A Potential Role for Drosophila Mucins in Development and Physiology

Vital vertebrate organs are protected from the external environment by a barrier that to a large extent consists of mucins. These proteins are characterized by poorly conserved repeated sequences that are rich in prolines and potentially glycosylated threonines and serines (PTS). We have now used the characteristics of the PTS repeat domain to identify Drosophila mucins in a simple bioinformatics approach. Searching the predicted protein database for proteins with at least 4 repeats and a high ST content, more than 30 mucin-like proteins were identified, ranging from 300–23000 amino acids in length. We find that Drosophila mucins are present at all stages of the fly life cycle, and that their transcripts localize to selective organs analogous to sites of vertebrate mucin expression. The results could allow for addressing basic questions about human mucin-related diseases in this model system. Additionally, many of the mucins are expressed in selective tissues during embryogenesis, thus revealing new potential functions for mucins as apical matrix components during organ morphogenesis

Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

An engineered protein prevents aggregation of the Aβ peptide and facilitates clearance of Aβ from the brain in a fruit fly model of Alzheimer's disease

Human antibody repertoires in normal physiology and in autoimmune disease

The immune system has to balance the need for a broad protection from infectious agents against the risk of developing autoimmune disease. The elements of the immune system should provide clues about conditions under which physiological autoreactivity may develop into autoaggressive immunity, if analyzed against the background of the immune system in healthy individuals. A possible object for this analysis is the antibody repertoire, since antibodies have been implicated in the effector phase and etiology of several autoimmune diseases. Therefore, we studied utilization of the genes coding for the variable part of the immunoglobulin molecule in healthy individuals as well as in patients with autoimmune disease, using RNA in situ hybridization and a newly developed competitive quantitative DNA-based PCR. For the analysis of gene utilization and mutational frequencies, we used a VH-family specific PCR in combination with cloning and nucleotide sequence analysis. Our results demonstrate that in adults the VH gene family repertoire is remarkably stable in time, as well as very similar between individuals of similar genetic backgrounds. We also found differences in VH, D and JH expression between lymphocytes from the spleen and from peripheral blood. On the level of the single gene we found, in healthy individuals, an age-related differential utilization of D and JH genes and an impaired affinity maturation in VH6-containing rearrangements. The number of cells carrying mutations decreased with age, as did the number of mutations per gene. To learn more about the possible role of B cell repertoires in the pathogenesis of autoimmune aggression, we studied two autoimmune diseases: IDDM (insulin dependent diabetes mellitus) as a model for a T cell mediated disease, and AITP (autoimmune idiopathic thrombocytopenic purpura) as a model for an antibody mediated disease. On VH gene family level, our results show an increase in VH6 gene utilization in the spleen of AITP patients. Interestingly, this change occurs in small resting lymphocytes rather than in naturally activated ones, indicating the absence of selection and actual utilization of the VH6 gene in these patients. This was confirmed by a sequence analysis of recombinations containing the VH6 gene, in which we found low mutational frequencies in functional VH6-containing recombinations both in AITP and in IDDM patients. Among non- functional rearrangements, however, we observed an increase in mutational frequencies in VH6-containing recombinations that appeared to be based in part on different mechanisms. In IDDM patients, the higher mutational frequencies were due both to an increase in the relative number of VH6 genes carrying mutations and to an actual increase in the number of mutations per gene, which suggests the presence of a high number of activated and differentiated B cells in the periphery. In AITP patients, however, only the number of clones that carried mutations was increased. In conclusion, the VH gene family repertoire appears to be remarkably stable both in time and between individuals, indicating a strict, genetic control. Deviations found in patients with autoimmune disease indicate a modified selection which, however, does not affect the actual VH gene family repertoire of these patients, Therefore, these deviations are more likely a consequence than a cause of the autoimmune process. The age- related changes found in our studies do not correlate with the changes observed in our two models of autoimmune disease and are therefore not likely to be related to the reported increase in autoreactivity in elderly individuals

Neurocognitive function and mortality in patients with schizophrenia spectrum disorders

Individuals with schizophrenia spectrum disorders (SSD) have significantly lower life-expectancy than healthy people. Previously, we have identified baseline neurocognitive function in general and verbal memory and executive function in particular as related to mortality nearly two decades later. In this study, we aim to replicate these findings with a larger and age-matched sample. The patient group consisted of 252 individuals, 44 of whom were deceased and 206 alive. Neurocognition was assessed with a comprehensive battery. Results showed that the deceased group, compared to the living group, had significantly more severe neurocognitive deficits across nearly all domains. There were no differences in sex, remission status, psychosis symptoms, or function level between the groups. Immediate verbal memory and executive function were the strongest predictors of survival status. These results were nearly identical to our previous studies, and we conclude that baseline neurocognitive function is an important predictor for mortality in SSD. Clinicians should be mindful of this relationship in patients with significant cognitive deficits

A luminal glycoprotein drives dose-dependent diameter expansion of the Drosophila melanogaster hindgut tube.

International audienceAn important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein

Tnc is required for hindgut lumen diameter expansion.

