Enhanced long term potentiation and decreased AMPA receptor desensitization in the acute period following a single kainate induced early life seizure.

Neonatal seizures are associated with long term disabilities including epilepsy and cognitive deficits. Using a neonatal seizure rat model that does not develop epilepsy, but develops a phenotype consistent with other models of intellectual disability (ID) and autism spectrum disorders (ASD), we sought to isolate the acute effects of a single episode of early life seizure on hippocampal CA1 synaptic development and plasticity. We have previously shown chronic changes in glutamatergic synapses, loss of long term potentiation (LTP) and enhanced long term depression (LTD), in the adult male rat ~50days following kainic acid (KA) induced early life seizure (KA-ELS) in post-natal (P) 7day old male Sprague-Dawley rats. In the present work, we examined the electrophysiological properties and expression levels of glutamate receptors in the acute period, 2 and 7days, post KA-ELS. Our results show for the first time enhanced LTP 7days after KA-ELS, but no change 2days post KA-ELS. Additionally, we report that ionotropic α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid type glutamate receptor (AMPAR) desensitization is decreased in the same time frame, with no changes in AMPAR expression, phosphorylation, or membrane insertion. Inappropriate enhancement of the synaptic connections in the acute period after the seizure could alter the normal patterning of synaptic development in the hippocampus during this critical period and contribute to learning deficits. Thus, this study demonstrates a novel mechanism by which KA-ELS alters early network properties that potentially lead to adverse outcomes.

Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1.

UNLABELLED: Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (ß-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the ß-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human ß-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of α-CoVs and ß-CoVs in groups B and C bind to their specific receptor proteins. Thus, two ß-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins. IMPORTANCE: Mouse hepatitis virus, a ß-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another ß-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory ß-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C domain neutralized virus infectivity. Thus, the receptor-binding domain of HKU1 spike glycoprotein is located in the C domain. Surprisingly, two ß-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.

Behavioral changes following a single episode of early-life seizures support the latent development of an autistic phenotype.

We probed the developmental and behavioral consequences of a single episode of kainic acid-induced early-life seizures (KA-ELS) in the rat on postnatal day 7. Correlates of developmental trajectory were not altered, demonstrating that long-term consequences following KA-ELS are not initiated by secondary causes, such as malnourishment or alterations in maternal care. We report reduced marble burying in adult rats, suggestive of restricted interests, a trait common to experimental and clinical autism. We did not detect increased repetitive grooming during habituated cage behavior. However, we did detect reduced grooming in adult KA-ELS rats in the presence of an unfamiliar rat, supporting altered social anxiety following KA-ELS. Reanalysis of a social approach task further indicated abnormal social interactions. Taken together with previous physiological and behavioral data, these data support the hypothesis that KA-ELS lead to a latent autistic phenotype in adult rats not attributable to other early alterations in development.

Phosphorylation of FMRP and alterations of FMRP complex underlie enhanced mLTD in adult rats triggered by early life seizures.

Outside of Fragile X syndrome (FXS), the role of Fragile-X Mental Retardation Protein (FMRP) in mediating neuropsychological abnormalities is not clear. FMRP, p70-S6 kinase (S6K) and protein phosphatase 2A (PP2A) are thought to cooperate as a dynamic signaling complex. In our prior work, adult rats have enhanced CA1 hippocampal long-term depression (LTD) following an early life seizure (ELS). We now show that mGluR-mediated LTD (mLTD) is specifically enhanced following ELS, similar to FMRP knock-outs. Total FMRP expression is unchanged but S6K is hyperphosphorylated, consistent with S6K overactivation. We postulated that either disruption of the FMRP-S6K-PP2A complex and/or removal of this complex from synapses could explain our findings. Using subcellular fractionation, we were surprised to find that concentrations of FMRP and PP2A were undisturbed in the synaptosomal compartment but reduced in parallel in the cytosolic compartment. Following ELS FMRP phosphorylation was reduced in the cytosolic compartment and increased in the synaptic compartment, in parallel with the compartmentalization of S6K activation. Furthermore, FMRP and PP2A remain bound following ELS. In contrast, the interaction of S6K with FMRP is reduced by ELS. Blockade of PP2A results in enhanced mLTD; this is occluded by ELS. This suggests a critical role for the location and function of the FMRP-S6K-PP2A signaling complex in limiting the amount of mLTD. Specifically, non-synaptic targeting and the function of the complex may influence the "set-point" for regulating mLTD. Consistent with this, striatal-enriched protein tyrosine phosphatase (STEP), an FMRP "target" which regulates mLTD expression, is specifically increased in the synaptosomal compartment following ELS. Further, we provide behavioral data to suggest that FMRP complex dysfunction may underlie altered socialization, a symptom associated and observed in other rodent models of autism, including FXS.

