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Objective The principal molecular targets of conventional antiseizure drugs consist of ligand-gated and voltage-gated ion channels and proteins subserving synaptic function. Inhibition of the receptor tyrosine kinase TrkB limits epileptogenesis, but its effect on individual seizures is unknown. We sought to determine whether inhibition of TrkB kinase exerts an antiseizure effect. Methods We utilized the kindling model in combination with an inducible conditional knockout of the TrkB gene ( Act-Cre ER TrkB flox/flox mice treated with tamoxifen), and also with a chemical-genetic approach in which mice carry a TrkB kinase with a phenylalanine to alanine substitution of residue 616 ( Trk B F 616A), which allows inhibition of the kinase by a blood-brain barrier permeable small molecule, 1′-naphthylmethyl-4-amino-1- tert-butyl-3-( p-methylphenyl)pyrazolo[3,4- d]pyrimidine (1 NMPP1). Results Following induction of kindling, reduction of TrkB protein levels in Act-Cre ER TrkB flox/flox mice treated with tamoxifen was associated with reduced severity of behavioral seizures evoked by stimulation. Treatment with 1 NMPP1 for 2 weeks following induction of kindling reversibly elevated both focal electrographic and generalized seizure thresholds in Trk B F 616A , but not wild-type ( WT), mice. In contrast to kindled animals, treatment of naive Trk B F 616A mice for 2 weeks had no detectable effect on electrographic seizure threshold ( EST). Significance This study provides proof of concept of a novel molecular target for antiseizure drugs, namely the receptor tyrosine kinase TrkB. A PowerPoint slide summarizing this article is available for download in the Supporting Information section . [ABSTRACT FROM AUTHOR]

Objective Elucidating molecular mechanisms underlying limbic epileptogenesis may reveal novel targets for preventive therapy. Studies of Trk B mutant mice led us to hypothesize that signaling through a specific phospholipase ( PLC), PLCγ1, promoted development of kindling. Methods To test this hypothesis, we examined the development of kindling in PLCγ1 heterozygous mice. We also examined the cellular and subcellular location of PLCγ1 in adult wild-type mice. Results The development of kindling was impaired in PLCγ1 heterozygous mice compared to wild-type controls. PLCγ1 immunoreactivity was localized to the soma and dendrites of both excitatory and inhibitory neurons in the hippocampus of adult mice. Significance This study implicates PLCγ1 signaling as the dominant pathway by which Trk B activation promotes limbic epileptogenesis. Its cellular localization places PLCγ1 in a position to modify the efficacy of both excitatory and inhibitory synaptic transmission. These findings advance PLCγ1 as a novel target for therapies aimed at preventing temporal lobe epilepsy induced by status epilepticus. [ABSTRACT FROM AUTHOR]

ABSTRACT Temporal lobe epilepsy is associated with changes in the morphology of hippocampal dentate granule cells. These changes are evident in numerous models that are associated with substantial neuron loss and spontaneous recurrent seizures. By contrast, previous studies have shown that in the kindling model, it is possible to administer a limited number of stimulations sufficient to produce a lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous seizures and minimal neuronal loss. Here we examined whether stimulation of the amygdala sufficient to evoke five convulsive seizures (class IV or greater on Racine's scale) produce morphological changes similar to those observed in models of epilepsy associated with substantial cell loss. The morphology of GFP-expressing granule cells from Thy-1 GFP mice was examined either 1 day or 1 month after the last evoked seizure. Interestingly, significant reductions in dendritic spine density were evident 1 day after the last seizure, the magnitude of which had diminished by 1 month. Further, there was an increase in the thickness of the granule cell layer 1 day after the last evoked seizure, which was absent a month later. We also observed an increase in the area of the proximal axon, which again returned to control levels a month later. No differences in the number of basal dendrites were detected at either time point. These findings demonstrate that the early stages of kindling epileptogenesis produce transient changes in the granule cell body layer thickness, molecular layer spine density, and axon proximal area, but do not produce striking rearrangements of granule cell structure. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Epilepsy Aphasia Syndromes (EAS) are a spectrum of childhood epileptic, cognitive, and language disorders of unknown etiology. CNKSR2 is a strong X-linked candidate gene implicated in EAS; however, there have been no studies of genetic models to dissect how its absence may lead to EAS. Here we develop a novel Cnksr2 KO mouse line and show that male mice exhibit increased neural activity and have spontaneous electrographic seizures. Cnksr2 KO mice also display significantly increased anxiety, impaired learning and memory, and a progressive and dramatic loss of ultrasonic vocalizations. We find that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at both excitatory and inhibitory postsynapses. Proteomics analysis reveals Cnksr2 anchors key binding partners at synapses, and its loss results in significant alterations of the synaptic proteome, including proteins implicated in epilepsy disorders. Our results validate that loss of CNKSR2 leads to EAS and highlights the roles of Cnksr2 in synaptic organization and neuronal network activity. [ABSTRACT FROM AUTHOR]

