mRNA translation in astrocytes controls hippocampal long-term synaptic plasticity and memory.

Activation of neuronal protein synthesis upon learning is critical for the formation of long-term memory. Here, we report that learning in the contextual fear conditioning paradigm engenders a decrease in eIF2α (eukaryotic translation initiation factor 2) phosphorylation in astrocytes in the hippocampal CA1 region, which promotes protein synthesis. Genetic reduction of eIF2α phosphorylation in hippocampal astrocytes enhanced contextual and spatial memory and lowered the threshold for the induction of long-lasting plasticity by modulating synaptic transmission. Thus, learning-induced dephosphorylation of eIF2α in astrocytes bolsters hippocampal synaptic plasticity and consolidation of long-term memories.

Excitatory neuron-specific suppression of the integrated stress response contributes to autism-related phenotypes in fragile X syndrome.

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold.

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as PTEN, TSC1/2, AKT3, PIK3CA, and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)-induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human PIK3CA mutation that enhances the activity of the PI3K-AKT pathway (Pik3caH1047R-Pvalb ) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.

eIF2α controls memory consolidation via excitatory and somatostatin neurons.

An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory1-4. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories5-10. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF211. Phosphorylation of the α-subunit of eIF2 (p-eIF2α), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds11-13. By contrast, inhibiting the ISR by mutating the eIF2α phosphorylation site, genetically11 and pharmacologically inhibiting the ISR kinases14-17, or mimicking reduced p-eIF2α with the ISR inhibitor ISRIB11, enhances long-term memory in health and disease18. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2α phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2α in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2α-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons.

Limited Proteolysis of Cyclooxygenase-2 Enhances Cell Proliferation.

Accumulating evidence suggests that the cyclooxygenase-2 (COX-2) enzyme has additional catalytic-independent functions. Here we show that COX-2 appears to be cleaved in mouse and human tumors, which led us to hypothesize that COX-2 proteolysis may play a role in cell proliferation. The data presented herein show that a K598R point mutation at the carboxyl-terminus of COX-2 causes the appearance of several COX-2 immunoreactive fragments in nuclear compartments, and significantly enhances cell proliferation. In contrast, insertion of additional mutations at the border of the membrane-binding and catalytic domains of K598R COX-2 blocks fragment formation and prevents the increase in proliferation. Transcriptomic analyses show that K598R COX-2 significantly affects the expression of genes involved in RNA metabolism, and subsequent proteomics suggest that it is associated with proteins that regulate mRNA processing. We observe a similar increase in proliferation by expressing just that catalytic domain of COX-2 (ΔNT- COX-2), which is completely devoid of catalytic activity in the absence of its other domains. Moreover, we show that the ΔNT- COX-2 protein also interacts in the nucleus with ß-catenin, a central regulator of gene transcription. Together these data suggest that the cleavage products of COX-2 can affect cell proliferation by mechanisms that are independent of prostaglandin synthesis.

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.

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.

Down-regulation of cyclooxygenase-2 by the carboxyl tail of the angiotensin II type 1 receptor.

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT1) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT1 also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or ß-arrestin-dependent signaling. Instead, AT1 enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequence alone is sufficient to down-regulate COX-2. Collectively these results propose a new role for AT1 in regulating COX-2 expression in a mechanism that deviates from its canonical signaling pathways. Down-regulation of COX-2 by a short peptide that originates from AT1 may present as a basis for novel therapeutic means of eliminating excess COX-2 protein.

Underwater trauma causes a long-term specific increase in the expression of cyclooxygenase-2 in the ventral CA1 of the hippocampus.

The pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is regularly expressed in the hippocampal neurons, but its role in emotional trauma is not known. Here we show that a single acute stress caused by a near-drowning experience results in heightened anxiety-like behavior one month after the trauma. Biochemical analyses of dorsal and ventral hippocampal CA1, CA3 and dentate gyrus revealed decreased ubiquitination and elevated levels of COX-2 in the traumatized animals only in the ventral CA1. To reveal the identity of the ubiquitin E3 ligase that targets COX-2, we tested the effect of several representative E3 ligases on COX-2 expression in vitro. We found that while AIP4 and Nedd4 had no effect, Mdm2 lowered COX-2 expression by nearly 50%, an effect that was not observed by its dominant negative form. To test whether this also occurs in the hippocampus, we immunoprecipitated Mdm2 from dorsal and ventral CA1 of traumatized and control animals and probed for the presence of COX-2. Our results showed that the levels of Mdm2 were not affected by the trauma but there was significantly less COX-2 associated with Mdm2 in the ventral but not dorsal CA1 of the traumatized animals. Together these data propose that an increase in COX-2 expression in ventral CA1 following trauma is likely due to its attenuated degradation. Unraveling the pathways and mechanisms that control hippocampal COX-2 degradation is important to boost the development of novel therapeutic approaches designed to treat stress-related pathologies.

