Suppression of PKR promotes network excitability and enhanced cognition by interferon-γ-mediated disinhibition.

The double-stranded RNA-activated protein kinase (PKR) was originally identified as a sensor of virus infection, but its function in the brain remains unknown. Here, we report that the lack of PKR enhances learning and memory in several behavioral tasks while increasing network excitability. In addition, loss of PKR increases the late phase of long-lasting synaptic potentiation (L-LTP) in hippocampal slices. These effects are caused by an interferon-γ (IFN-γ)-mediated selective reduction in GABAergic synaptic action. Together, our results reveal that PKR finely tunes the network activity that must be maintained while storing a given episode during learning. Because PKR activity is altered in several neurological disorders, this kinase presents a promising new target for the treatment of cognitive dysfunction. As a first step in this direction, we show that a selective PKR inhibitor replicates the Pkr(-/-) phenotype in WT mice, enhancing long-term memory storage and L-LTP.

A CRISPR toolbox for generating intersectional genetic mouse models for functional, molecular, and anatomical circuit mapping.

BACKGROUND: The functional understanding of genetic interaction networks and cellular mechanisms governing health and disease requires the dissection, and multifaceted study, of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic effector alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical studies. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with production of intersectional mouse lines hindering customized approaches to many researchers. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. RESULTS: Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. CONCLUSIONS: The lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of long-term neurological disability, yet the mechanisms underlying the chronic cognitive deficits associated with TBI remain unknown. Consequently, there are no effective treatments for patients suffering from the long-lasting symptoms of TBI. Here, we show that TBI persistently activates the integrated stress response (ISR), a universal intracellular signaling pathway that responds to a variety of cellular conditions and regulates protein translation via phosphorylation of the translation initiation factor eIF2α. Treatment with ISRIB, a potent drug-like small-molecule inhibitor of the ISR, reversed the hippocampal-dependent cognitive deficits induced by TBI in two different injury mouse models-focal contusion and diffuse concussive injury. Surprisingly, ISRIB corrected TBI-induced memory deficits when administered weeks after the initial injury and maintained cognitive improvement after treatment was terminated. At the physiological level, TBI suppressed long-term potentiation in the hippocampus, which was fully restored with ISRIB treatment. Our results indicate that ISR inhibition at time points late after injury can reverse memory deficits associated with TBI. As such, pharmacological inhibition of the ISR emerges as a promising avenue to combat head trauma-induced chronic cognitive deficits.

Melatonin Attenuates Myocardial Ischemia/Reperfusion Injury by Inhibiting Autophagy Via an AMPK/mTOR Signaling Pathway.

BACKGROUND/AIMS: Melatonin has been demonstrated to protect cardiac microvascular endothelial cells (CMECs) against ischemia/reperfusion injury (IRI). Autophagy plays different roles in the heart during ischemia and reperfusion. The AMP activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway is associated with autophagy. This study sought to explore whether melatonin regulates CMEC autophagy through the AMPK/mTOR signaling pathway. METHODS: The effects of melatonin in IRI were investigated in vivo rat models and in vitro neonatal CMECs. Myocardial infarct size was achieved by Evans blue and triphenyltetrazolium chloride staining. The severity of cell injury was evaluated by cell vitality and lactate dehydrogenase (LDH) release assays, and autophagy was evaluated by transmission electron microscopy and the assessment of autophagy-related gene expression, such as that of Beclin 1 and light chain 3-II. RESULTS: In vivo, melatonin markedly reduced infarcted area, improved cardiac function and decreased LDH release. However, the AMPK activator AICAR and the mTOR inhibitor rapamycin reduced the protective effects of melatonin on IRI. In vitro, Beclin1 and light chain 3-II protein were found to be down-regulated and autophagosomes were found to be reduced in response to melatonin, together with an increase in cell vitality and a decrease in LDH. Treatment with AICAR or rapamycin ablated the benefit observed with melatonin treatment. CONCLUSIONS: Melatonin played an important and protective role in CMECs by inhibiting autophagy against IRI via the AMPK/mTOR system.