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin-dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual, and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD has indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3ß, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3ß activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3ß activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3ß. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces postsynaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity, and human neuropathology.

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 ( CDKL5 ) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD have indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3b, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3b activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3b activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3b. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces post-synaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated, key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity and human neuropathology.

The CaV1.2 G406R mutation decreases synaptic inhibition and alters L-type Ca2+ channel-dependent LTP at hippocampal synapses in a mouse model of Timothy Syndrome.

Genetic alterations in autism spectrum disorders (ASD) frequently disrupt balance between synaptic excitation and inhibition and alter plasticity in the hippocampal CA1 region. Individuals with Timothy Syndrome (TS), a genetic disorder caused by CaV1.2 L-type Ca2+ channel (LTCC) gain-of function mutations, such as G406R, exhibit social deficits, repetitive behaviors, and cognitive impairments characteristic of ASD that are phenocopied in TS2-neo mice expressing G406R. Here, we characterized hippocampal CA1 synaptic function in male TS2-neo mice and found basal excitatory transmission was slightly increased and inhibitory transmission strongly decreased. We also found distinct impacts on two LTCC-dependent forms of long-term potentiation (LTP) synaptic plasticity that were not readily consistent with LTCC gain-of-function. LTP induced by high-frequency stimulation (HFS) was strongly impaired in TS2-neo mice, suggesting decreased LTCC function. Yet, CaV1.2 expression, basal phosphorylation, and current density were similar for WT and TS2-neo. However, this HFS-LTP also required GABAA receptor activity, and thus may be impaired in TS2-neo due to decreased inhibitory transmission. In contrast, LTP induced in WT mice by prolonged theta-train (PTT) stimulation in the presence of a ß-adrenergic receptor agonist to increase CaV1.2 phosphorylation was partially induced in TS2-neo mice by PTT stimulation alone, consistent with increased LTCC function. Overall, our findings provide insights regarding how altered CaV1.2 channel function disrupts basal transmission and plasticity that could be relevant for neurobehavioral alterations in ASD.

Characterization of a ViI-like phage specific to Escherichia coli O157:H7.

Phage vB_EcoM_CBA120 (CBA120), isolated against Escherichia coli O157:H7 from a cattle feedlot, is morphologically very similar to the classic phage ViI of Salmonella enterica serovar Typhi. Until recently, little was known genetically or physiologically about the ViI-like phages, and none targeting E. coli have been described in the literature. The genome of CBA120 has been fully sequenced and is highly similar to those of both ViI and the Shigella phage AG3. The core set of structural and replication-related proteins of CBA120 are homologous to those from T-even phages, but generally are more closely related to those from T4-like phages of Vibrio, Aeromonas and cyanobacteria than those of the Enterobacteriaceae. The baseplate and method of adhesion to the host are, however, very different from those of either T4 or the cyanophages. None of the outer baseplate proteins are conserved. Instead of T4's long and short tail fibers, CBA120, like ViI, encodes tail spikes related to those normally seen on podoviruses. The 158 kb genome, like that of T4, is circularly permuted and terminally redundant, but unlike T4 CBA120 does not substitute hmdCyt for cytosine in its DNA. However, in contrast to other coliphages, CBA120 and related coliphages we have isolated cannot incorporate 3H-thymidine (3H-dThd) into their DNA. Protein sequence comparisons cluster the putative "thymidylate synthase" of CBA120, ViI and AG3 much more closely with those of Delftia phage φW-14, Bacillus subtilis phage SPO1, and Pseudomonas phage YuA, all known to produce and incorporate hydroxymethyluracil (hmdUra).

