Down-regulation of the non-neuronal acetylcholine synthesis and release machinery in acute allergic airway inflammation of rat and mouse

Acetylcholine (ACh), derived both from nerve fibres and from non-neuronal sources such as epithelial cells, is a major regulator of airway function. There is evidence that dysfunction of the neuronal cholinergic system is involved in the pathogenesis of asthma. Here, we asked whether the pulmonary non-neuronal ACh-synthesis and release machinery is altered in a rat and a mouse model of allergic airway disease. Animals were sensitized against ovalbumin, challenged by allergen inhalation, and sacrificed 24 or 48 h later. Targets of investigation were the high-affinity choline transporter-1 (CHT1), that mediates cellular uptake of choline, the ACh-synthesizing enzyme choline acetyltransferase (ChAT), the vesicular ACh transporter (VAChT), and the polyspecific organic cation transporters (OCT1-3), which are able to translocate choline and ACh across the plasma membrane. With cell-type specific distribution patterns, immunohistochemistry identified these proteins in airway epithelial cells and alveolar macrophages. Real-time RT-PCR revealed significant decreases in ChAT-, CHT1-, VAChT-, OCT-mRNA in the lung of sensitized and allergen challenged animals. These data were supported by immunohistochemistry, demonstrating reduced labeling intensity of airway epithelial cells. ChAT-, CHT1-, VAChT-, and OCT1-mRNA were also significantly reduced in cells recovered by bronchoalveolar lavage from sensitized and challenged rats. In conclusion, the pulmonary non-neuronal cholinergic system is down-regulated in acute allergic airway inflammation. In view of the role of ACh in maintenance of cell-cell-contacts, stimulation of fluid-secretion and of ciliary beat frequency, this down-regulation may contribute to epithelial shedding and ciliated cell dysfunction that occur in this pathological condition.     

Semen-derived amyloid fibrils drastically enhance HIV infection

Sexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.     

Acquisition of obligate mutualist symbionts during the larval stage is not beneficial for a coral host

Theory suggests that the direct transmission of beneficial endosymbionts (mutualists) from parents to offspring (vertical transmission) in animal hosts is advantageous and evolutionarily stable, yet many host species instead acquire their symbionts from the environment (horizontal acquisition). An outstanding question in marine biology is why some scleractinian corals do not provision their eggs and larvae with the endosymbiotic dinoflagellates that are necessary for a juvenile's ultimate survival. We tested whether the acquisition of photosynthetic endosymbionts (family Symbiodiniaceae) during the planktonic larval stage was advantageous, as is widely assumed, in the ecologically important and threatened Caribbean reef‐building coral Orbicella faveolata. Following larval acquisition, similar changes occurred in host energetic lipid use and gene expression regardless of whether their symbionts were photosynthesizing, suggesting the symbionts did not provide the energetic benefit characteristic of the mutualism in adults. Larvae that acquired photosymbionts isolated from conspecific adults on their natal reef exhibited a reduction in swimming, which may interfere with their ability to find suitable settlement substrate, and also a decrease in survival. Larvae exposed to two cultured algal species did not exhibit differences in survival, but decreased their swimming activity in response to one species. We conclude that acquiring photosymbionts during the larval stage confers no advantages and can in fact be disadvantageous to this coral host. The timing of symbiont acquisition appears to be a critical component of a host's life history strategy and overall reproductive fitness, and this timing itself appears to be under selective pressure.     

Stability of proICA512/IA-2 and Its Targeting to Insulin Secretory Granules Require β4-Sheet-Mediated Dimerization of Its Ectodomain in the Endoplasmic Reticulum

The type 1 diabetes autoantigen ICA512/IA-2/RPTPN is a receptor protein tyrosine phosphatase of the insulin secretory granules (SGs) which regulates the size of granule stores, possibly via cleavage/signaling of its cytosolic tail. The role of its extracellular region remains unknown. Structural studies indicated that β2- or β4-strands in the mature ectodomain (ME ICA512) form dimers in vitro. Here we show that ME ICA512 prompts proICA512 dimerization in the endoplasmic reticulum. Perturbation of ME ICA512 β2-strand N-glycosylation upon S508A replacement allows for proICA512 dimerization, O-glycosylation, targeting to granules, and conversion, which are instead precluded upon G553D replacement in the ME ICA512 β4-strand. S508A/G553D and N506A/G553D double mutants dimerize but remain in the endoplasmic reticulum. Removal of the N-terminal fragment (ICA512-NTF) preceding ME ICA512 allows an ICA512-ΔNTF G553D mutant to exit the endoplasmic reticulum, and ICA512-ΔNTF is constitutively delivered to the cell surface. The signal for SG sorting is located within the NTF RESP18 homology domain (RESP18-HD), whereas soluble NTF is retained in the endoplasmic reticulum. Hence, we propose that the ME ICA512 β2-strand fosters proICA512 dimerization until NTF prevents N506 glycosylation. Removal of this constraint allows for proICA512 β4-strand-induced dimerization, exit from the endoplasmic reticulum, O-glycosylation, and RESP18-HD-mediated targeting to granules.     

Innate Responses Induced by Whole Inactivated Virus or Subunit Influenza Vaccines in Cultured Dendritic Cells Correlate with Immune Responses In Vivo

Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates.     

Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity

Magnesium (Mg²⁺) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg²⁺ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg²⁺ (re)absorption in kidney and intestine. The apical entry of Mg²⁺ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway.     

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition,but Causes a Transient Reduction of Articular Cartilage Thickness

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.     

Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling

The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetase^L274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.