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.

IMPORTANCEMouse hepatitis virus, a ␤-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another ␤-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 ␤-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 ␤-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors. C oronaviruses (CoVs) primarily cause respiratory and enteric diseases in humans, animals, and birds, and some CoVs also cause systemic diseases, including hepatitis or neurological diseases (1). Since the 2002-2003 epidemic of severe acute respiratory syndrome (SARS), intensive surveillance of humans and animals has led to the discovery of numerous other CoVs (2, 3). Phylogenetically, CoVs now are divided into four genera, called the ␣-, ␤-, ␥-, and ␦-CoVs (4). Currently there are six CoVs known to infect humans: two ␣-CoVs, 229E and NL63; two ␤-CoVs in group A, OC43 and HKU1; one ␤-CoV in group B, SARS-CoV; and one ␤-CoV in group C, Middle East respiratory syndrome coronavirus (MERS-CoV), that currently is causing an epidemic with an ϳ30% fatality rate (5-12). While the first four of these human CoVs circulate only in humans and predominately cause mild respiratory diseases, SARS-CoV and MERS-CoV are zoonoses associated with episodically emerging epidemics of severe respiratory infection, including pneumonia, the acute respiratory distress syndrome (ARDS), and death in about 10% to 30% of cases (12,13).The large spikes on the envelope of CoV virions consist of trimers of the ϳ200-kDa spike (S) glycoprotein that bind to hostspecific receptors; mediate virus entry, tissue tropism, and host range; and can affect virus virulence. S protein is the target for CoV neutralizing antibodies and is an essential component of CoV vaccines and vaccine candidates. CoV S proteins are class I viral fusion proteins, like influenza virus hemagglutinin (HA), HIV Env, Ebola virus G, and paramyxovirus F glycoproteins (14). CoV S proteins contain two subunits, called S1 and S2, which are

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).

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 Ca2+/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.