Truncation of Ube3a-ATS unsilences paternal Ube3a and ameliorates behavioral defects in the Angelman syndrome mouse model.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal deficiency of the imprinted gene UBE3A. Individuals with AS suffer from intellectual disability, speech impairment, and motor dysfunction. Currently there is no cure for the disease. Here, we evaluated the phenotypic effect of activating the silenced paternal allele of Ube3a by depleting its antisense RNA Ube3a-ATS in mice. Premature termination of Ube3a-ATS by poly(A) cassette insertion activates expression of Ube3a from the paternal chromosome, and ameliorates many disease-related symptoms in the AS mouse model, including motor coordination defects, cognitive deficit, and impaired long-term potentiation. Studies on the imprinting mechanism of Ube3a revealed a pattern of biallelic transcription initiation with suppressed elongation of paternal Ube3a, implicating transcriptional collision between sense and antisense polymerases. These studies demonstrate the feasibility and utility of unsilencing the paternal copy of Ube3a via targeting Ube3a-ATS as a treatment for Angelman syndrome.

Selective pharmacogenetic inhibition of mammalian target of Rapamycin complex I (mTORC1) blocks long-term synaptic plasticity and memory storage.

Both the formation of long-term memory (LTM) and late-long-term potentiation (L-LTP), which is thought to represent the cellular model of learning and memory, require de novo protein synthesis. The mammalian target of Rapamycin (mTOR) complex I (mTORC1) integrates information from various synaptic inputs and its best characterized function is the regulation of translation. Although initial studies have shown that rapamycin reduces L-LTP and partially blocks LTM, recent genetic and pharmacological evidence indicating that mTORC1 promotes L-LTP and LTM is controversial. Thus, the role of mTORC1 in L-LTP and LTM is unclear. To selectively inhibit mTORC1 activity in the adult brain, we used a "pharmacogenetic" approach that relies on the synergistic action of a drug (rapamycin) and a genetic manipulation (mTOR heterozygotes, mTOR(+/-) mice) on the same target (mTORC1). Although L-LTP and LTM are normal in mTOR(+/-) mice, application of a low concentration of rapamycin-one that is subthreshold for WT mice-prevented L-LTP and LTM only in mTOR(+/-) mice. Furthermore, we found that mTORC1-mediated translational control is required for memory reconsolidation. We provide here direct genetic evidence supporting the role of mTORC1 in L-LTP and behavioral memory.

Developmental alteration of endocannabinoid retrograde signaling in the hippocampus.

Endocannabinoids are lipid derivatives that mediate paracrine and juxtacrine signaling between cells. In the hippocampal CA1 region, a retrograde endocannabinoid signal suppresses GABA release by acting on presynaptic cannabinoid receptor-1 (CB1) and can be functionally manifested as depolarization-induced suppression of inhibition (DSI). In the present study, whole cell patch-clamp recordings in hippocampal slices were made to examine DSI in rats from P7-P21. Robust DSI develops in rat hippocampus at postnatal ages greater than two weeks, but only modest DSI is observed in P7-9 rat. DSI in neonatal rats can be enhanced by activation of group I metabotropic glutamate receptors (mGluRs) or muscarinic acetylcholine receptors in those neonatal rats. The DSI is also enhanced by sustained low-frequency (1 Hz) stimulation (5 min). This stimulus-enhanced DSI was prevented in the presence of 6-methyl-2-(phenylethynyl)-pyridine (10 microM), a group I mGluR antagonist. WIN55212-2, a synthetic CB1 agonist, produced a similar level of inhibition of GABAergic synaptic transmission at different postnatal time points. Therefore postsynaptic mechanisms appear to be mainly responsible for developmental changes in DSI, although presynaptic mechanisms cannot be ruled out entirely. We have also obtained evidence that tonic endocannabinoid release suppresses GABAergic transmission in the mature but not the neonatal hippocampus. The differential DSI magnitude at different stages of maturation could alter synaptic plasticity and learning and memory during hippocampal development.

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death.

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a beta-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.

Off-Target Effects of Clozapine-N-Oxide on the Chemosensory Reflex Are Masked by High Stress Levels.

