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Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

Treatment of the epilepsies have benefitted immensely from study of animal models, most notably in the development of diverse anti-seizure medications in current clinical use. However, available drugs provide only symptomatic relief from seizures and are often ineffective. As a result, a critical need remains for developing improved symptomatic or disease-modifying therapies - or ideally, preventive therapies. Animal models will undoubtedly play a central role in such efforts. To ensure success moving forward, a critical question arises, namely "How does one make laboratory models relevant to our clinical understanding and treatment?" Our answer to this question: It all begins with a detailed understanding of the clinical phenotype one seeks to model. To make our case, we point to two examples - Fragile X syndrome and status epilepticus-induced mesial temporal lobe epilepsy - and examine how development of animal models for these distinct syndromes is based upon observations by astute clinicians and systematic study of the disorder. We conclude that the continuous and effective interaction of skilled clinicians and bench scientists is critical to the optimal design and study of animal models to facilitate insight into the nature of human disorders and enhance likelihood of improved therapies.

We analyzed four families that presented with a similar condition characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. We show that recessive mutations in the ASNS gene are responsible for this syndrome. Two of the identified missense mutations dramatically reduce ASNS protein abundance, suggesting that the mutations cause loss of function. Hypomorphic Asns mutant mice have structural brain abnormalities, including enlarged ventricles and reduced cortical thickness, and show deficits in learning and memory mimicking aspects of the patient phenotype. ASNS encodes asparagine synthetase, which catalyzes the synthesis of asparagine from glutamine and aspartate. The neurological impairment resulting from ASNS deficiency may be explained by asparagine depletion in the brain or by accumulation of aspartate/glutamate leading to enhanced excitability and neuronal damage. Our study thus indicates that asparagine synthesis is essential for the development and function of the brain but not for that of other organs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The necessity for, or redundancy of, distinctive KChIP proteins is not known., Results: Deletion of KChIP2 leads to increased susceptibility to epilepsy and to a reduction in IA and increased excitability in pyramidal hippocampal neurons., Conclusion: KChIP2 is essential for homeostasis in hippocampal neurons., Significance: Mutations in K(A) channel auxiliary subunits may be loci for epilepsy. 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 amplitude and shift in the V½ for steady-state inactivation to hyperpolarized potentials when compared with WT neurons. 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 with WT controls. Together, these data demonstrate that the KChIP2 is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons.

Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ( "QUSIM" ) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo .

Purpose:   Vagus nerve stimulation (VNS) provides partial relief of medically refractory partial seizures in a subset of patients. The optimal pattern of stimulation and the mechanism of the antiseizure effects are uncertain. Establishing the efficacy of VNS in an animal model of epilepsy would provide an experimental preparation with which to address these questions. We sought to determine whether VNS exerted antiseizure effects in the kindling model of epilepsy., Methods:   We implanted adult rats with bipolar stimulating electrodes in the right amygdala and VNS devices around the left vagus nerve. Following induction of kindling, electrographic seizure threshold (EST) was determined by quantifying the amygdala electrode current required to evoke a seizure. Once stable ESTs were established, VNS devices were programmed to deliver U.S. Food and Drug Administration (FDA)-approved, clinically used (standard) or an experimental (microburst) pattern of stimulation of variable intensity. VNS devices were programmed identically in control animals except that no current was delivered. EST was examined at 60 min and 1 week in the control and vagus nerve stimulated groups., Key Findings:   Significant reductions of EST values were detected in control animals when tested both 60 min and 1 week following device programming. Both clinically used and experimental patterns of VNS prevented the reduction of EST evident in control animals when tested either 60 min or 1 week after device programming., Significance:   These findings establish an experimental preparation with which to elucidate the antiseizure mechanisms of VNS and to determine patterns of VNS most effective at elevating seizure threshold., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

Idiopathic generalized epilepsy (IGE) is a complex disease with high heritability, but little is known about its genetic architecture. Rare copy-number variants have been found to explain nearly 3% of individuals with IGE; however, it remains unclear whether variants with moderate effect size and frequencies below what are reliably detected with genome-wide association studies contribute significantly to disease risk. In this study, we compare the exome sequences of 118 individuals with IGE and 242 controls of European ancestry by using next-generation sequencing. The exome-sequenced epilepsy cases include study subjects with two forms of IGE, including juvenile myoclonic epilepsy (n = 93) and absence epilepsy (n = 25). However, our discovery strategy did not assume common genetic control between the subtypes of IGE considered. In the sequence data, as expected, no variants were significantly associated with the IGE phenotype or more specific IGE diagnoses. We then selected 3,897 candidate epilepsy-susceptibility variants from the sequence data and genotyped them in a larger set of 878 individuals with IGE and 1,830 controls. Again, no variant achieved statistical significance. However, 1,935 variants were observed exclusively in cases either as heterozygous or homozygous genotypes. It is likely that this set of variants includes real risk factors. The lack of significant association evidence of single variants with disease in this two-stage approach emphasizes the high genetic heterogeneity of epilepsy disorders, suggests that the impact of any individual single-nucleotide variant in this disease is small, and indicates that gene-based approaches might be more successful for future sequencing studies of epilepsy predisposition., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

