Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS.

Atomic force microscopy analysis of central nervous system cell morphology on silicon carbide and diamond substrates.

Brain machine interface (BMI) devices offer a platform that can be used to assist people with extreme disabilities, such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease. Silicon (Si) has been the material of choice used for the manufacture of BMI devices due to its mechanical strength, its electrical properties and multiple fabrication techniques; however, chronically implanted BMI devices have usually failed within months of implantation due to biocompatibility issues and the fact that Si does not withstand the harsh environment of the body. Single crystal cubic silicon carbide (3C-SiC) and nanocrystalline diamond (NCD) are semiconductor materials that have previously shown good biocompatibility with skin and bone cells. Like Si, these materials have excellent physical characteristics, good electrical properties, but unlike Si, they are chemically inert. We have performed a study to evaluate the general biocompatibility levels of all of these materials through the use of in vitro techniques. H4 human neuroglioma and PC12 rat pheochromocytoma cell lines were used for the study, and polystyrene (PSt) and amorphous glass were used as controls or for morphological comparison. MTT [3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide] assays were performed to determine general cell viability with each substrate and atomic force microscopy (AFM) was used to quantify the general cell morphology on the substrate surface along with the substrate permissiveness to lamellipodia extension. 3C-SiC was the only substrate tested to have good viability and superior lamellipodia permissiveness with both cell lines, while NCD showed a good level of viability with the neural H4 line but a poor viability with the PC12 line and lower permissiveness than 3C-SiC. Explanations pertaining to the performance of each substrate with both cell lines are presented and discussed along with future work that must be performed to further evaluate specific cell reactions on these substrates.

Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aß ligation to ß1-integrin conformers.

Correction to: Cell Death and Differentiation (2015) 22, 921­934; doi:10.1038/cdd.2015.5; published online 20 February 2015. Since the publication of this paper, the authors have noticed the y-axis label of Figure 7e was incorrect. It should be % of the fESP slope. This has now been rectified and the corrected article appears in this issue together with this corrigendum.

Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Aß ligation to ß1-integrin conformers.

The accumulation of amyloid-ß protein (Aß) is an early event associated with synaptic and mitochondrial damage in Alzheimer's disease (AD). Recent studies have implicated the filamentous actin (F-actin) severing protein, Cofilin, in synaptic remodeling, mitochondrial dysfunction, and AD pathogenesis. However, whether Cofilin is an essential component of the AD pathogenic process and how Aß impinges its signals to Cofilin from the neuronal surface are unknown. In this study, we found that Aß42 oligomers (Aß42O, amyloid-ß protein 1-42 oligomers) bind with high affinity to low or intermediate activation conformers of ß1-integrin, resulting in the loss of surface ß1-integrin and activation of Cofilin via Slingshot homology-1 (SSH1) activation. Specifically, conditional loss of ß1-integrin prevented Aß42O-induced Cofilin activation, and allosteric modulation or activation of ß1-integrin significantly reduced Aß42O binding to neurons while blocking Aß42O-induced reactive oxygen species (ROS) production, mitochondrial dysfunction, depletion of F-actin/focal Vinculin, and apoptosis. Cofilin, in turn, was required for Aß42O-induced loss of cell surface ß1-integrin, disruption of F-actin/focal Talin-Vinculin, and depletion of F-actin-associated postsynaptic proteins. SSH1 reduction, which mitigated Cofilin activation, prevented Aß42O-induced mitochondrial Cofilin translocation and apoptosis, while AD brain mitochondria contained significantly increased activated/oxidized Cofilin. In mechanistic support in vivo, AD mouse model (APP (amyloid precursor protein)/PS1) brains contained increased SSH1/Cofilin and decreased SSH1/14-3-3 complexes, indicative of SSH1-Cofilin activation via release of SSH1 from 14-3-3. Finally, genetic reduction in Cofilin rescued APP/Aß-induced synaptic protein loss and gliosis in vivo as well as deficits in long-term potentiation (LTP) and contextual memory in APP/PS1 mice. These novel findings therefore implicate the essential involvement of the ß1-integrin-SSH1-Cofilin pathway in mitochondrial and synaptic dysfunction in AD.