Objective: Temporal lobe epilepsy (TLE) is a devastating disease in which seizures persist in 35% of patients despite optimal use of antiseizure drugs. Clinical and preclinical evidence implicates seizures themselves as one factor promoting epilepsy progression. What is the molecular consequence of a seizure that promotes progression? Evidence from preclinical studies led us to hypothesize that activation of tropomyosin kinase B (TrkB)-phospholipase-C-gamma-1 (PLCγ1) signaling induced by a seizure promotes epileptogenesis.Methods: To examine the effects of inhibiting TrkB signaling on epileptogenesis following an isolated seizure, we implemented a modified kindling model in which we induced a seizure through amygdala stimulation and then used either a chemical-genetic strategy or pharmacologic methods to disrupt signaling for 2 days following the seizure. The severity of a subsequent seizure was assessed by behavioral and electrographic measures.Results: Transient inhibition of TrkB-PLCγ1 signaling initiated after an isolated seizure limited progression of epileptogenesis, evidenced by the reduced severity and duration of subsequent seizures. Unexpectedly, transient inhibition of TrkB-PLCγ1 signaling initiated following a seizure also reverted a subset of animals to an earlier state of epileptogenesis. Remarkably, inhibition of TrkB-PLCγ1 signaling in the absence of a recent seizure did not reduce severity of subsequent seizures.Interpretation: These results suggest a novel strategy for limiting progression or potentially ameliorating severity of TLE whereby transient inhibition of TrkB-PLCγ1 signaling is initiated following a seizure. ANN NEUROL 2019;86:939-950. [ABSTRACT FROM AUTHOR]

Temporal lobe epilepsy (TLE) is a common and commonly devastating form of human epilepsy for which only symptomatic therapy is available. One cause of TLE is an episode of de novo prolonged seizures [status epilepticus (SE)J. Understanding the molecular signaling mechanisms by which SE transforms a brain from normal to epileptic may reveal novel targets for preventive and disease-modifying therapies. SE-induced activation of the BDNF receptor tyrosine kinase, TrkB, is one signaling pathway by which SE induces TLE. Although activation of TrkB signaling promotes development of epilepsy in this context, it also reduces SE-induced neuronal death. This led us to hypothesize that distinct signaling pathways downstream of TrkB mediate the desirable (neuroprotective) and undesirable (epileptogenesis) consequences. We subsequently demonstrated that TrkB-mediated activation of phospholipase Cyl is required for epileptogenesis. Here we tested the hypothesis that the TrkB-Shc-Akt signaling pathway mediates the neuroprotective consequences of TrkB activation following SE. We studied measures of molecular signaling and cell death in a model of SE in mice of both sexes, including wild-type and TrkBshdShc mutant mice in which a point mutation (Y515F) of TrkB prevents the binding of She to activated TrkB kinase. Genetic disruption of TrkB-Shc signaling had no effect on severity of SE yet partially inhibited activation of the prosurvival adaptor protein Akt. Importantly, genetic disruption of TrkB-Shc signaling exacerbated hippocampal neuronal death induced by SE. We conclude that therapies targeting TrkB signaling for preventing epilepsy should spare TrkB-Shc-Akt signaling and thereby preserve the neuroprotective benefits. [ABSTRACT FROM AUTHOR]

The lack of preventive and disease modifying therapies for temporal lobe epilepsy (TLE) is a major unmet medical need. Search for such therapies utilize mouse models and require detection of seizures in electroencephalography (EEG) recordings. The labor-intensive nature of reviewing EEGs spanning many weeks underscores the need for a method of automated detection. Here we report a simple automated method of detecting seizures in long term EEG recordings from electrodes implanted in the hippocampus in animal models of TLE. We utilize a 2-pronged approach that relies on the increase in power within the gamma band range (20–50hz) during the seizure followed by suppression of activity following the seizure (post-ictal suppression [PIS]). We demonstrate the utility of this method for detecting seizures in hippocampal and amygdala EEG recordings from multiple models of TLE. • Seizure detection in mouse models of epilepsy is essential for assessing putative preventive and disease modifying therapies. • The gold standard method has been analyses of EEG recordings by skilled humans, a labor intensive and time-consuming task. • We validated a simple, automated method for detecting seizures in long term recordings of awake, tethered adult mice in five models. • This method will be freely available to all investigators and easily adaptable across different software platforms. [ABSTRACT FROM AUTHOR]

Vagal Nerve Stimulation ( VNS) Therapy® is a United States Food and Drug Administration approved neurotherapeutic for medically refractory partial epilepsy and treatment-resistant depression. The molecular mechanisms underlying its beneficial effects are unclear. We hypothesized that one mechanism involves neuronal activity-dependent modifications of central nervous system excitatory synapses. To begin to test this hypothesis, we asked whether VNS modifies the activity of neurons in amygdala and hippocampus. Neuronal recordings from adult, freely moving rats revealed that activity in both amygdala and hippocampus was modified by VNS immediately after its application, and changes were detected following 1 week of stimulation. To investigate whether VNS modifies the proteome of excitatory synapses, we established a label-free, quantitative liquid chromatography-tandem mass spectrometry workflow that enables global analysis of the constituents of the postsynaptic density ( PSD) proteome. PSD proteins were biochemically purified from amygdala/piriform cortex of VNS- or dummy-treated rats following 1-week stimulation, and individual PSD protein levels were quantified by liquid chromatography-tandem mass spectrometry analysis. We identified 1899 unique peptides corresponding to 425 proteins in PSD fractions, of which expression levels of 22 proteins were differentially regulated by VNS with changes greater than 150%. Changes in a subset of these proteins, including significantly increased expression of neurexin-1α, cadherin 13 and voltage-dependent calcium channel α2δ1, the primary target of the antiepileptic drug gabapentin, and decreased expression of voltage-dependent calcium channel γ3, were confirmed by western blot analysis of PSD samples. These results demonstrate that VNS modulates excitatory synapses through regulating a subset of the PSD proteome. Our study reveals molecular targets of VNS and point to possible mechanisms underlying its beneficial effects, including activity-dependent formation of excitatory synapses. [ABSTRACT FROM AUTHOR]