Upregulation of prostaglandin receptor EP1 expression involves its association with cyclooxygenase-2.

While many signals cause upregulation of the pro-inflammatory enzyme cyclooxygenase -2 (COX-2), much less is known about mechanisms that actively downregulate its expression. We have recently shown that the prostaglandin EP1 receptor reduces the expression of COX-2 in a pathway that facilitates its ubiquitination and degradation via the 26S proteasome. Here we show that an elevation of COX-2 intracellular levels causes an increase in the endogenous expression of prostaglandin EP1. The increase in EP1 levels does not occur at the transcriptional level, but is rather associated with complex formation between the receptor and COX-2, which occurs both in vitro and in mammalian tissues. The EP1-COX-2 complex is disrupted following binding of arachidonic acid to COX-2 and accompanied by a parallel reduction in EP1 levels. We propose that a transient interaction between COX-2 and EP1 constitutes a feedback loop whereby an increase in COX-2 expression elevates EP1, which ultimately acts to downregulate COX-2 by expediting its proteasomal degradation. Such a post translational mechanism may serve to control both the ligand-generating system of COX-2 and its reception system.

Prostaglandin receptor EP1-mediated differential degradation of cyclooxygenases involves a specific lysine residue.

The cyclooxygenase (COX) enzyme isoforms COX-1 and COX-2 catalyze the main step in the generation of prostanoids that mediate major physiological functions. Whereas COX-1 is a ubiquitously expressed stable protein, COX-2 is transiently upregulated in many pathologies and is often associated with a poor prognostic outcome. We have recently shown that an interaction of COX-2 with the prostaglandin EP1 receptor accelerates its degradation via a mechanism that augments its level of ubiquitination. Here we show that the sensitivity of both COX-1 and COX-2 to EP1 is altered upon modification of one lysine residue. A point mutation of lysine to-arginine in position 432 of COX-2 (K432R) yields an enzyme with decreased sensitivity to EP1 -mediated degradation. In contrast, insertion of a putative ubiquitination site into the corresponding position of COX-1 (H446K') yields an enzyme with higher levels of ubiquitination and reduced expression. Furthermore, compared to wild type COX-1, H446K' is significantly more sensitive to downregulation by EP1 . Together these data suggest that distinctive ubiquitination of COX-1 and COX-2 may be responsible for their different sensitivity to EP1 -mediated degradation.

DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

Selective increase in the association of the ß2 adrenergic receptor, ß Arrestin-1 and p53 with Mdm2 in the ventral hippocampus one month after underwater trauma.

Chronic infusion of mice with a ß2 adrenergic receptor (ß2AR) analog was shown to cause long-term DNA damage in a pathway which involves ß Arresin-1-mediated activation of Mdm2 and subsequent degradation of the tumor suppressor protein p53. The objective of the present study was to test whether a single acute stress, which manifests long lasting changes in behavior, affects the interaction of Mdm2 with p53, ß2AR, and ß Arrestin-1 in the dorsal and ventral hippocampal CA1. Adult rats were subject to underwater trauma, a brief forceful submersion under water and tested a month later for behavioral and biochemical changes. Elevated plus maze tests confirmed that animals that experienced the threat of drowning present heightened levels of anxiety one month after trauma. An examination of the CA1 hippocampal areas of the same rats showed that underwater trauma caused a significant increase in the association of Mdm2 with ß2AR, ß Arrestin-1, and p53 in the ventral but not dorsal CA1. Our results provide support for the idea that stress-related events may result in biochemical changes restricted to the ventral 'emotion-related' parts of the hippocampus.

Prostaglandin EP1 receptor down-regulates expression of cyclooxygenase-2 by facilitating its proteasomal degradation.

The enzyme cyclooxygenase-2 (COX-2) is rapidly and transiently up-regulated by a large variety of signals and implicated in pathologies such as inflammation and tumorigenesis. Although many signals cause COX-2 up-regulation, much less is known about mechanisms that actively down-regulate its expression. Here we show that the G protein-coupled receptor prostaglandin E(1) (EP(1)) reduces the expression of COX-2 in a concentration-dependent manner through a mechanism that does not require receptor activation. The reduction in COX-2 protein is not due to decreased protein synthesis and occurs because of enhancement of substrate-independent COX-2 proteolysis. Although EP(1) does not interfere with the entry of COX-2 into the endoplasmic reticulum-associated degradation cascade, it facilitates COX-2 ubiquitination through complex formation. Blockade of proteasomal activity results in degradation of the receptor and concomitant recovery in the expression of COX-2, suggesting that EP(1) may scaffold an unknown E3 ligase that ubiquitinates COX-2. These findings propose a new role for the EP(1) receptor in resolving inflammation through down-regulation of COX-2.