RanBP9 at the intersection between cofilin and Aß pathologies: rescue of neurodegenerative changes by RanBP9 reduction.

Molecular pathways underlying the neurotoxicity and production of amyloid ß protein (Aß) represent potentially promising therapeutic targets for Alzheimer's disease (AD). We recently found that overexpression of the scaffolding protein RanBP9 increases Aß production in cell lines and in transgenic mice while promoting cofilin activation and mitochondrial dysfunction. Translocation of cofilin to mitochondria and induction of cofilin-actin pathology require the activation/dephosphorylation of cofilin by Slingshot homolog 1 (SSH1) and cysteine oxidation of cofilin. In this study, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates Aß-induced translocation of cofilin to mitochondria and induction of cofilin-actin pathology in cultured cells, primary neurons, and in vivo. Endogenous level of RanBP9 was also required for Aß-induced collapse of growth cones in immature neurons (days in vitro 9 (DIV9)) and depletion of synaptic proteins in mature neurons (DIV21). In vivo, amyloid precursor protein (APP)/presenilin-1 (PS1) mice exhibited 3.5-fold increased RanBP9 levels, and RanBP9 reduction protected against cofilin-actin pathology, synaptic damage, gliosis, and Aß accumulation associated with APP/PS1 mice. Brains slices derived from APP/PS1 mice showed significantly impaired long-term potentiation (LTP), and RanBP9 reduction significantly enhanced paired pulse facilitation and LTP, as well as partially rescued contextual memory deficits associated with APP/PS1 mice. Therefore, these results underscore the critical importance of endogenous RanBP9 not only in Aß accumulation but also in mediating the neurotoxic actions of Aß at the level of synaptic plasticity, mitochondria, and cofilin-actin pathology via control of the SSH1-cofilin pathway in vivo.

Extracellular proteolysis of reelin by tissue plasminogen activator following synaptic potentiation.

The secreted glycoprotein reelin plays an indispensable role in neuronal migration during development and in regulating adult synaptic functions. The upstream mechanisms responsible for initiating and regulating the duration and magnitude of reelin signaling are largely unknown. Here we report that reelin is cleaved between EGF-like repeats 6-7 (R6-7) by tissue plasminogen activator (tPA) under cell-free conditions. No changes were detected in the level of reelin and its fragments in the brains of tPA knockouts, implying that other unknown proteases are responsible for generating reelin fragments found constitutively in the adult brain. Induction of NMDAR-independent long-term potentiation with the potassium channel blocker tetraethylammonium chloride (TEA-Cl) led to a specific up-regulation of reelin processing at R6-7 in wild-type mice. In contrast, no changes in reelin expression and processing were observed in tPA knockouts following TEA-Cl treatment. These results demonstrate that synaptic potentiation results in tPA-dependent reelin processing and suggest that extracellular proteolysis of reelin may regulate reelin signaling in the adult brain.

Impaired hippocampal synaptic function in secretin deficient mice.

Secretin is a gut peptide hormone that is also expressed in the CNS. To explore the potential neuroactive role of secretin in the brain, we have generated secretin deficient mice. Secretin deficient mice demonstrated impairment in synaptic plasticity (significant reduction in long term potentiation (LTP) induction and LTP maintenance) in the CA1 area of the hippocampus. Using a beta-galactosidase (lacZ) reporter in the targeted allele and secretin antibody staining, we have detected secretin gene expression in the hippocampus, cerebellum, and the brain stem in adult mouse brain. In the hippocampus, secretin was expressed in the dentate gyrus, the hilus, and the molecular layer. These findings suggest that secretin is involved in synaptic function in the adult brain.

Effects of prenatal ethanol exposure on phospholipase C-beta 1 and phospholipase A2 in hippocampus and medial frontal cortex of adult rat offspring.

