Increased anxiety-like behavior and enhanced synaptic efficacy in the amygdala of GluR5 knockout mice

GABAergic transmission in the amygdala modulates the expression of anxiety. Understanding the interplay between GABAergic transmission and excitatory circuits in the amygdala is, therefore, critical for understanding the neurobiological basis of anxiety. Here, we used a multi-disciplinary approach to demonstrate that GluR5-containing kainate receptors regulate local inhibitory circuits, modulate the excitatory transmission from the basolateral amygdala to the central amygdala, and control behavioral anxiety. Genetic deletion of GluR5 or local injection of a GluR5 antagonist into the basolateral amygdala increases anxiety-like behavior. Activation of GluR5 selectively depolarized inhibitory neurons, thereby increasing GABA release and contributing to tonic GABA current in the basolateral amygdala. The enhanced GABAergic transmission leads to reduced excitatory inputs in the central amygdala. Our results suggest that GluR5 is a key regulator of inhibitory circuits in the amygdala and highlight the potential use of GluR5-specific drugs in the treatment of pathological anxiety.     

Ring-expanded analogues of natural oxetanocin: (+) and (-) hydroxymethyl isodideoxyadenosine

New enantiomeric isonucleoside analogues related to natural oxetanocin have been synthesized from D-glucosamine and D-glucose. The structures of the target compounds were confirmed by NMR, HRMS, UV, single crystal X-ray, and optical rotation data. Stability studies with respect to purine nucleoside phosphorylase and adenosine deaminase show that these compounds are not substrates. Antiviral results are discussed.     

Selective activation of microglia in spinal cord but not higher cortical regions following nerve injury in adult mouse

Neuronal plasticity along the pathway for sensory transmission including the spinal cord and cortex plays an important role in chronic pain, including inflammatory and neuropathic pain. While recent studies indicate that microglia in the spinal cord are involved in neuropathic pain, a systematic study has not been performed in other regions of the central nervous system (CNS). In the present study, we used heterozygous Cx3cr1 ^GFP/+mice to characterize the morphological phenotypes of microglia following common peroneal nerve (CPN) ligation. We found that microglia showed a uniform distribution throughout the CNS, and peripheral nerve injury selectively activated microglia in the spinal cord dorsal horn and related ventral horn. In contrast, microglia was not activated in supraspinal regions of the CNS, including the anterior cingulate cortex (ACC), prefrontal cortex (PFC), primary and secondary somatosensory cortex (S1 and S2), insular cortex (IC), amygdala, hippocampus, periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Our results provide strong evidence that nerve injury primarily activates microglia in the spinal cord of adult mice, and pain-related cortical plasticity is likely mediated by neurons.     

Sensory nerve induced inflammation contributes to heterotopic ossification

Heterotopic ossification (HO), or bone formation in soft tissues, is often the result of traumatic injury. Much evidence has linked the release of BMPs (bone morphogenetic proteins) upon injury to this process. HO was once thought to be a rare occurrence, but recent statistics from the military suggest that as many as 60% of traumatic injuries, resulting from bomb blasts, have associated HO. In this study, we attempt to define the role of peripheral nerves in this process. Since BMP2 has been shown previously to induce release of the neuroinflammatory molecules, substance P (SP) and calcitonin gene related peptide (CGRP), from peripheral, sensory neurons, we examined this process in vivo. SP and CGRP are rapidly expressed upon delivery of BMP2 and remain elevated throughout bone formation. In animals lacking functional sensory neurons (TRPV1(-/-) ), BMP2-mediated increases in SP and CGRP were suppressed as compared to the normal animals, and HO was dramatically inhibited in these deficient mice, suggesting that neuroinflammation plays a functional role. Mast cells, known to be recruited by SP and CGRP, were elevated after BMP2 induction. These mast cells were localized to the nerve structures and underwent degranulation. When degranulation was inhibited using cromolyn, HO was again reduced significantly. Immunohistochemical analysis revealed nerves expressing the stem cell markers nanog and Klf4, as well as the osteoblast marker osterix, after BMP2 induction, in mice treated with cromolyn. The data collectively suggest that BMP2 can act directly on sensory neurons to induce neurogenic inflammation, resulting in nerve remodeling and the migration/release of osteogenic and other stem cells from the nerve. Further, blocking this process significantly reduces HO, suggesting that the stem cell population contributes to bone formation.     