Mutation of fibroblast growth factor 13 ( FGF13) has recently been implicated in genetic epilepsy with febrile seizures plus (GEFS+) in a single family segregating a balanced translocation with a breakpoint in this X chromosome gene, predicting a partial knockout involving 3 of 5 known FGF13 isoforms. Investigation of a mouse model of complete Fgf13 knock-out revealed increased susceptibility to hyperthermia-induced seizures and epilepsy. Here we investigated whether mutation of FGF13 would explain other cases of GEFS+ compatible with X-linked inheritance. We screened the coding and splice site regions of the FGF13 gene in a sample of 45 unrelated probands where GEFS+ segregated in an X-linked pattern. We subsequently identified a de novo FGF13 missense variant in an additional patient with febrile seizures and facial edema. Our data suggests FGF13 is not a common cause of GEFS+. [ABSTRACT FROM AUTHOR]

Summary The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans. [ABSTRACT FROM AUTHOR]

We identified a family in which a translocation between chromosomes X and 14 was associated with cognitive impairment and a complex genetic disorder termed "Genetic Epilepsy and Febrile Seizures Plus" (GEFS+). We demonstrate that the breakpoint on the X chromosome disrupted a gene that encodes an auxiliary protein of voltage-gated Na+ channels, fibroblast growth factor 13 (Fgf13). Female mice in which one Fgf13 allele was deleted exhibited hyperthermia-induced seizures and epilepsy. Anatomic studies revealed expression of Fgf13 mRNA in both excitatory and inhibitory neurons of hippocampus. Electrophysiological recordings revealed decreased inhibitory and increased excitatory synaptic inputs in hippocampal neurons of Fgf13 mutants. We speculate that reduced expression of Fgf13 impairs excitability of inhibitory interneurons, resulting in enhanced excitability within local circuits of hippocampus and the clinical phenotype of epilepsy. These findings reveal a novel cause of this syndrome and underscore the powerful role of FGF13 in control of neuronal excitability. [ABSTRACT FROM AUTHOR]

Technologies that enable the rapid detection and localization of bacterial infections in living animals could address an unmet need for infectious disease diagnostics. We describe a molecular imaging approach for the specific, noninvasive detection of S. aureus based on the activity of the S. aureus secreted nuclease, micrococcal nuclease (MN). Several short synthetic oligonucleotides, rendered resistant to mammalian serum nucleases by various chemical modifications and flanked with a fluorophore and quencher, were activated upon degradation by purified MN and in S. aureus culture supernatants. A probe consisting of a pair of deoxythymidines flanked by several 2′-O-methyl-modified nucleotides was activated in culture supernatants of S. aureus but not in culture supernatants of several other pathogenic bacteria. Systemic administration of this probe to mice bearing S. aureus muscle infections resulted in probe activation at the infection sites in an MN-dependent manner. This new bacterial imaging approach has potential clinical applicability for infections with S. aureus and several other medically important pathogens. [ABSTRACT FROM AUTHOR]

Summary: Temporal lobe epilepsy is the most common and often devastating form of human epilepsy. The molecular mechanism underlying the development of temporal lobe epilepsy remains largely unknown. Emerging evidence suggests that activation of the BDNF receptor TrkB promotes epileptogenesis caused by status epilepticus. We investigated a mouse model in which a brief episode of status epilepticus results in chronic recurrent seizures, anxiety-like behavior, and destruction of hippocampal neurons. We used a chemical-genetic approach to selectively inhibit activation of TrkB. We demonstrate that inhibition of TrkB commencing after status epilepticus and continued for 2 weeks prevents recurrent seizures, ameliorates anxiety-like behavior, and limits loss of hippocampal neurons when tested weeks to months later. That transient inhibition commencing after status epilepticus can prevent these long-lasting devastating consequences establishes TrkB signaling as an attractive target for developing preventive treatments of epilepsy in humans. [ABSTRACT FROM AUTHOR]

The somatodendritic IA (A-type)K+ current underlies neuronal excitability, and loss of IA has been associated with the development of epilepsy. Whether any one of the four auxiliary potassium channel interacting proteins (KChIPs), KChIP1-KChIP4, in specific neuronal populations is critical for IA is not known. Here we show that KChIP2, which is abundantly expressed in hippocampal pyramidal cells, is essential for IA regulation in hippocampal neurons and that deletion of Kchip2 affects susceptibility to limbic seizures. The specific effects of Kchip2 deletion on IA recorded from isolated hippocampal pyramidal neurons were a reduction in amplitudeandshift in theV1/2 for steady-state inactivation to hyperpolarized potentialswhencompared withWTneurons. Consistent with the relative loss of IA, hippocampal neurons from Kchip2-/- mice showed increased excitability. WT cultured neurons fired only occasional single action potentials, but the average spontaneous firing rate (spikes/s) was almost 10-fold greater in Kchip2-/- neurons. In slice preparations, spontaneous firing was detected in CA1 pyramidal neurons from Kchip2-/- mice but not from WT. Additionally, when seizures were induced by kindling, the number of stimulations required to evoke an initial class 4 or 5 seizure was decreased, and the average duration of electrographic seizures was longer in Kchip2-/- mice compared withWTcontrols. Together, these data demonstrate that theKChIP2is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons. [ABSTRACT FROM AUTHOR]