Interleukin-10 (IL-10) is a key suppressor of inflammation in chronic infections and in cancer. In mice, the inability of the immune system to clear viral infections or inhibit tumor growth can be reversed by antibody-mediated blockade of IL-10 action. We used a modified selection protocol to isolate RNA-based, nuclease-resistant, aptamers that bind to the murine IL-10 receptor. After 5 rounds of selection high-throughput sequencing (HTS) was used to analyze the library. Using distribution statistics on about 11 million sequences, aptamers were identified which bound to IL-10 receptor in solution with low K(d). After 12 rounds of selection the predominant IL-10 receptor-binding aptamer identified in the earlier rounds remained, whereas other high-affinity aptamers were not detected. Prevalence of certain nucleotide (nt) substitutions in the sequence of a high-affinity aptamer present in round 5 was used to deduce its secondary structure and guide the truncation of the aptamer resulting in a shortened 48-nt long aptamer with increased affinity. The aptamer also bound to IL-10 receptor on the cell surface and blocked IL-10 function in vitro. Systemic administration of the truncated aptamer was capable of inhibiting tumor growth in mice to an extent comparable to that of an anti- IL-10 receptor antibody.

The paucity of costimulation at the tumor site compromises the ability of tumor-specific T cells to eliminate the tumor. Here, we show that bi-specific oligonucleotide aptamer conjugates can deliver costimulatory ligands to tumor cells in situ and enhance antitumor immunity. In poorly immunogenic subcutaneously implanted tumor and lung metastasis models, systemic delivery of an agonistic 4-1BB aptamer ligand conjugated to a prostate specific membrane antigen (PSMA)-binding tumor-targeting aptamer led to inhibition of tumor growth, was more effective than, and synergized with, vaccination, and exhibited a superior therapeutic index compared to costimulation with 4-1BB antibodies. Tumor inhibition was dependent on homing to PSMA-expressing tumor cells and 4-1BB costimulation. Aptamer targeted costimulation is a broadly applicable and clinically feasible approach to enhance the costimulatory environment of disseminated tumor lesions. This study suggests that potentiating naturally occurring antitumor immunity via tumor-targeted costimulation could be an effective approach to elicit protective immunity to control tumor progression in cancer patients.

The neurotrophin receptor tyrosine kinase TrkB is critical to diverse biological processes. We investigated the interplay of Src family kinases (SFKs) and TrkB to better understand mechanisms of TrkB signaling in physiological and pathological conditions. We compared and contrasted the role of SFKs in TrkB signaling following activation of TrkB by two mechanisms, its transactivation by zinc, and its activation by its prototypic neurotrophin ligand, brain-derived neurotrophic factor (BDNF). Using biochemical, pharmacological, and chemical genetic studies of cultured rodent neurons, we found that zinc promotes preferential phosphorylation of Tyr-705/Tyr-706 of TrkB by a SFK-dependent but TrkB kinase-independent mechanism, a signaling event critical for transactivation of TrkB by zinc. By contrast, SFK activity is not essential for BDNF-mediated activation of TrkB, yet SFK activity is increased as a consequence of TrkB activation by BDNF. Moreover, BDNF-induced phosphorylation of Tyr-705/Tyr-706 of TrkB was inhibited by SFK inhibitors, implicating a regulatory role of SFKs in TrkB activation by BDNF. In sum, SFKs are activated by TrkB and, in turn, SFKs can promote TrkB activation. We propose models depicting the mutual regulation of SFKs and TrkB following activation of TrkB by zinc and BDNF.

Many signalling proteins involved in diverse functions such as cell growth and differentiation can act as oncogenes and cause cellular transformation. These molecules represent attractive targets for cancer diagnosis or therapy and therefore are subject to intensive investigation. Aptamers are small, highly structured nucleic acid molecules, isolated from combinatorial libraries by a procedure termed SELEX. Aptamers bind to a target molecule by providing a limited number of specific contact points imbedded in a larger, defined three-dimensional structure. Recently, aptamers have been selected against whole living cells, opening a new path which presents three major advantages: (1) direct selection without prior purification of membrane-bound targets, (2) access to membrane proteins in their native conformation similar to the in vivo conditions and (3) identification of (new) targets related to a specific phenotype. The ability to raise aptamers against living cells opens some attractive possibilities for new therapeutic and delivery approaches. In this chapter, the most recent advances in the field will be reviewed together with detailed descriptions of the relevant experimental approaches.