Activated ADP-ribosylation factor assembles distinct pools of actin on golgi membranes

The small GTP-binding protein ADP-ribosylation factor (ARF) has been shown to regulate the interaction of actin and actin-binding proteins with the Golgi apparatus. Here we report that ARF activation stimulates the assembly of distinct pools of actin on Golgi membranes. One pool of actin cofractionates with coatomer (COPI)- coated vesicles and is sensitive to salt extraction and the plus end actin-binding toxin cytochalasin D. A second ARF-dependent actin pool remains on the Golgi membranes following vesicle extraction and is insensitive to cytochalasin D. Isolation of the salt-extractable ARF-dependent actin from the Golgi reveals that it is bound to a distinct repertoire of actin-binding proteins. The two abundant actin-binding proteins of the ARF-dependent actin complex are identified as spectrin and drebrin. We show that drebrin is a specific component of the cytochalasin D-sensitive, ARF-dependent actin pool on the Golgi. Finally, we show that depolymerization of this actin pool with cytochalasin D increases the extent of the salt-dependent release of COPI-coated vesicles from the Golgi following cell-free budding reactions. Together these data suggest that regulation of the actin-based cytoskeleton may play an important role during ARF-mediated transport vesicle assembly or release on the Golgi.     

Optimization of quorum quenching mediated bacterial attenuation of Solanum torvum root extract by response surface modelling through Box-Behnken approach

The present study was intended to optimize the quorum sensing inhibitory action of Solanum torvum root extract against Chromobacterium violaceum. Factors such as bacterial density, frequency of administration and concentration of extract were analysed. Plant samples were collected from Thrissur District, Kerala, India. Response surface modelling of factors by Box-Behnken approach was employed for optimizing quorum quenching activity of extract. The adequacy of mathematical model was verified by ANOVA and Cook’s distance table. Results revealed that quorum quenching property of Solanum torvum root extract is highly influenced by variables studied whereas maximum activity was found during administration of 300?µg/ml extract thrice in a day. It was also understood that extract does not possess any bactericidal activity wherein it only silence its quorum sensing mediated functions. This observations can be further used in quorum quenching studies.     

Type I Phosphatidylinositol Phosphate Kinase Beta Regulates Focal Adhesion Disassembly by Promoting β1 Integrin Endocytosis