Summary: The neurotrophin receptor, tropomyosin-related kinase B (TrkB), is required for epileptogenesis in the kindling model. The role of a closely related neurotrophin receptor, TrkC, in limbic epileptogenesis is unknown. We examined limbic epileptogenesis in the kindling model in TrkC conditional null mice, using a strategy that previously established a critical role of TrkB. Despite elimination of TrkC mRNA, no differences in development of kindling were detected between TrkC conditional null and wild type control mice. These findings reinforce the central role of TrkB as the principal neurotrophin receptor involved in limbic epileptogenesis. [Copyright &y& Elsevier]

Summary: The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits a form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions, vesicular zinc is critical to proper function of hippocampal circuitry in health and disease. [ABSTRACT FROM AUTHOR]

Abstract: Exogenous brain-derived neurotrophic factor (BDNF) can regulate behavioral sensitization and conditioned place preference (CPP) when animals are exposed to repeated cocaine administration. However, it is unclear whether BDNF signaling through the TrkB receptor can mediate these behavioral responses when animals are given a single cocaine exposure. Because TrkB knockout mice die as neonates, we engineered a transgenic mouse that expressed a dominant negative form of TrkB (dnTrkB) in a conditional and reversible manner. We assessed also activation of endogenous TrkB by quantifying levels of phosphorylated TrkB (p-TrkB) in the nucleus accumbens (NAc). We found that a single exposure to cocaine was sufficient to increase p-TrkB within the NAc 9–12h after administration. Expression of the dnTrkB transgene not only prevented the acute cocaine-induced increase in p-TrkB, but it also prevented behavioral sensitization and CPP following a single cocaine injection. These findings demonstrate that TrkB activation is required both for behavioral sensitization and CPP to a single cocaine exposure. The fact that enhanced TrkB activation is induced within 9h of a single injection of cocaine suggests that inhibition of TrkB signaling commencing hours after cocaine exposure may prevent at least the initial antecedents to the sensitizing and reinforcing effects of this psychostimulant. [Copyright &y& Elsevier]

Summary: Examining the behavioral consequences of selective CNS neuronal activation is a powerful tool for elucidating mammalian brain function in health and disease. Newly developed genetic, pharmacological, and optical tools allow activation of neurons with exquisite spatiotemporal resolution; however, the inaccessibility to light of widely distributed neuronal populations and the invasiveness required for activation by light or infused ligands limit the utility of these methods. To overcome these barriers, we created transgenic mice expressing an evolved G protein-coupled receptor (hM3Dq) selectively activated by the pharmacologically inert, orally bioavailable drug clozapine-N-oxide (CNO). Here, we expressed hM3Dq in forebrain principal neurons. Local field potential and single-neuron recordings revealed that peripheral administration of CNO activated hippocampal neurons selectively in hM3Dq-expressing mice. Behavioral correlates of neuronal activation included increased locomotion, stereotypy, and limbic seizures. These results demonstrate a powerful chemical-genetic tool for remotely controlling the activity of discrete populations of neurons in vivo. [Copyright &y& Elsevier]

Dentate granule cells play a critical role in the function of the entorhinal-hippocampal circuitry in health and disease. Dentate granule cells are situated to regulate the flow of information into the hippocampus, a structure required for normal learning and memory. Correspondingly, impaired granule cell function leads to memory deficits, and, interestingly, altered granule cell connectivity may contribute to the hyperexcitability of limbic epilepsy. It is important, therefore, to understand the molecular determinants of synaptic connectivity of these neurons. Brain-derived neurotrophic factor and its receptor TrkB are expressed at high levels in the dentate gyrus (DG) of the hippocampus, and are implicated in regulating neuronal development, neuronal plasticity, learning, and the development of epilepsy. Whether and how TrkB regulates granule cell structure, however, is incompletely understood. To begin to elucidate the role of TrkB in regulating granule cell morphology, here we examine conditional TrkB knockout mice crossed to mice expressing green fluorescent protein in subsets of dentate granule cells. In stratum lucidum, where granule cell mossy fiber axons project, the density of giant mossy fiber boutons was unchanged, suggesting similar output to CA3 pyramidal cell targets. However, filopodial extensions of giant boutons, which contact inhibitory interneurons, were increased in number in TrkB knockout mice relative to wildtype controls, predicting enhanced feedforward inhibition of CA3 pyramidal cells. In knockout animals, dentate granule cells possessed fewer primary dendrites and enlarged dendritic spines, indicative of disrupted excitatory synaptic input to the granule cells. Together, these findings demonstrate that TrkB is required for development and/or maintenance of normal synaptic connectivity of the granule cells, thereby implying an important role for TrkB in the function of the granule cells and hippocampal circuitry. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Summary: The receptor tyrosine kinase, TrkB, is critical to diverse functions of the mammalian nervous system in health and disease. Evidence of TrkB activation during epileptogenesis in vivo despite genetic deletion of its prototypic neurotrophin ligands led us to hypothesize that a non-neurotrophin, the divalent cation zinc, can transactivate TrkB. We found that zinc activates TrkB through increasing Src family kinase activity by an activity-regulated mechanism independent of neurotrophins. One subcellular locale at which zinc activates TrkB is the postsynaptic density of excitatory synapses. Exogenous zinc potentiates the efficacy of the hippocampal mossy fiber (mf)-CA3 pyramid synapse by a TrkB-requiring mechanism. Long-term potentiation of this synapse is impaired by deletion of TrkB, inhibition of TrkB kinase activity, and by CaEDTA, a selective chelator of zinc. The activity-dependent activation of synaptic TrkB in a neurotrophin-independent manner provides a mechanism by which this receptor can regulate synaptic plasticity. [Copyright &y& Elsevier]

