The long noncoding RNA Synage regulates synapse stability and neuronal function in the cerebellum

The brain is known to express many long noncoding RNAs (lncRNAs); however, whether and how these lncRNAs function in modulating synaptic stability remains unclear. Here, we report a cerebellum highly expressed lncRNA, Synage, regulating synaptic stability via at least two mechanisms. One is through the function of Synage as a sponge for the microRNA miR-325-3p, to regulate expression of the known cerebellar synapse organizer Cbln1. The other function is to serve as a scaffold for organizing the assembly of the LRP1-HSP90AA1-PSD-95 complex in PF-PC synapses. Although somewhat divergent in its mature mRNA sequence, the locus encoding Synage is positioned adjacent to the Cbln1 loci in mouse, rhesus macaque, and human, and Synage is highly expressed in the cerebella of all three species. Synage deletion causes a full-spectrum cerebellar ablation phenotype that proceeds from cerebellar atrophy, through neuron loss, on to synapse density reduction, synaptic vesicle loss, and finally to a reduction in synaptic activity during cerebellar development; these deficits are accompanied by motor dysfunction in adult mice, which can be rescued by AAV-mediated Synage overexpression from birth. Thus, our study demonstrates roles for the lncRNA Synage in regulating synaptic stability and function during cerebellar development.Subject terms: RNA, Cell biology, Molecular biology, Neuroscience     

Nckbeta adapter controls neuritogenesis by maintaining the cellular paxillin level

The SH2/SH3 adapter Nck has an evolutionarily conserved role in neurons, linking the cell surface signals to actin cytoskeleton-mediated responses. The mechanism, however, remains poorly understood. We have investigated the role of Nck/Nckalpha/Nck1 versus Grb4/Nckbeta/Nck2 side-by-side in the process of mammalian neuritogenesis. Here we show that permanent genetic silencing of Nckbeta, but not Nckalpha, completely blocked nerve growth factor-induced neurite outgrowth in PC12 cells and dramatically disrupted the axon and dendrite tree in primary rat cortical neurons. By screening for changes among the components reportedly present in complex with Nck, we found that the steady-state level of paxillin was significantly reduced in Nckbeta knockdown, but not Nckalpha knockdown, neurons. Interestingly, Nckbeta knockdown did not affect the paxillin level in glial cells and several other cell types of various tissue origins. Genetic silencing of paxillin blocked neuritogenesis, just like Nckbeta knockdown. Reintroducing a nondegradable Nckbeta into Nckbeta short interfering RNA-expressing PC12 cells rescued paxillin from down-regulation and allowed the resumption of neuritogenesis. Forced expression of paxillin in Nckbeta knockdown PC12 also rescued its capacity for neuritogenesis. Finally, Nckbeta, but not Nckalpha, binds strongly to paxillin and treatment of the neurons with proteosome inhibitors prevented paxillin down-regulation in Nckbeta knockdown neurons. Thus, Nckbeta maintains paxillin stability during neuritogenesis.     

Phosphorylation of a carboxyl-terminal serine within the kappa-opioid receptor produces desensitization and internalization

G-protein receptor kinase and beta-arrestin mediated desensitization of the rat kappa-opioid receptor (KOR) was previously shown using Xenopus oocyte expression to require serine 369 within the C terminus of KOR. To define the effects of phosphorylation of this residue in desensitization and internalization processes in mammalian expression systems, wild-type KOR-green fluorescent protein (KOR-GFP) and KOR(S369A)-GFP were stably expressed in AtT-20 and HEK293 cells. Using whole-cell patch clamp recording in transfected AtT-20 cells, agonist activation of either kappa receptor form produced equivalent activation of the intrinsic G-protein-gated inwardly rectifying potassium channel. Incubation for 60 min with the kappa agonist U50,488 (100 nm) desensitized the response in cells expressing wild-type KOR-GFP by 86% but had no effect on KOR(S369A)-GFP-expressing cells. Phosphorylation of serine 369 was detected using a phosphospecific antibody (KOR-P) able to distinguish the phosphorylated form of the receptor. The agonist-induced increase in KOR-P labeling was dose-dependent, blocked by co-treatment with the kappa antagonist norbinaltorphimine, and prevented by co-expression of the dominant negative form of the G-protein receptor kinase, GRK2(K220R). In contrast, agonist-induced increase in KOR-P labeling was not evident in KOR(S369A) expressing cells. Prolonged activation resulted in receptor internalization that was also blocked by KOR(S369A) substitution, but interestingly, KOR-P labeling was evident at lower agonist concentrations than required to induce internalization. Following the removal of agonist, receptor dephosphorylation detected by loss of KOR-P labeling was complete within 60 min, could be blocked by okadaic acid, and was not blocked by sucrose inhibition of receptor internalization. These results demonstrate that GRK-mediated phosphorylation of serine 369 mediates rat KOR desensitization and internalization.     

Preliminary evaluation of the sural nerve using 22-MHz ultrasound: a new approach for evaluation of diabetic cutaneous neuropathy

Background

The application of 22-MHz high-frequency ultrasound allows for visualization of the inner part of the sural nerve. The aim of this study was to evaluate the morphological changes of sural nerves in patients with type 2 diabetes mellitus using ultrasound.

