, along with two new alternatively transcribed exons (1A, and 1BC) in the 5' untranslated region. Transcripts initiating in Exon 1A are ubiquitous, whereas exon 1BC-initiated transcripts are non-neuronal. Only the sequence surrounding exon 1A, which includes homologous sequences to the human PSEN2 promoter, harbored significant promoter activity. Sequences upstream of exon 1A and a downstream enhancer were specifically important in neuronal cells, but similar to the human promoter, the murine promoter was characteristic of a housekeeping gene, and its activity depended on Sp1 binding. Egr-1 did not bind the murine promoter. Egr-1 over-expression and down-regulation, as well as in-vivo examination of Egr-1 and Psen2 expression during fear conditioning in mice, showed that Egr-1 does not regulate the murine Psen2 promoter. Differential Psen2 regulation in human and mouse has implications for Alzheimer disease mouse models.