4-1BB is a major costimulatory receptor that promotes the survival and expansion of activated T cells. Administration of agonistic anti-4-1BB Abs has been previously shown to enhance tumor immunity in mice. Abs are cell-based products posing significant cost, manufacturing, and regulatory challenges. Aptamers are oligonucleotide-based ligands that exhibit specificity and avidity comparable to, or exceeding, that of Abs. To date, various aptamers have been shown to inhibit the function of their cognate target. Here, we have described the development of an aptamer that binds 4-1BB expressed on the surface of activated mouse T cells and shown that multivalent configurations of the aptamer costimulated T cell activation in vitro and mediated tumor rejection in mice. Because aptamers can be chemically synthesized, manufacturing and the regulatory approval process should be substantially simpler and less costly than for Abs. Agonistic aptamers could therefore represent a superior alternative to Abs for the therapeutic manipulation of the immune system. [ABSTRACT FROM AUTHOR]

Abstract: αvβ3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. αvβ3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of αvβ3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-αvβ3 that binds recombinant αvβ3 integrin, for its ability to bind endogenous αvβ3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-αvβ3 binds αvβ3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-αvβ3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-αvβ3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-αvβ3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation. [Copyright &y& Elsevier]

Epileptogenesis is the process whereby a normal brain becomes epileptic. We hypothesized that the neurotrophin brain-derived neurotrophic factor (BDNF) activates its receptor, TrkB, in the hippocampus during epileptogenesis and that BDNF-mediated activation of TrkB is required for epileptogenesis. We tested these hypotheses in Synapsin-Cre conditional BDNF-/- and TrkB-/- mice using the kindling model. Despite marked reductions of BDNF expression, only a modest impairment of epileptogenesis and increased hippocampal TrkB activation were detected in BDNF-/- mice. In contrast, reductions of electrophysiological measures and no behavioral evidence of epileptogenesis were detected in TrkB-/- mice. Importantly, TrkB-/- mice exhibited behavioral endpoints of epileptogenesis, tonic-clonic seizures. Whereas TrkB can be activated, and epileptogenesis develops in BDNF-/- mice, the plasticity of epileptogenesis is eliminated in TrkB-/- mice. Its requirement for epileptogenesis in kindling implicates TrkB and downstream signaling pathways as attractive molecular targets for drugs for preventing epilepsy. [Copyright &y& Elsevier]

We set out to establish a novel model of temporal lobe epilepsy (TLE) in a mouse. We sought to induce TLE through the injection of kainic acid (KA) into the tail vein with subsequent development of status epilepticus (SE). Using C57BL/6 mice, we implanted hippocampal EEG recording electrodes before or after injection of KA or phosphate buffered saline (PBS). Video and EEG analysis were conducted to evaluate for SE and development of recurrent seizures, the hallmark of TLE. All mice injected with KA developed SE while those who were injected with PBS did not. Of the animals injected with KA monitored for recurrent seizures following SE, 33% developed spontaneous recurrent seizures while those injected with PBS did not. Injection of KA through the tail vein of a mouse reliably and rapidly induces SE which remits spontaneously and leads to the development of TLE in a subset of mice. • Injection of kainic acid in a mouse via the tail vein rapidly and reliably induces status epilepticus. • Systemic injection of kainic acid via the tail vein in mice shows heterogeneity in SE and seizure development. • Surgical implantations of EEG electrodes prior to kainic acid infusion increases mortality. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) and status epilepticus (SE) have both been linked to development of human epilepsy. Although distinct etiologies, current research has suggested the convergence of molecular mechanisms underlying epileptogenesis following these insults. One such mechanism involves the neurotrophin brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyosin related kinase B (TrkB). In this review, we focus on currently available data regarding the pathophysiologic role of BDNF/TrkB signaling in epilepsy development. We specifically examine the axonal injury and SE epilepsy models, two animal models that recapitulate many aspects of TBI- and SE-induced epilepsy in humans respectively. Thereafter, we discuss aspiring strategies for targeting BDNF/TrkB signaling so as to prevent epilepsy following an insult or suppress its expression once developed. This article is part of the special issue entitled 'New Epilepsy Therapies for the 21st Century – From Antiseizure Drugs to Prevention, Modification and Cure of Epilepsy'. • BDNF/TrkB signaling plays a pathophysiologic role in TBI- and SE-induced epilepsies. • Contribution of BDNF/TrkB signaling to epileptogenesis in axonal transection models is opposite in lesions of neocortex versus hippocampus. • Peptide inhibitor is a valuable tool for inhibiting BDNF/TrkB signaling. [ABSTRACT FROM AUTHOR]

Purpose: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the 'smart-activatable' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus., Methods: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria., Results: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min., Conclusion: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials., (© 2023. The Author(s).)

