UNC-6/netrin and SLT-1/slit guidance cues orient axon outgrowth mediated by MIG-10/RIAM/lamellipodin

BACKGROUND: Axon migrations are guided by extracellular cues that can act as repellants or attractants. However, the logic underlying the manner through which attractive and repulsive responses are determined is unclear. Many extracellular guidance cues, and the cellular components that mediate their signals, have been implicated in both types of responses. RESULTS: Genetic analyses indicate that MIG-10/RIAM/lamellipodin, a cytoplasmic adaptor protein, functions downstream of the attractive guidance cue UNC-6/netrin and the repulsive guidance cue SLT-1/slit to direct the ventral migration of the AVM and PVM axons in C. elegans. Furthermore, overexpression of MIG-10 in the absence of UNC-6 and SLT-1 induces a multipolar phenotype with undirected outgrowths. Addition of either UNC-6 or SLT-1 causes the neurons to become monopolar. Moreover, the ability of UNC-6 or SLT-1 to direct the axon ventrally is enhanced by the MIG-10 overexpression. We also demonstrate that an interaction between MIG-10 and UNC-34, a protein that promotes actin-filament extension, is important in the response to guidance cues and that MIG-10 colocalizes with actin in cultured cells, where it can induce the formation of lamellipodia. CONCLUSIONS: We conclude that MIG-10 mediates the guidance of AVM and PVM axons in response to the extracellular UNC-6 and SLT-1 guidance cues. The attractive and repulsive guidance cues orient MIG-10-dependant axon outgrowth to cause a directional response.     

A role for foxd3 and sox10 in the differentiation of gonadotropin-releasing hormone (GnRH) cells in the zebrafish Danio rerio

Gonadotropin-releasing hormone (GnRH) is found in a wide range of vertebrate tissues, including the nervous system. In general, GnRH has two functions: endocrine, acting as a releasing hormone; and neuromodulatory, affecting neural activity in the peripheral and central nervous system. The best understood population of GnRH cells is that of the hypothalamus, which is essential for reproduction. Less well understood are the populations of GnRH cells found in the terminal nerve and midbrain, which appear to be neuromodulatory in function. The GnRH-containing cells of the midbrain are proposed to arise from the mesencephalic region of the neural tube. Previously, we showed that neuromodulatory GnRH cells of the terminal nerve arise from cranial neural crest. To test the hypothesis that neuromodulatory GnRH cells of the midbrain also arise from neural crest, we used gene knockdown experiments in zebrafish to disrupt neural crest development. We demonstrate that decrement of the function of foxd3 and/or sox10, two genes important for the development and specification of neural crest, resulted in a reduction and/or loss of GnRH cells of the midbrain, as well as a reduction in the number of terminal nerve GnRH cells. Therefore, our data support a neural crest origin for midbrain GnRH cells. Additionally, we demonstrate that knockdown of kallmann gene function resulted in the loss of endocrine GnRH cells of the hypothalamus, but not of neuromodulatory GnRH cells of the midbrain and terminal nerve, thus providing additional evidence for separate pathways controlling the development of neuromodulatory and endocrine GnRH cells.     

The morphology of the fourth abdominal ganglion of the hermit crab: A light microscope study

The hermit crab, Pagurus pollicarus, has the same organization in its fourth abdominal ganglion as its macruran relatives in spite of the reduction in abdominal muscles, sensory receptors, and appendages. Connective axons are grouped into discrete bundles between which five groups of commissural fibers run to connect left and right sides. The neurites of ventral cell bodies run dorsally in characteristic groups between the connective bundles. The hermit crab fourth ganglion has two thirds as many cells as the crayfish and is laterally compressed. This reduction appears related to the reduction in the sizes of the ganglionic roots. The ventral fine fibered neuropil is larger on the left than the right side reflecting the loss of pleopods on the right side. The basic organization of decapod abdominal ganglia appears to permit considerable integrative flexibility within a relatively conservative morphological framework.     

Pharmacological inhibition of Carbonic Anhydrase IX and XII to enhance targeting of acute myeloid leukaemia cells under hypoxic conditions

Acute myeloid leukaemia (AML) is an aggressive form of blood cancer that carries a dismal prognosis. Several studies suggest that the poor outcome is due to a small fraction of leukaemic cells that elude treatment and survive in specialised, oxygen (O₂)-deprived niches of the bone marrow. Although several AML drug targets such as FLT3, IDH1/2 and CD33 have been established in recent years, survival rates remain unsatisfactory, which indicates that other, yet unrecognized, mechanisms influence the ability of AML cells to escape cell death and to proliferate in hypoxic environments. Our data illustrates that Carbonic Anhydrases IX and XII (CA IX/XII) are critical for leukaemic cell survival in the O₂-deprived milieu. CA IX and XII function as transmembrane proteins that mediate intracellular pH under low O₂ conditions. Because maintaining a neutral pH represents a key survival mechanism for tumour cells in O₂-deprived settings, we sought to elucidate the role of dual CA IX/XII inhibition as a novel strategy to eliminate AML cells under hypoxic conditions. Our findings demonstrate that the dual CA IX/XII inhibitor FC531 may prove to be of value as an adjunct to chemotherapy for the treatment of AML.     

Human Cytomegalovirus Vaccine Based on the Envelope gH/gL Pentamer Complex

Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral responses that neutralize HCMV infection of EC, placental macrophages and fibroblasts, properties of potential value in a prophylactic vaccine.     

Differential α4(+)/(?)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)₂(β2)₃ and (α4)₃(β2)₂ subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with ¹²⁵I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.     

Characterization of modified antisense oligonucleotides in Xenopus laevis embryos

A wide variety of modified oligonucleotides have been tested as antisense agents. Each chemical modification produces a distinct profile of potency, toxicity, and specificity. Novel cationic phosphoramidate-modified antisense oligonucleotides have been developed recently that have unique and interesting properties. We compared the relative potency and specificity of a variety of established antisense oligonucleotides, including phosphorothioates (PS), 2'-O-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), and neutral methoxyethyl (MEA) phosphoramidates with new cationic N,N-dimethylethylenediamine (DMED) phosphoramidate-modified antisense oligonucleotides. A series of oligonucleotides was synthesized that targeted two sites in the Xenopus laevis survivin gene and were introduced into Xenopus embryos by microinjection. Effects on survivin gene expression were examined using quantitative real-time PCR. Of the various modified oligonucleotide designs tested, LNA/PS chimeras (which showed the highest melting temperature) and DMED/phosphodiester chimeras (which showed protection of neighboring phosphate bonds) were potent in reducing gene expression. At 40 nM, overall specificity was superior for the LNA/PS-modified compounds compared with the DMED-modified oligonucleotides. However, at 400 nM, both of these compounds led to significant degradation of survivin mRNA, even when up to three mismatches were present in the heteroduplex.     

NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

Background

Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.

Methodology/Principal Findings

We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47^phox−/− mice and gp91^phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-κB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation.

Conclusions/Significance

These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.     

Synthesis of desformylflustrabromine and its evaluation as an alpha4beta2 and alpha7 nACh receptor modulator

Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of alpha4beta2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.