Previous studies in our laboratory using a rat model of fetal alcohol exposure (FAE) suggest that FAE-induced behavioral deficits are, in part, linked to neurochemical and electrophysiological deficits in long-term potentiation (LTP) in the entorhinal cortical perforant path projection to the hippocampal formation. Several findings suggest that signal-activated phospholipase C (PLC) and phospholipase A2 (PLA2) are critical to the induction and maintenance of LTP. Thus, alterations in phospholipid metabolism may play a significant role in the LTP deficits observed in FAE offspring. To test this hypothesis, we measured PLC-beta 1 and PLA2 activities in the hippocampus and medial frontal cortex of adult rats prenatally exposed to ethanol. PLC-beta 1 activities were significantly decreased by 20 to 30% in both the hippocampus and medial frontal cortex of FAE rats, compared with ad libitum and pair-fed controls. Total Ca(2+)-dependent PLA2 activity was 25% lower in the medial frontal cortex of FAE rats, but did not significantly differ from controls in the hippocampal formation. Approximately 30% of the measured activity in both the medial frontal cortex and hippocampal formation of ad libitum and pair-fed animals was associated with an 85 kDa cytosolic PLA2 form. Cytosolic PLA2 activities were significantly reduced in both the medial frontal cortex and hippocampal formation of FAE rats, compared with controls. These changes in Ca(2+)-dependent PLA 2 and PLC-beta 1 activities, coupled with reports of FAE-induced deficits in protein kinase C activity, indicate that prenatal exposure to moderate quantities of ethanol causes profound and long-lasting deficits in the cellular signaling mechanisms associated with activity-dependent synaptic plasticity and memory formation.

Fear conditioning-induced alterations of phospholipase C-beta1a protein level and enzyme activity in rat hippocampal formation and medial frontal cortex.

We investigated the effects of one-trial fear conditioning on phospholipase C-beta1a catalytic activity and protein level in hippocampal formation and medial frontal cortex of untreated control rats and rats prenatally exposed to ethanol. One hour following fear conditioning of untreated control rats, phospholipase C-beta1a protein level was increased in the hippocampal cytosolic fraction and decreased in the hippocampal membrane and cortical cytosolic and cortical membrane fractions. Twenty-four hours after fear conditioning, phospholipase C-beta1a protein level was reduced in the hippocampal cytosolic fraction and elevated in the cortical nuclear fraction; in addition, 24 h after conditioning, phospholipase C-beta1a activity in the cortical cytosolic fraction was increased. Rats that were exposed prenatally to ethanol displayed attenuated contextual fear conditioning, whereas conditioning to the acoustic-conditioned stimulus was not different from controls. In behavioral control (unconditioned) rats, fetal ethanol exposure was associated with reduced phospholipase C-beta1a enzyme activity in the hippocampal nuclear, cortical cytosolic, and cortical membrane fractions and increased phospholipase C-beta1a protein level in the hippocampal membrane and cortical cytosolic fractions. In certain cases, prenatal ethanol exposure modified the relationship between fear conditioning and changes in phospholipase C-beta1a protein level and/or activity. The majority of these effects occurred 1 h, rather than 24 h, after fear conditioning. Multivariate analysis of variance revealed interactions between fear conditioning, subcellular fraction, and prenatal ethanol exposure for measures of phospholipase C-beta1a protein level in hippocampal formation and phospholipase C-beta1a enzyme activity in medial frontal cortex. In the majority of cases, fear conditioning-induced changes in hippocampal phospholipase C-beta1a protein level were augmented in rats prenatally exposed to ethanol. In contrast, fear conditioning-induced changes in cortical phospholipase C-beta1a activity were, often, in opposite directions in prenatal ethanol-exposed compared to diet control rats. We speculate that alterations in subcellular phospholipase C-beta1a catalytic activity and protein level contribute to contextual fear conditioning and that learning deficits observed in rats exposed prenatally to ethanol result, in part, from dysfunctions in phospholipase C-beta1a signal transduction.

Protein kinase inhibition by omega-3 fatty acids.