Respiratory chemosensory circuits are implicated in several physiological and behavioral disorders ranging from sudden infant death syndrome to panic disorder. Thus, a comprehensive map of the chemosensory network would be of significant value. To delineate chemosensory neuronal populations, we have utilized pharmacogenetic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) perturbations for acute neuronal perturbations in respiratory circuit mapping. Recent studies show that the biologically inert DREADD ligand clozapine-N-oxide (CNO) is back-metabolized into the bioactive compound clozapine in rodents, emphasizing the need for CNO-only DREADD-free controls, which have been carried out in several studies. However, we show that high CNO doses used in several chemosensory circuit mapping studies nonetheless affect the chemosensory ventilatory reflexes in control mice, which is unmasked by extensive habituation. Here, unhabituated control animals showed no differences in respiratory parameters after CNO administration, whereas habituated animals receiving the commonly used dose of 10 mg/kg of CNO show a deficit in the hypercapnic (high CO2) chemosensory reflex, which is not present in 1 mg/kg CNO treated or saline control groups. Our findings indicate that even in appropriately controlled studies, additional masked CNO off-target effects may exist and underscore the importance of using minimal doses of activating ligand in combination with high levels of habituation.

Activation of the ISR mediates the behavioral and neurophysiological abnormalities in Down syndrome.

Down syndrome (DS) is the most common genetic cause of intellectual disability. Protein homeostasis is essential for normal brain function, but little is known about its role in DS pathophysiology. In this study, we found that the integrated stress response (ISR)-a signaling network that maintains proteostasis-was activated in the brains of DS mice and individuals with DS, reprogramming translation. Genetic and pharmacological suppression of the ISR, by inhibiting the ISR-inducing double-stranded RNA-activated protein kinase or boosting the function of the eukaryotic translation initiation factor eIF2-eIF2B complex, reversed the changes in translation and inhibitory synaptic transmission and rescued the synaptic plasticity and long-term memory deficits in DS mice. Thus, the ISR plays a crucial role in DS, which suggests that tuning of the ISR may provide a promising therapeutic intervention.

Inhibition of Upf2-Dependent Nonsense-Mediated Decay Leads to Behavioral and Neurophysiological Abnormalities by Activating the Immune Response.

In humans, disruption of nonsense-mediated decay (NMD) has been associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorder and intellectual disability. However, the mechanism by which deficient NMD leads to neurodevelopmental dysfunction remains unknown, preventing development of targeted therapies. Here we identified novel protein-coding UPF2 (UP-Frameshift 2) variants in humans with NDD, including speech and language deficits. In parallel, we found that mice lacking Upf2 in the forebrain (Upf2 fb-KO mice) show impaired NMD, memory deficits, abnormal long-term potentiation (LTP), and social and communication deficits. Surprisingly, Upf2 fb-KO mice exhibit elevated expression of immune genes and brain inflammation. More importantly, treatment with two FDA-approved anti-inflammatory drugs reduced brain inflammation, restored LTP and long-term memory, and reversed social and communication deficits. Collectively, our findings indicate that impaired UPF2-dependent NMD leads to neurodevelopmental dysfunction and suggest that anti-inflammatory agents may prove effective for treatment of disorders with impaired NMD.

A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS.

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.

mTORC2, but not mTORC1, is required for hippocampal mGluR-LTD and associated behaviors.

The mechanistic target of rapamycin complex 1 (mTORC1) has been reported to be necessary for metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD). Here we found that mTORC1-deficient mice exhibit normal hippocampal mGluR-LTD and associated behaviors. Moreover, rapamycin blocks mGluR-LTD in mTORC1-deficient mice. However, both rapamycin and mGluR activation regulate mTOR complex 2 (mTORC2) activity, and mTORC2-deficient mice show impaired mGluR-LTD and associated behaviors. Thus, mTORC2 is a major regulator of mGluR-LTD.

Liraglutide directly protects cardiomyocytes against reperfusion injury possibly via modulation of intracellular calcium homeostasis.