Competing Interests: Declaration of interests J.O.M. is the founder and CEO of Nuclease Probe Technologies, Inc., a molecular diagnostics startup company in Lowell, MA (USA). P.H.G. is an employee of Eleven Therapeutics, Inc.; she was previously employed at Moderna (Division of Rare Diseases).

Insight into the cellular and circuit mechanisms underlying development of temporal lobe epilepsy (TLE) will provide a foundation for improved therapies. We studied a model in which an episode of prolonged seizures is followed by recovery lasting two weeks before emergence of spontaneous recurrent seizures. We focused on the interval between the prolonged seizures and the late onset recurrent seizures. We investigated the hippocampal mossy fiber CA3 pyramidal cell microcircuit in models spanning in vitro , in vivo , and ex vivo preparations. Expression of channelrhodopsin-2 in the dentate granule cells of DGC ChR mice enabled the selective activation of mossy fiber axons. In vivo studies revealed marked potentiation of mossy fiber evoked field potentials in hippocampal CA3 beginning within hours following seizures, a potentiation which persisted at least 7 d. Stimulation of mossy fibers in hippocampal slices in vitro using patterns of activity mimicking seizures induced LTP not only of the monosynaptic EPSC but also of the disynaptic IPSC of CA3 pyramidal cells. Ex vivo studies of slices isolated following seizures revealed evidence of LTP of mossy fiber evoked EPSC and disynaptic IPSC of CA3 pyramidal cells. We suggest that activation of dentate granule cells during seizures induces these plasticities in vivo and the retained balance of synaptic excitation and inhibition limits excessive activation of CA3 pyramidal cells, thereby protecting animals from spontaneous recurrent seizures at this interval following status epilepticus., (Copyright © 2022 Pan et al.)

Staphylococcus aureus bacteraemia (SAB) is associated with high mortality and morbidity rates. Yet, there is currently no adequate diagnostic test for early and rapid diagnosis of SAB. Therefore, this study was aimed at exploring the potential for clinical implementation of a nuclease-activatable fluorescent probe for early diagnosis of SAB. To this end, clinical blood culture samples from patients with bloodstream infections were incubated for 1 h with the "smart" activatable P2&3TT probe, the total assay time being less than 2 h. Cleavage of this probe by the secreted S. aureus enzyme micrococcal nuclease results in emission of a readily detectable fluorescence signal. Incubation of S. aureus-positive blood culture samples with the P2&3TT probe resulted in 50-fold higher fluorescence intensity levels than incubation with culture-negative samples. Moreover, incubation of the probe with non-S. aureus-positive blood cultures yielded essentially background fluorescence intensity levels for cultures with Gram-negative bacteria, and only ~ 3.5-fold increased fluorescence intensity levels over background for cultures with non-S. aureus Gram-positive bacteria. Importantly, the measured fluorescence intensities were dose-dependent, and a positive signal was clearly detectable for S. aureus-positive blood cultures with bacterial loads as low as ~ 7,000 colony-forming units/mL. Thus, the nuclease-activatable P2&3TT probe distinguishes clinical S. aureus-positive blood cultures from non-S. aureus-positive blood cultures and culture-negative blood, accurately, rapidly and with high sensitivity. We conclude that this probe may enhance the diagnosis of SAB.

While many neuroscience questions aim to understand the human brain, much current knowledge has been gained using animal models, which replicate genetic, structural, and connectivity aspects of the human brain. While voxel-based analysis (VBA) of preclinical magnetic resonance images is widely-used, a thorough examination of the statistical robustness, stability, and error rates is hindered by high computational demands of processing large arrays, and the many parameters involved therein. Thus, workflows are often based on intuition or experience, while preclinical validation studies remain scarce. To increase throughput and reproducibility of quantitative small animal brain studies, we have developed a publicly shared, high throughput VBA pipeline in a high-performance computing environment, called SAMBA. The increased computational efficiency allowed large multidimensional arrays to be processed in 1-3 days-a task that previously took ~1 month. To quantify the variability and reliability of preclinical VBA in rodent models, we propose a validation framework consisting of morphological phantoms, and four metrics. This addresses several sources that impact VBA results, including registration and template construction strategies. We have used this framework to inform the VBA workflow parameters in a VBA study for a mouse model of epilepsy. We also present initial efforts towards standardizing small animal neuroimaging data in a similar fashion with human neuroimaging. We conclude that verifying the accuracy of VBA merits attention, and should be the focus of a broader effort within the community. The proposed framework promotes consistent quality assurance of VBA in preclinical neuroimaging, thus facilitating the creation and communication of robust results.