Recent data suggest that omega-3 fatty acids may be effective in epilepsy, cardiovascular disorders, arthritis, and as mood stabilizers for bipolar disorder; however, the mechanism of action of these compounds is unknown. Based on earlier studies implicating omega-3 fatty acids as inhibitors of protein kinase C activity in intact cells, we hypothesized that omega-3 fatty acids may act through direct inhibition of second messenger-regulated kinases and sought to determine whether the omega-3 double bond might uniquely confer pharmacologic efficacy and potency for fatty acids of this type. In our studies we observed that omega-3 fatty acids inhibited the in vitro activities of cAMP-dependent protein kinase, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and the mitogen-activated protein kinase (MAPK). Our results with a series of long-chain fatty acid structural homologs suggest an important role for the omega-3 double bond in conferring inhibitory efficacy. To assess whether omega-3 fatty acids were capable of inhibiting protein kinases in living neurons, we evaluated their effect on signal transduction pathways in the hippocampus. We found that omega-3 fatty acids could prevent serotonin receptor-induced MAPK activation in hippocampal slice preparations. In addition, we evaluated the effect of omega-3 fatty acids on hippocampal long-term potentiation, a form of synaptic plasticity known to be dependent on protein kinase activation. We observed that omega-3 fatty acids blocked long-term potentiation induction without inhibiting basal synaptic transmission. Overall, our results from both in vitro and live cell preparations suggest that inhibition of second messenger-regulated protein kinases is one locus of action of omega-3 fatty acids.

Inhibitory effects of omega-3 fatty acids on protein kinase C activity in vitro.

Preliminary clinical data indicate that omega-3 fatty acids may be effective mood stabilizers for patients with bipolar disorder. Both lithium and valproic acid are known to inhibit protein kinase C (PKC) activity after subchronic administration in cell culture and in vivo. The current study was undertaken to determine the effects of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on protein kinase C phosphotransferase activity in vitro. Various concentrations of DHA, EPA, and arachidonic acid (AA) were incubated with the catalytic domain of protein kinase C beta from rat brain. Protein kinase C activity was measured by quantifying incorporation of (32)P-PO(4) into a synthetic peptide substrate. Both DHA and EPA, as well as the combination of DHA and EPA, inhibited PKC activity at concentrations as low as 10 micromol l(-1). In contrast, arachidonic acid had no effect on PKC activity. Thus, PKC represents a potential site of action of omega-3 fatty acids in their effects on the treatment of bipolar disorder.

Leitmotifs in the biochemistry of LTP induction: amplification, integration and coordination.

Hippocampal long-term potentiation (LTP) is a robust and long-lasting form of synaptic plasticity that is the leading candidate for a cellular mechanism contributing to mammalian learning and memory. Investigations over the past decade have revealed that the biochemistry of LTP induction involves mechanisms of great subtlety and complexity. This review highlights themes that have emerged as a result of our increased knowledge of the signal transduction pathways involved in the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus. Among these themes are signal amplification, signal integration and signal coordination. Here we use these themes as an organizing context for reviewing the profusion of signaling mechanisms involved in the induction of LTP.

A role for the beta isoform of protein kinase C in fear conditioning.

The protein kinase C family of enzymes has been implicated in synaptic plasticity and memory in a wide range of animal species, but to date little information has been available concerning specific roles for individual isoforms of this category of kinases. To investigate the role of the beta isoform of PKC in mammalian learning, we characterized mice deficient in the PKC beta gene using anatomical, biochemical, physiological, and behavioral approaches. In our studies we observed that PKC beta was predominantly expressed in the neocortex, in area CA1 of the hippocampus, and in the basolateral nucleus of the amygdala. Mice deficient in PKC beta showed normal brain anatomy and normal hippocampal synaptic transmission, paired pulse facilitation, and long-term potentiation and normal sensory and motor responses. The PKC beta knock-out animals exhibited a loss of learning, however; they suffered deficits in both cued and contextual fear conditioning. The PKC expression pattern and behavioral phenotype in the PKC beta knock-out animals indicate a critical role for the beta isoform of PKC in learning-related signal transduction mechanisms, potentially in the basolateral nucleus of the amygdala.