BACKGROUND: Liraglutide is glucagon-like peptide-1 receptor agonist for treating patients with type 2 diabetes mellitus. Our previous studies have demonstrated that liraglutide protects cardiac function through improving endothelial function in patients with acute myocardial infarction undergoing percutaneous coronary intervention. The present study will investigate whether liraglutide can perform direct protective effects on cardiomyocytes against reperfusion injury. METHODS: In vitro experiments were performed using H9C2 cells and neonatal rat ventricular cadiomyocytes undergoing simulative hypoxia/reoxygenation (H/R) induction. Cardiomyocytes apoptosis was detected by fluorescence TUNEL. Mitochondrial membrane potential (ΔΨm) and intracellular reactive oxygen species (ROS) was assessed by JC-1 and DHE, respectively. Fura-2/AM was used to measure intracellular Ca2+ concentration and calcium transient. Immunofluorescence staining was used to assess the expression level of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a). In vivo experiments, myocardial apoptosis and expression of SERCA2a were detected by colorimetric TUNEL and by immunofluorescence staining, respectively. RESULTS: In vitro liraglutide inhibited cardiomyotes apoptosis against H/R. ΔΨm of cardiomyocytes was higher in liraglutide group than H/R group. H/R increased ROS production in H9C2 cells which was attenuated by liraglutide. Liraglutide significantly lowered Ca2+ overload and improved calcium transient compared with H/R group. Immunofluorescence staining results showed liraglutide promoted SERCA2a expression which was decreased in H/R group. In ischemia/reperfusion rat hearts, apoptosis was significantly attenuated and SERCA2a expression was increased by liraglutide compared with H/R group. CONCLUSIONS: Liraglutide can directly protect cardiomyocytes against reperfusion injury which is possibly through modulation of intracellular calcium homeostasis.

Retrograde endocannabinoid signaling in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala.

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in the brain. In the basolateral amygdala (BLA), endocannabinoid signaling has been implicated in learning and memory, specifically in extinction of aversive memories. To examine retrograde endocannabinoid signaling in this brain region, BLA neurons were freshly isolated using an enzyme-free procedure. These isolated neurons retain attached functional excitatory and inhibitory synaptic boutons. Spontaneous GABAergic IPSCs (sIPSCs) were isolated from these freshly isolated neurons and a 4 s step of depolarization from -60 to 0 mV produced suppression of sIPSC frequency and amplitude. A similar depolarization-induced suppression of inhibition (DSI) was observed in neurons in BLA slices. DSI in the single-cell preparation was abolished by the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, and DSI duration was shortened in the presence of 2-methyl-6-(phenylethynyl) pyridine, an mGluR5 (metabotropic glutamate receptor 5) antagonist. The initial decrease in sIPSCs induced by the DSI procedure was greatly attenuated in recordings with 20 mm BAPTA containing postsynaptic internal solution, but a delayed-onset decrease was observed under this recording condition. A CB1 agonist decreased sIPSC frequency and amplitude, whereas CB1 antagonists increased these responses. The antagonist-induced increase was abolished in 20 mm BAPTA-filled cells. These data provide solid evidence for retrograde endocannabinoid signaling in the BLA and also indicate that this retrograde signaling requires only a postsynaptic neuron and attached synaptic boutons.

Endocannabinoid signaling and synaptic plasticity in the brain.

Repetitive firing neuron or activation of synaptic transmission plays an important role in the modulation of synaptic efficacy, such as long-term potentiation (LTP) and long-term depression (LTD). These activity-dependent changes in synaptic efficacy are thought to be critical to learning and memory; however, the underlying mechanisms remain to be defined. Endogenous cannabinoids (eCBs) are diffusible modulators that are released from depolarized postsynaptic neurons and act on presynaptic terminals. Persistent release of eCBs can lead to long-term modulation of synaptic plasticity in the brain. Given a broad distribution of eCB receptors in the brain, the eCB signaling system could contribute to use-dependent modification of brain functions.

mTORC2 controls actin polymerization required for consolidation of long-term memory.