Temporal lobe epilepsy (TLE) is a common and commonly devastating form of human epilepsy for which only symptomatic therapy is available. One cause of TLE is an episode of de novo prolonged seizures [status epilepticus (SE)]. Understanding the molecular signaling mechanisms by which SE transforms a brain from normal to epileptic may reveal novel targets for preventive and disease-modifying therapies. SE-induced activation of the BDNF receptor tyrosine kinase, TrkB, is one signaling pathway by which SE induces TLE. Although activation of TrkB signaling promotes development of epilepsy in this context, it also reduces SE-induced neuronal death. This led us to hypothesize that distinct signaling pathways downstream of TrkB mediate the desirable (neuroprotective) and undesirable (epileptogenesis) consequences. We subsequently demonstrated that TrkB-mediated activation of phospholipase Cγ1 is required for epileptogenesis. Here we tested the hypothesis that the TrkB-Shc-Akt signaling pathway mediates the neuroprotective consequences of TrkB activation following SE. We studied measures of molecular signaling and cell death in a model of SE in mice of both sexes, including wild-type and TrkB Shc/Shc mutant mice in which a point mutation (Y515F) of TrkB prevents the binding of Shc to activated TrkB kinase. Genetic disruption of TrkB-Shc signaling had no effect on severity of SE yet partially inhibited activation of the prosurvival adaptor protein Akt. Importantly, genetic disruption of TrkB-Shc signaling exacerbated hippocampal neuronal death induced by SE. We conclude that therapies targeting TrkB signaling for preventing epilepsy should spare TrkB-Shc-Akt signaling and thereby preserve the neuroprotective benefits. SIGNIFICANCE STATEMENT Temporal lobe epilepsy (TLE) is a common and devastating form of human epilepsy that lacks preventive therapies. Understanding the molecular signaling mechanisms underlying the development of TLE may identify novel therapeutic targets. BDNF signaling thru TrkB receptor tyrosine kinase is one molecular mechanism promoting TLE. We previously discovered that TrkB-mediated activation of phospholipase Cγ1 promotes epileptogenesis. Here we reveal that TrkB-mediated activation of Akt protects against hippocampal neuronal death in vivo following status epilepticus. These findings strengthen the evidence that desirable and undesirable consequences of status epilepticus-induced TrkB activation are mediated by distinct signaling pathways downstream of this receptor. These results provide a strong rationale for a novel therapeutic strategy selectively targeting individual signaling pathways downstream of TrkB for preventing epilepsy., (Copyright © 2019 the authors.)

Hippocampal mossy fiber axons simultaneously activate CA3 pyramidal cells and stratum lucidum interneurons (SLINs), the latter providing feedforward inhibition to control CA3 pyramidal cell excitability. Filopodial extensions of giant boutons of mossy fibers provide excitatory synaptic input to the SLIN. These filopodia undergo extraordinary structural plasticity causally linked to execution of memory tasks, leading us to seek the mechanisms by which activity regulates these synapses. High-frequency stimulation of the mossy fibers induces long-term depression (LTD) of their calcium-permeable AMPA receptor synapses with SLINs; previous work localized the site of induction to be postsynaptic and the site of expression to be presynaptic. Yet, the underlying signaling events and the identity of the retrograde signal are incompletely understood. We used whole cell recordings of SLINs in hippocampal slices from wild-type and mutant mice to explore the mechanisms. Genetic and pharmacologic perturbations revealed a requirement for both the receptor tyrosine kinase TrkB and its agonist, brain-derived neurotrophic factor (BDNF), for induction of LTD. Inclusion of inhibitors of Trk receptor kinase and PLC in the patch pipette prevented LTD. Endocannabinoid receptor antagonists and genetic deletion of the CB1 receptor prevented LTD. We propose a model whereby release of BDNF from mossy fiber filopodia activates TrkB and PLCγ1 signaling postsynaptically within SLINs, triggering synthesis and release of an endocannabinoid that serves as a retrograde signal, culminating in reduced glutamate release. Insights into the signaling pathways by which activity modifies function of these synapses will facilitate an understanding of their contribution to the local circuit and behavioral consequences of hippocampal granule cell activity. NEW & NOTEWORTHY We investigated signaling mechanisms underlying plasticity of the hippocampal mossy fiber filopodial synapse with interneurons in stratum lucidum. High-frequency stimulation of the mossy fibers induces long-term depression of this synapse. Our findings are consistent with a model in which brain-derived neurotrophic factor released from filopodia activates TrkB of a stratum lucidum interneuron; the ensuing activation of PLCγ1 induces synthesis of an endocannabinoid, which provides a retrograde signal leading to reduced release of glutamate presynaptically.

The key to effective treatment of bacterial infections is a swift and reliable diagnosis. Current clinical standards of bacterial diagnosis are slow and laborious. There are several anatomical imaging modalities that can detect inflammation, but none can distinguish between bacterial and sterile inflammation. Novel tracers such as smart activatable fluorescent probes represent a promising development that allow fast and specific testing without the use of ionizing radiation. Previously, a smart activatable probe was developed that is a substrate for the micrococcal nuclease as produced by Staphylococcus aureus. In the present study, the function of this probe was validated. Practical applicability in terms of sensitivity was assessed by incubation of the probe with 26 clinical S. aureus isolates, and probe specificity was verified by incubation with 30 clinical isolates and laboratory strains of various bacterial pathogens. The results show that the nuclease-specific probe was activated by all tested S. aureus isolates and laboratory strains with a threshold of ~10 6 -10 7 cells/mL. The probe was also activated by certain opportunistic staphylococci. We therefore propose that the studied nuclease probe represents a significant step forward to address the need for a rapid, practical, and precise method to detect infections caused by S. aureus.