<p>(A–C) Wild type embryos were labelled for Crb (magenta) and Dg (green) to visualize the apical and basal surfaces of the hindgut epithelium. Arrows point to anterior and posterior Li borders, and stippled lines mark outer tube diameter. Crb-staining only is shown in A′–C′. At stage 13 (A, lateral view) the hindgut is narrow with an anterior hook pointing ventral. By stage 14, (B, dorsal view) the anterior hook has turned right and Crb-expressing border cells demarcate hindgut subdomains. From stage 14 to stage 16 (C, dorsal view) the hindgut expands in diameter and length. (D–F) <i>tnc^13c</i> mutant embryos stained for Crb revealed a normal hindgut lumen at stage 13 (D), slight reduction in lumen diameter at stage 14 (E) and a clear reduction in lumen diameter at stage 16 (F), compared to the wild type. White and open arrowheads in (C′) and (F) illustrate lumen diameter of Li and Si, respectively. (G) Drawings of the hindgut lumen at stages 14 and 16 with Si in red and Li in blue. Border cells (black lines) mark anterior and posterior boundaries of Li and separates dorsal Li (dLi) and ventral Li (vLi). (H) The graphs show mean lumen diameter of Si and Li at stages 14 and 16 in the wild type and at stage 16 in <i>tnc^13c</i> mutants. Li expands in diameter from 6.3 (+/−0.09) µm to 8.8 (+/−0.16) µm, and Si from 8.9 (+/−0.52) µm to 17.3 (+/−0.56) µm. * = P-value<0.05. Bars represent standard error of mean (n>5). Scale bar: 10 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s002" target="_blank">Figures S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s003" target="_blank">S3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s004" target="_blank">S4</a>.</p

Tnc is a glycosylated intralumial matrix-component.

<p>(A) Tnc from wild type and <i>tnc^13c</i> mutant embryos and larvae (l) were detected on western blot and resided as high-molecular weight species in the stacking gel (stippled line indicates end of stacking gel). Anti-α-Tubulin was used as loading control. (B) Protein extracts from stage 16 wild type embryos were subjected to deglycosylation (no enz = no enzyme, N-Gly = N-Glycanase (PNGase F), O-Gly = O-Glycanase, O-Gly+ = O-Glycanase+Sialidase+β(1-4) Galactosidase+β-N-Acetylglucosaminidase). Addition of O-glycanase caused slightly faster migration of Tnc. (C–H) Embryos were co-labelled for the Tn antigen (green) and Crb (magenta) (C, D, F and G) or with VVA (E and H). Anti-Tn stains the wild type lumen with highest intensity in Si at stages 15 and 16 (C and D). The staining is reduced in mutant embryos (F and G). Arrows point to the Si/Li border. VVA also labels the wild type lumen (E) stronger than the mutant lumen (H). The embryos were processed in parallel and the hindgut was imaged at similar views with identical confocal settings. (I) Wild type embryos were prepared with Clark's fixation and stained for Tnc (I, green) and the Tn antigen (I′, magenta). The merged image (I″) shows partial overlap of the staining. Arrow points to the Si/Li border. (J) High magnification of the hindgut in (I), showing a striated pattern of Tnc-staining. Scale bars: 10 µm in I, 5 µm in J.</p

Tnc acts non-cell-autonomously.

<p>(A) <i>enGAL4</i> drives expression of <i>UAS-GFP</i> in the dorsal Li (bracket), as seen by labelling with anti-GFP (green) and anti-Crb (magenta). (B and C) Stage 16 embryos stained for DECad and Crb show that <i>enGAL4</i>-driven expression of <i>tnc</i> in dorsal Li (bracket) caused enlarged apical cell circumferences in both dorsal and ventral Li (C), when compared to embryos that only express <i>enGAL4</i> (B). (D–G) <i>enGAL4</i> drives expression of <i>UAS-GFP</i> (green) in a cluster of cells in the anterior salivary gland (D, stage 13). <i>enGAL4</i>-driven expression of Tnc in salivary glands resulted in local tube dilation (E). By merging serial z-stacked images, the apical cell circumferences were visualized (F). Note that <i>enGAL4</i> drives expression in one side of the tube, but all cells at the perimeter show enlarged apical cell circumference. Co-staining for Tnc (green) and Crb (magenta) shows that luminal Tnc localizes to the dilated part of the salivary gland lumen (G). (H) A possible model for the function of Tnc during lumen dilation. Expression of <i>tnc</i> in the tubular epithelium causes tube dilation according to the level of expression (“low" and “high") and causes differential dilation along the tube. Once inside the lumen, Tnc acts on surrounding cells, possibly by generating a mechanical pressure, to expand the tube wall.</p

Loss of <i>tnc</i> causes reduced apical cell circumferences, altered cell arrangement, and a smaller outer tube diameter.

<p>(A–H) Embryos were labelled with anti-DECad, and serial z-stacked images spanning the upper half of the hindgut tube were merged to reveal apical cell circumferences. Arrows point to the Si/Li border. The hindgut of a wild type and <i>tnc</i> mutant embryo at dorsal view (A and B) is shown together with high magnifications of respective Si (in C and D) and posterior Li (in E and F). Identically sized squares (in C and D) span ∼10 cells of the wild type Si and ∼18 cells of the mutant Si. Brackets of identical size (in E and F) illustrate that cells are more elongated along the lumen perimeter in the wild type compared to the mutant. In the dorsal Li (G and H, ventral view), identically sized brackets span 9 or 11 cells along the border cells in the wild type and mutant hindgut, respectively. The mutant hindgut also has fewer cells at the dorsal lumen perimeter. (I and J) The outer hindgut diameter, visualized by labelling for Dg (green) and merging serial z-stacked images that span the entire hindgut, is reduced in the mutants. Stippled and full lines represent wild type Si and Li diameter, respectively. (K and L) Labelling with anti-Fas3 (green) reveal comparable level and distribution of Fas3 (bracket) in the hindgut epithelium of wild type (K) and <i>tnc</i> mutant (L) embryos. Co-staining for Tnc (magenta) shows the absence of Tnc in the mutant hindgut. All images are of stage 16 embryos. Scale bars: 10 µm in A (A and B), 10 µm in C (C–H), 10 µm in I (I and J), 10 µm in K (K and L).</p