Cell migration requires the regulated disassembly of focal adhesions, but the underlying mechanisms remain poorly defined. We have previously shown that focal adhesion disassembly requires the dynamin 2- and clathrin-dependent endocytosis of ligand-activated β1 integrins. Here, we identify type I phosphatidylinositol phosphate kinase beta (PIPKIβ), an enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI4,5P₂), as a key regulator of this process. We found that knockdown of PIPKIβ by RNA interference blocks the internalization of active β1 integrins and impairs focal adhesion turnover and cell migration. These defects are caused by the failure to target the endocytic machinery, including clathrin adaptors and dynamin 2, to focal adhesion sites. As a consequence, depletion of PIPKIβ blocks clathrin assembly at adhesion plaques and prevents complex formation between dynamin 2 and focal adhesion kinase (FAK), a critical step in focal adhesion turnover. Together, our findings identify PIPKIβ as a novel regulator of focal adhesion disassembly and suggest that PIPKIβ spatially regulates integrin endocytosis at adhesion sites to control cell migration.Cell migration is a highly dynamic process that depends on the ability of a cell to adhere to and deadhere from the extracellular matrix in a coordinated manner. Adhesion is mediated through focal adhesion sites, which assemble in response to activation and clustering of integrin receptors and comprise signaling and scaffolding proteins, such as focal adhesion kinase (FAK), talin, vinculin, paxillin, and zyxin (9, 55). These complexes anchor the extracellular matrix to the actin cytoskeleton and also serve as signaling platforms (9, 55). The formation of adhesive complexes is essential for the stabilization of membrane protrusions and to provide the tensile forces for migration (9, 55). However, rapid cell movement requires that focal adhesions not only be continuously formed, but also disassembled (9, 56). The coordinated control of cell adhesion, and release thereof, is therefore a critical regulatory function for migrating cells. However, while much has been learned about the mechanisms underlying focal adhesion assembly, comparatively little is known about how the turnover of adhesion sites is regulated, despite the importance of this process for cell migration.Recently, the protease calpain, FAK, and phosphatases and kinases that control the activity of FAK, as well as microtubules and the large GTPase dynamin 2, have been identified as regulators of focal adhesion disassembly (9, 10, 21, 22). In particular, a pathway has been defined in which microtubule targeting of focal adhesions leads to their disassembly (21). A critical step in this process is the formation of a protein complex between FAK and dynamin 2, a key regulator of endocytosis (21). Dynamin 2, together with components of the clathrin machinery, then mediates the turnover of focal adhesions by promoting the internalization of β1 integrins (14, 41). Notably, dynamin 2 and clathrin adaptors become enriched at focal adhesion sites prior to their disassembly (14, 21). Therefore, mechanisms that control the recruitment of the endocytic machinery to focal adhesion sites must exist. However, how this process is regulated during focal adhesion turnover remains unknown.Phosphatidylinositol-4,5-bisphosphate (PI4,5P₂) has recently emerged as an important regulator of focal adhesion dynamics (38, 47, 51, 57). In addition to serving as the precursor to other second messengers, PI4,5P₂ directly binds and modulates many focal adhesion components, including talin, vinculin, and α-actinin, that regulate adhesion assembly and their linkage to the actin cytoskeleton (38, 47, 51, 57). Adhesion to the extracellular matrix stimulates the synthesis of PI4,5P₂, and the general paradigm has been that the resulting local increase in PI4,5P₂ levels promotes focal adhesion assembly (23, 38, 40). Intriguingly, emerging evidence suggests that PI4,5P₂ also promotes the disassembly of focal adhesions (13, 46). This finding implies that PI4,5P₂ levels at adhesion sites must be tightly regulated, both spatially and temporally, to elicit its specific, yet inverse, effects on focal adhesion dynamics.The generation of PI4,5P₂ at specific subcellular sites is modulated in part by the selective targeting and activation of specific type I phosphatidylinositol phosphate kinases (PIPKI), which synthesize PI4,5P₂ (38). Three related PIPKI isoforms, designated PIPKIα, PIPKIβ, and PIPKIγ, and multiple splice variants are present in mammalian cells (27, 28, 39, 49). Recent studies have shown that the increase of PI4,5P₂ synthesis leading to focal adhesion assembly is mediated through the specific recruitment of PIPKIγ661, a splice variant of PIPKIγ, to focal adhesions (18, 37). However, whether PIPKIγ661 or another member of the PIPKI family is responsible for synthesizing the PI4,5P₂ pool regulating focal adhesion disassembly is currently unknown, and the molecular mechanisms whereby PI4,5P₂ regulates this process are not well defined.Coincidently, PI4,5P₂ is also an important organizer of clathrin assembly at the plasma membrane (17). In this study, we therefore set out to determine whether PI4,5P₂ promotes focal adhesion disassembly through its effects on endocytosis and to identify the PIPKI isoform involved in generating this pool of PI4,5P₂. We show that knockdown of one specific PIPKI isoform, PIPKIβ, blocks adhesion turnover leading to the inhibition of cell migration. We further show that PIPKIβ is necessary for the uptake of activated β1 integrins and provide evidence that PI4,5P₂ produced by PIPKIβ orchestrates the recruitment of components of the endocytic machinery to adhesion sites. Together, these studies define the role of PI4,5P₂ in the regulation of focal adhesion disassembly and identify PIPKIβ as the enzyme synthesizing this pool of PI4,5P₂.     

A Specialized Microvascular Domain in the Mouse Neural Stem Cell Niche

The microenvironment of the subependymal zone (SEZ) neural stem cell niche is necessary for regulating adult neurogenesis. In particular, signaling from the microvasculature is essential for adult neural stem cell maintenance, but microvascular structure and blood flow dynamics in the SEZ are not well understood. In this work, we show that the mouse SEZ constitutes a specialized microvascular domain defined by unique vessel architecture and reduced rates of blood flow. Additionally, we demonstrate that hypoxic conditions are detectable in the ependymal layer that lines the ventricle, and in a subpopulation of neurons throughout the SEZ and striatum. Together, these data highlight previously unidentified features of the SEZ neural stem cell niche, and further demonstrate the extent of microvascular specialization in the SEZ microenvironment.     

Genetic evidence for adenylyl cyclase 1 as a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.