Materials and Methods

The thickness/width (T/W) ratio, the cross-sectional area (CSA) of the sural nerves and the maximum thickness (MT) of the nerve fascicles were measured in 100 patients with type 2 diabetes mellitus and 50 healthy volunteers using 22-MHz ultrasound. Receiver operating characteristic (ROC) curves were plotted to determine the optimal cut-off values as well as the sensitivities and specificities. All parameters were significantly different between the subject and control groups. The ROC curves demonstrated that the MT was the most predictive of diabetic cutaneous neuropathy, with an optimal cut-off value of 0.365 mm that yielded a sensitivity of 90.3% and a specificity of 87.7%.

Conclusions

The results of this study suggest that 22-MHz ultrasound may be a valuable tool for evaluating diabetic cutaneous nerve neuropathy.     

SYSTEMATICS OF THE CALCIFIED GENERA OF THE GALAXAURACEAE (NEMALIALES,RHODOPHYTA) WITH AN EMPHASIS ON TAIWAN SPECIES1

The current separation of the calcified genera Actinotrichia Decaisne, Galaxaura Lamouroux, and Tricleocarpa Huisman et Borowitzka in the Galaxauraceae is largely based on type of life history (whether the gametophytes and tetrasporophytes are isomorphic or heteromorphic with respect to gross morphology or cortical‐cell features) and on features of postfertilization development (such as the composition of the pericarp). We reexamined the phylogenetic relationships of these genera based on comparative rbcL sequence analysis, types of life cycle, and cystocarp development. Four distinct assemblages have been identified: an Actinotrichia clade, a Tricleocarpa clade, a Galaxaura clade (containing the type species), and a Dichotomaria clade made of a number of formerly Galaxaura species (D. obtusata [Ellis et Solander] Lamarck, D. marginata [Ellis et Solander] Lamarck, and D. diesingiana [Zanardini] Huisman, Harper and Saunders). Key differences between Dichotomaria and Galaxaura include the habit of the gametophytic and tetrasporophytic generations (isomorphic in Dichotomaria and dimorphic in Galaxaura) as well as the presence or absence of a persistent pericarp in the cystocarp (present in Dichotomaria and absent in Galaxaura). Molecular data do not support monophyly for the putatively pantropical species Galaxaura rugosa, Dichotomaria obtusata, and D. marginata, all of which we conclude are in need of taxonomic revision.     

Stimulation of regional lymphatic and blood flow by epicutaneous oxazolone.

The application of the epicutaneous antigen oxazolone results in persistent induration and erythema; however, the relative changes in lymph and blood flow in the inflammatory skin are largely unknown. To define the contribution of lymph and blood flow to the clinical appearance of cutaneous inflammation, we studied the sheep ear after the application of oxazolone. As a model for the study of these changes, the sheep ear had several experimental advantages: 1) a simplified superficial vascular network, 2) defined lymphatic drainage, and 3) an avascular and alymphatic cartilaginous barrier. Lymph flow was continuously monitored by cannulation of the prescapular efferent lymph duct. Blood flow, as reflected by cutaneous erythema, was noninvasively measured by use of a visible-spectrum spectrophotometer. The application of the epicutaneous oxazolone resulted in increased ear thickness for >7 days. The lymph flow from the oxazolone-stimulated ear peaked between 24 and 48 h after oxazolone stimulation. Spectrophotometric evaluation indicated that the cutaneous erythema peaked 72-96 h after application of oxazolone. Corrosion casting and scanning electron microscopy of the microcirculation at 96 h after antigen stimulation demonstrated significant dilatation of the superficial vascular network. These results suggest a biphasic response to oxazolone stimulation: 1) an early increase in vascular permeability associated with increased lymph flow and 2) a subsequent increase in relative blood flow associated with a dilated inflammatory microcirculation.     

Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy-brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.     

Phenylbutyrates as potent,orally bioavailable vitronectin receptor (integrin alphavbeta3) antagonists

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.     

Cloning and characterization of Cbl-associated protein splicing isoforms

Cbl-associated protein (CAP) is an adaptor protein that plays important roles in both signal transduction and cytoskeleton rearrangement. Alternative splicing of the gene SORBS1 results in multiple isoforms of CAP protein. We report here the cloning of 3 new CAP isoforms, CAP2, CAP3, and CAP4, from mouse adipose tissue. RT-PCR analyses reveal that the isoform mRNAs are differentially expressed. CAP2, CAP3, and CAP4 contain a coiled-coil domain. In addition, CAP4 contains a proline-rich region, part of which exists in CAP3. Coimmunoprecipitation experiments show that CAP4 forms a homodimeric complex. While these new isoforms similarly interact with Cbl, they exhibit varied binding specificity toward vinculin. In contrast to CAP1 and CAP2, CAP4 does not interact with vinculin, and CAP3 binds with low affinity. Immunofluorescence analysis demonstrates differential subcellular localization of Myc-tagged CAP isoforms in 3T3-L1 adipocytes. These results suggest that these new isoforms of CAP might play different signaling roles.