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.

O-glycosylation regulates polarized secretion by modulating Tango1 stability.

Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. Here, we demonstrate that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. Our studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, our data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases.

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

An 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.

Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo.

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta) peptide by using a small engineered binding protein (Z(Abeta3)) that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3) in the brains of Drosophila melanogaster expressing either Abeta(42) or the aggressive familial associated E22G variant of Abeta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3) not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

Localization of O-glycans in MUC1 glycoproteins using electron-capture dissociation fragmentation mass spectrometry.

MUC1 is a mucin glycoprotein containing multiple tandem repeats of 20 amino acids, with five serines and threonines that can be O-glycosylated. Here, we investigated the O-glycosylation site occupancy in MUC1 glycoproteins produced in two mutant CHO cell lines, Lec3.2.8.1 and ldlD. We found that the average site occupancy was higher in MUC1 from Lec3.2.8.1 than from ldlD and that the occupancy increased with the number of tandem repeats in the protein and also depended on the culture conditions used for production. Moreover, we describe the successful use of electron-capture dissociation (ECD) fragmentation, coupled to online liquid chromatography mass spectrometry, to determine the glycosylation of individual sites in recombinant MUC1 proteins with 16 tandem repeats. We analyzed MUC1 tandem repeat peptides with 1-5 GalNAc residues by ECD fragmentation and found that the first site to be glycosylated was either Ser-5 or Thr-6, with the addition of a second GalNAc at Thr-14. For peptides with three GalNAc residues, several different variants of glycopeptides were found, indicating a heterogeneous order of glycosylation at this stage. In contrast, only one variant was found for peptides with four GalNAc residues, where Thr-19 in the PDTR motif was left unglycosylated, indicating that this site is glycosylated last. The results gave novel insight into the order of GalNAc substitution in MUC1 in vivo.

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.

ATM is not required in somatic hypermutation of VH, but is involved in the introduction of mutations in the switch mu region.

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes that share common key factors such as activation-induced cytidine deaminase. We have previously shown a role for ATM (mutated in ataxia-telangiectasia) in CSR. In this paper we show that the frequency, distribution, and nature of base pair substitutions in the Ig variable (V) heavy chain genes in ataxia-telangiectasia patients are largely similar to those in normal donors, suggesting a normal SHM process. Characterization of the third complementarity-determining region in B cells from ataxia-telangiectasia patients also shows a normal V(D)J recombination process. SHM-like mutations could be identified in the switch (S) mu region (up to several hundred base pairs upstream of the S mu -S(alpha) breakpoints) in normal in vivo switched human B cells. In the absence of ATM, mutations can still be found in this region, but at less than half the frequency of that in normal donors. The latter mutations are mainly due to transitions (86% compared with 58% in controls) and are biased to A or T nucleotides. An ATM-dependent mechanism, different from that generating SHM in V genes, is therefore likely to be involved in introducing SHM-like mutations in the S region. ATM may thus be one of the factors that is not shared by the CSR and SHM processes.

Long-term kinetics of adult human antibody repertoires.

In healthy humans, antibody repertoires change during ontogeny and senescence. The dynamics of antibody repertoires among adults over a longer period of time in one and the same individual has, however, not been extensively studied. In this study we analysed peripheral blood samples from five healthy adults, taken over a period of 10 weeks and once 9 years later. A competitive, quantitative polymerase chain reaction (PCR) was developed to investigate short and long-term variations in VH gene family repertoires. Serum antibody levels to common self and non-self antigens were determined in samples taken at the same time-points as the cell samples to analyse possible correlations between molecular and serological expression profiles. We found a high degree of stability in the VH gene family repertoire over time as well as between individuals with a Caucasian background. A specific change in the usage of primarily the VH3 and VH5 gene families was observed in one individual at one time-point. The deviating pattern resembled the VH gene family utilization pattern observed in naturally activated B lymphocytes. The fluctuations in VH3 and VH5 gene family expression correlated with the presence of rheumatoid factor in serum. We discuss the possible influence of polyclonal, transient stimulation of B cells on VH gene repertoires, as measured in circulating B cells.