Maintenance of synaptic plasticity requires protein translation. Because changes in synaptic strength are regulated at the level of individual synapses, a mechanism is required for newly translated proteins to specifically and persistently modify only a subset of synapses. Evidence suggests this may be accomplished through local translation of proteins at or near synapses in response to plasticity-inducing patterns of activity. A number of proteins important for synaptic function are integral membrane proteins, which require a specialized group of organelles, proteins and enzymatic activities for proper synthesis. Dendrites appear to contain machinery necessary for the proper production of these proteins, and mRNAs for integral membrane proteins have been found localized to dendrites. Experiments are described that investigate the local translation of membrane proteins in the dendrites of cultured rat hippocampal neurons, using fluorescence recovery after photobleaching. Neurons were transfected with cDNAs encoding a fluorescently labeled transmembrane protein, TGN-38. Under conditions where the transport of this reporter construct was inhibited, the appearance of newly synthesized protein was observed via fluorescent microscopy. The dendritic translation of this protein required activation of glutamate receptors. The results demonstrate a functional capacity for activity-dependent synthesis of integral membrane proteins for distal dendrites in hippocampal neurons.

Protein transport to and from the postsynaptic plasma membrane is thought to be of central importance for synaptic plasticity. However, the molecular details of such processes are poorly understood. One mechanism by which membrane and secretory proteins may be transported to and from postsynaptic membranes is via cargo receptors. We studied the dendritic transport of TGN38, a putative cargo receptor thought to mediate protein transport between the trans-Golgi network (TGN), endosomes, and the plasma membrane. With fluorescence time-lapse imaging of neurons expressing a TGN38-green fluorescent protein fusion protein (GFP-TGN38), we observed rapid bidirectional dynamics of the protein in dendritic shafts. In addition, the protein was present on the surface and on intracellular membranes of dendrites and dendritic spines. Finally, GFP-TGN38 was found to cycle rapidly between the plasma membrane and intracellular membranes within dendrites, including those of spines. Together, our results suggest a role for TGN38 in facilitating rapid changes in the protein composition of postsynaptic membranes.

Insidious attack of cortical neurons by complement has been implicated in Alzheimer's and other neurodegenerative diseases. Excitotoxicity, triggered by excessive activation of glutamate receptors, has been implicated in neuronal death following diverse insults, including ischemia and seizures. Clinical studies suggested that a minimal excitotoxic insult might sensitize neurons to complement attack. We found that fleeting activation of ionotropic glutamate receptors sensitizes neurons but not astrocytes to complement attack. The complement molecule effecting cytotoxicity was the membrane attack complex. The site within the complement cascade at which sensitization was effected was the membrane attack pathway. Sensitization mediated by glutamate receptor activation required Ca(2+)(o) and generation of reactive oxygen species. These in vitro findings predict that a fleeting excitotoxic insult could act synergistically with complement to destroy cortical neurons and accelerate neurological deterioration.

The molecular basis of the selective death of motor neurons in amyotrophic lateral sclerosis (ALS) has been an enigma since its description by Charcot in 1869. In this issue of Neuron, demonstrate a motor neuron-specific death pathway which involves Fas and NO. Remarkably, motor neurons from mice carrying ALS-linked mutant forms of superoxide dismutase 1 (SOD1) exhibit an increased sensitivity to death triggered by Fas but not other insults. These data suggest new insights into the mechanisms of, and potential therapeutic strategies for, death of motor neurons in ALS.

Genetic and pharmacological perturbations suggest that tyrosine receptor kinase B (trkB) receptor activation promotes limbic epileptogenesis, but whether or where trkB activation occurs during epileptogenesis is uncertain. Because activation of trk receptors involves phosphorylation of specific tyrosine residues (Segal et al., 1996), the availability of antibodies that selectively recognize the phosphorylated form of trk receptors at the Shc site permits an immunohistochemical assessment of trk receptor activation. We reported previously increased phospho-specific trk (p-trk) immunoreactivity in the mossy fiber pathway of the hippocampus during epileptogenesis in rats (Binder et al., 1999b). Because the p-trk antibody does not distinguish among trkA, trkB, and trkC, the identity of the neurotrophin receptor(s) undergoing phosphorylation was uncertain. The development of mice carrying a point mutation of the Shc binding site (Y515F) in the trkB gene (trkB(shc)) provided an opportunity to test the hypothesis that trkB is the neurotrophin receptor undergoing phosphorylation. Epileptogenesis in wild-type (WT) mice was associated with increased p-trk immunoreactivity in both the mossy fiber pathway and CA3 stratum oriens of hippocampus. In contrast, the epileptogenesis-associated increase of p-trk immunoreactivity was reduced in trkB(shc) mutant mice. The development of epileptogenesis as measured by electrophysiological and behavioral indices did not differ between trkB(shc) mutant and WT mice. These data demonstrate that the neurotrophin receptor trkB undergoes phosphorylation in the mossy fiber pathway and CA3 stratum oriens of the hippocampus during limbic epileptogenesis. In addition, the signaling pathways activated by the Shc site of trkB exert no detectable regulatory effects on limbic epileptogenesis.