A challenge for circulating tumor cell (CTC)-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1) their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2) their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Current water quality monitoring methods rely on growth-based measurements to detect fecal indicator bacteria, such as Escherichia coli and enterococci, and Staphylococcus aureus (S. aureus). These growth-based measurements, however, can take days to complete. This is a significant limitation in the evaluation of contaminated food and water sources. Various methods for selective in vitro detection of S. aureus have also been reported; however, these strategies, such as ELISA, agar-diffusion, PCR, or liquid chromatography-tandem mass spectrometry, all require overnight culturing or sophisticated instrumentation. There is a pressing need for a portable, simple diagnostic for S. aureus. Here, we demonstrate that oligonucleotide-functionalized gold nanoparticles (Oligo-AuNPs) can be designed to rapidly and selectively detect S. aureus with a colorimetric readout. We have functionalized a chemically modified 11-mer sequence onto AuNPs and have found that aggregation occurs in the presence of S. aureus supernantants. The particles can be stored as a lyophilized powder and reconstituted at time of use, and this has been tested in biologically relevant samples such as creek and ocean water. This approach requires minimal sample preparation and requires no extraneous instrumentation, leading to a rapid and simple diagnostic read-out that could be used in field tests to monitor food and water sources.

The Rho GTPase proteins Rac1, RhoA and Cdc42 have a central role in regulating the actin cytoskeleton in dendritic spines, thereby exerting control over the structural and functional plasticity of spines and, ultimately, learning and memory. Although previous work has shown that precise spatiotemporal coordination of these GTPases is crucial for some forms of cell morphogenesis, the nature of such coordination during structural spine plasticity is unclear. Here we describe a three-molecule model of structural long-term potentiation (sLTP) of murine dendritic spines, implicating the localized, coincident activation of Rac1, RhoA and Cdc42 as a causal signal of sLTP. This model posits that complete tripartite signal overlap in spines confers sLTP, but that partial overlap primes spines for structural plasticity. By monitoring the spatiotemporal activation patterns of these GTPases during sLTP, we find that such spatiotemporal signal complementation simultaneously explains three integral features of plasticity: the facilitation of plasticity by brain-derived neurotrophic factor (BDNF), the postsynaptic source of which activates Cdc42 and Rac1, but not RhoA; heterosynaptic facilitation of sLTP, which is conveyed by diffusive Rac1 and RhoA activity; and input specificity, which is afforded by spine-restricted Cdc42 activity. Thus, we present a form of biochemical computation in dendrites involving the controlled complementation of three molecules that simultaneously ensures signal specificity and primes the system for plasticity.

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are crucial for many forms of neuronal plasticity, including structural long-term potentiation (sLTP), which is a correlate of an animal's learning. However, it is unknown whether BDNF release and TrkB activation occur during sLTP, and if so, when and where. Here, using a fluorescence resonance energy transfer-based sensor for TrkB and two-photon fluorescence lifetime imaging microscopy, we monitor TrkB activity in single dendritic spines of CA1 pyramidal neurons in cultured murine hippocampal slices. In response to sLTP induction, we find fast (onset < 1 min) and sustained (>20 min) activation of TrkB in the stimulated spine that depends on NMDAR (N-methyl- d -aspartate receptor) and CaMKII signalling and on postsynaptically synthesized BDNF. We confirm the presence of postsynaptic BDNF using electron microscopy to localize endogenous BDNF to dendrites and spines of hippocampal CA1 pyramidal neurons. Consistent with these findings, we also show rapid, glutamate-uncaging-evoked, time-locked BDNF release from single dendritic spines using BDNF fused to superecliptic pHluorin. We demonstrate that this postsynaptic BDNF-TrkB signalling pathway is necessary for both structural and functional LTP. Together, these findings reveal a spine-autonomous, autocrine signalling mechanism involving NMDAR-CaMKII-dependent BDNF release from stimulated dendritic spines and subsequent TrkB activation on these same spines that is crucial for structural and functional plasticity.

S. aureus bacteremia (SAB) is a common condition with high rates of morbidity and mortality. Current methods used to diagnose SAB take at least a day, and often longer. Patients with suspected bacteremia must therefore be empirically treated, often unnecessarily, while assay results are pending. In this proof-of-concept study, we describe an inexpensive assay that detects SAB via the detection of micrococcal nuclease (an enzyme secreted by S. aureus) in patient plasma samples in less than three hours. In total, 17 patient plasma samples from culture-confirmed S. aureus bacteremic individuals were tested. 16 of these yielded greater nuclease assay signals than samples from uninfected controls or individuals with non-S. aureus bacteremia. These results suggest that a nuclease-detecting assay may enable the rapid and inexpensive diagnosis of SAB, which is expected to substantially reduce the mortality and morbidity that result from this condition.