Transcriptome analysis of rat dorsal hippocampal CA1 after an early life seizure induced by kainic acid.

Seizures that occur during early development are associated with adverse neurodevelopmental outcomes. Causation and mechanisms are currently under investigation. Induction of an early life seizure by kainic acid (KA) in immature rats on post-natal day (P) 7 results in behavioral changes in the adult rat that reflect social and intellectual deficits without overt cellular damage. Our previous work also demonstrated increased expression of CA1 hippocampal long-term potentiation (LTP) and reduced desensitization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic glutamate receptors (AMPA-R) one week following a kainic acid induced seizure (KA-ELS). Here we used RNA sequencing (RNAseq) of mRNA from dorsal hippocampal CA1 to probe changes in mRNA levels one week following KA-ELS as a means to investigate the mechanisms for these functional changes. Ingenuity pathway analysis (IPA) confirmed our previous results by predicting an up-regulation of the synaptic LTP pathway. Differential gene expression results revealed significant differences in 7 gene isoforms. Additional assessments included AMPA-R splice variants and adenosine deaminase acting on RNA 2 (ADAR2) editing sites as a means to determine the mechanism for reduced AMPA-R desensitization. Splice variant analysis demonstrated that KA-ELS result in a small, but significant decrease in the "flop" isoform of Gria3, and editing site analysis revealed significant changes in the editing of a kainate receptor subunit, Grik2, and a serotonin receptor, Htr2c. While these specific changes may not account for altered AMPA-R desensitization, the differences indicate that KA-ELS alters gene expression in the hippocampal CA1 one week after the insult.

Necessary, but not sufficient: insights into the mechanisms of mGluR mediated long-term depression from a rat model of early life seizures.

Using the rat model of early life seizures (ELS), which has exaggerated mGluR mediated long-term depression of synaptic strength (mGluR-LTD) in adulthood, we probed the signaling cascades underlying mGluR-LTD induction. Several inhibitors completely blocked mGluR-LTD in control but not in ELS rats: the proteasome, the mammalian target of rapamycin (mTOR), S6 kinase (S6K), or L-type voltage-gated calcium channels (L-type VGCC). Inhibition of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) resulted in a near complete block of mGluR-LTD in control rats and a slight reduction of mGluR-LTD in ELS rats. "Autonomous" CaMKII was found to be upregulated in ELS rats, while elevated S6K activity, which is stimulated by mTOR, was described previously. Thus, modulation of each of these factors was necessary for mGluR-LTD induction in control rats, but even their combined, permanent activation in the ELS rats was not sufficient to individually support mGluR-LTD induction following ELS. This implies that while these factors may act sequentially in controls to mediate mGluR-LTD, this is no longer the case after ELS. In contrast, activated ERK was found to be significantly down-regulated in ELS rats. Inhibition of MEK/ERK activation in control rats elevated mGluR-LTD to the exaggerated levels seen in ELS rats. Together, these results elucidate both the mechanisms that persistently enhance mGluR-LTD after ELS and the mechanisms underlying normal mGluR-LTD by providing evidence for multiple, convergent pathways that mediate mGluR-LTD induction. With our prior work, this ties these signaling cascades to the ELS behavioral phenotype that includes abnormal working memory, fear conditioning and socialization.