A major goal of biomedical research is the identification of molecular and cellular mechanisms that underlie memory storage. Here we report a previously unknown signaling pathway that is necessary for the conversion from short- to long-term memory. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2), which contains the regulatory protein Rictor (rapamycin-insensitive companion of mTOR), was discovered only recently and little is known about its function. We found that conditional deletion of Rictor in the postnatal murine forebrain greatly reduced mTORC2 activity and selectively impaired both long-term memory (LTM) and the late phase of hippocampal long-term potentiation (L-LTP). We also found a comparable impairment of LTM in dTORC2-deficient flies, highlighting the evolutionary conservation of this pathway. Actin polymerization was reduced in the hippocampus of mTORC2-deficient mice and its restoration rescued both L-LTP and LTM. Moreover, a compound that promoted mTORC2 activity converted early LTP into late LTP and enhanced LTM. Thus, mTORC2 could be a therapeutic target for the treatment of cognitive dysfunction.

Persistent synaptic activity produces long-lasting enhancement of endocannabinoid modulation and alters long-term synaptic plasticity.

Learning and memory are thought to involve activity-dependent changes in synaptic efficacy such as long-term potentiation (LTP) and long-term depression (LTD). Recent studies have indicated that endocannabinoid-dependent modulation of inhibitory transmission facilitates induction of hippocampal LTP and that endocannabinoids play a key role in certain forms of LTD. Here, we show that repetitive low-frequency synaptic stimulation (LFS) produces persistent up-regulation of endocannabinoid signaling at hippocampal CA1 GABAergic synapses. This LFS also produces LTD of inhibitory synapses and facilitates LTP at excitatory, glutamatergic synapses. These endocannabinoid-mediated plastic changes could contribute to information storage within the brain.

Ethanol potentiates GABAergic synaptic transmission in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala.

Interactions between ethanol and synaptic transmission mediated by gamma -amino-N-butyric acid (GABA) have been suggested to contribute to alcohol intoxication. Ethanol effects on postsynaptic GABAA receptors have been the major focus of this line of research. There is increasing evidence that ethanol potentiation of GABAergic transmission involves increased GABA release from presynaptic terminals. In the present study, a mechanically isolated neuron/bouton preparation from the basolateral amygdala was used to examine the effects of ethanol on spontaneous GABAergic synaptic currents elicited by GABA release from the presynaptic terminals. We found that ethanol application produced a rapid increase in the frequency of spontaneous GABAergic synaptic currents. An acute tolerance to ethanol was also observed, and this tolerance involved GABAB receptor activation. The ethanol-induced potentiation did not involve alterations in the function of postsynaptic GABAA receptors and was independent of presynaptic action potential firing. These findings indicate that ethanol potentiates GABA release, most likely via a direct action on presynaptic boutons.

Actions of acute and chronic ethanol on presynaptic terminals.

This article presents the proceedings of a symposium entitled "The Tipsy Terminal: Presynaptic Effects of Ethanol" (held at the annual meeting of the Research Society on Alcoholism, in Santa Barbara, CA, June 27, 2005). The objective of this symposium was to focus on a cellular site of ethanol action underrepresented in the alcohol literature, but quickly becoming a "hot" topic. The chairs of the session were Marisa Roberto and George Robert Siggins. Our speakers were chosen on the basis of the diverse electrophysiological and other methods used to discern the effects of acute and chronic ethanol on presynaptic terminals and on the basis of significant insights that their data provide for understanding ethanol actions on neurons in general, as mechanisms underlying problematic behavioral effects of alcohol. The 5 presenters drew from their recent studies examining the effects of acute and chronic ethanol using a range of sophisticated methods from electrophysiological analysis of paired-pulse facilitation and spontaneous and miniature synaptic currents (Drs. Weiner, Valenzuela, Zhu, and Morrisett), to direct recording of ion channel activity and peptide release from acutely isolated synaptic terminals (Dr. Treistman), to direct microscopic observation of vesicular release (Dr. Morrisett). They showed that ethanol administration could both increase and decrease the probability of release of different transmitters from synaptic terminals. The effects of ethanol on synaptic terminals could often be correlated with important behavioral or developmental actions of alcohol. These and other novel findings suggest that future analyses of synaptic effects of ethanol should attempt to ascertain, in multiple brain regions, the role of presynaptic terminals, relevant presynaptic receptors and signal transduction linkages, exocytotic mechanisms, and their involvement in alcohol's behavioral actions. Such studies could lead to new treatment strategies for alcohol intoxication, alcohol abuse, and alcoholism.