p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.     

Chronic Filarial Infection Provides Protection against Bacterial Sepsis by Functionally Reprogramming Macrophages

Helminths immunomodulate their hosts and induce a regulatory, anti-inflammatory milieu that prevents allergies and autoimmune diseases. Helminth immunomodulation may benefit sepsis outcome by preventing exacerbated inflammation and severe pathology, but the influence on bacterial clearance remains unclear. To address this, mice were chronically infected with the filarial nematode Litomosoides sigmodontis (L.s.) and the outcome of acute systemic inflammation caused by i.p. Escherichia coli injection was determined. L.s. infection significantly improved E. coli-induced hypothermia, bacterial clearance and sepsis survival and correlated with reduced concentrations of associated pro-inflammatory cytokines/chemokines and a less pronounced pro-inflammatory macrophage gene expression profile. Improved sepsis outcome in L.s.-infected animals was mediated by macrophages, but independent of the alternatively activated macrophage subset. Endosymbiotic Wolbachia bacteria that are present in most human pathogenic filariae, as well as L.s., signal via TLR2 and modulate macrophage function. Here, gene expression profiles of peritoneal macrophages from L.s.-infected mice revealed a downregulation of genes involved in TLR signaling, and pulsing of macrophages in vitro with L.s. extract reduced LPS-triggered activation. Subsequent transfer improved sepsis outcome in naïve mice in a Wolbachia- and TLR2-dependent manner. In vivo, phagocytosis was increased in macrophages from L.s.-infected wild type, but not TLR2-deficient animals. In association, L.s. infection neither improved bacterial clearance in TLR2-deficient animals nor ameliorated E. coli-induced hypothermia and sepsis survival. These results indicate that chronic L.s. infection has a dual beneficial effect on bacterial sepsis, reducing pro-inflammatory immune responses and improving bacterial control. Thus, helminths and their antigens may not only improve the outcome of autoimmune and allergic diseases, but may also present new therapeutic approaches for acute inflammatory diseases that do not impair bacterial control.     

Fine Mapping of a GWAS-Derived Obesity Candidate Region on Chromosome 16p11.2

IntroductionLarge-scale genome-wide association studies (GWASs) have identified 97 chromosomal loci associated with increased body mass index in population-based studies on adults. One of these SNPs, rs7359397, tags a large region (approx. 1MB) with high linkage disequilibrium (r²>0.7), which comprises five genes (SH2B1, APOBR, sulfotransferases: SULT1A1 and SULT1A2, TUFM). We had previously described a rare mutation in SH2B1 solely identified in extremely obese individuals but not in lean controls.MethodsThe coding regions of the genes APOBR, SULT1A1, SULT1A2, and TUFM were screened for mutations (dHPLC, SSCP, Sanger re-sequencing) in 95 extremely obese children and adolescents. Detected non-synonymous variants were genotyped (TaqMan SNP Genotyping, MALDI TOF, PCR-RFLP) in independent large study groups (up to 3,210 extremely obese/overweight cases, 485 lean controls and 615 obesity trios). In silico tools were used for the prediction of potential functional effects of detected variants.ResultsExcept for TUFM we detected non-synonymous variants in all screened genes. Two polymorphisms rs180743 (APOBR p.Pro428Ala) and rs3833080 (APOBR p.Gly369_Asp370del9) showed nominal association to (extreme) obesity (uncorrected p = 0.003 and p = 0.002, respectively). In silico analyses predicted a functional implication for rs180743 (APOBR p.Pro428Ala). Both APOBR variants are located in the repetitive region with unknown function.ConclusionVariants in APOBR contributed as strongly as variants in SH2B1 to the association with extreme obesity in the chromosomal region chr16p11.2. In silico analyses implied no functional effect of several of the detected variants. Further in vitro or in vivo analyses on the functional implications of the obesity associated variants are warranted.     

Deceit pollination of Orchis spitzelii (Orchidaceae) on the Island of Gotland in the Baltic: a suboptimal system

Interactions between anthophilous insects and Orchis spitzelii were studied on the island of Gotland, in the Baltic Sea of southern Sweden. The plant is nectar - less and thus deceives its nectar-seeking pollinators. No specific nectar-plant model exists for mimetic resemblance, but the orchid produces an exuberant floral display. Pollinators comprise mainly bumble-bee queens ( Bombus spp.) that transport 90% of the pollinaria. The remaining pollinators are cuckoo bumble-bee females ( Psithyrus spp.). Pollinating bees make exploratory visits primarily to only a few of the lowermost flowers in inflorescences. Many-flowered individuals are more attractive and have a higher fitness in terms of fruit set. Bees quickly learn to avoid the non-rewarding orchid. Arctostaphylos uva-ursi (Ericacae) and Rubus saxatilis (Rosaceae) are the most important subsidiary nectar plants for the bees during the flowering of O. spitzelii. Phenological, morphological and functional data indicate that the anth-ecological adaptation to the main pollen vectors is suboptimal. Moreover, experiments indicate that O. spitzelii is an obligate outcrosser and lacks an effective isolating mechanism against the abundant and concurrently blooming Orchis mascula. It is therefore proposed that O. spitzelii has spread to Gotland in recent time.     

Levels of selenium in plasma and glutathione peroxidase in erythrocytes and the risk of breast cancer

Plasma selenium and glutathione peroxidase in erythrocytes were analyzed in a case-control study encompassing 441 cases with breast cancer and 191 controls with benign breast disease. No difference in mean serum selenium level between cases and controls on supplementary selenium intake was seen. If only individuals without supplementary intake, 278 cases and 135 controls, were considered a preventive effect was found increasing with selenium level. This finding was significant among women 50 years old or more with Mantel-Haenszel odds ratio=0.16 for individuals with serum selenium >1.21 μmol/L. Also for subjects with serum selenium in the range 1.00–1.21 μmol/L a significant preventive effect was seen with odds ratio=0.38. For women under 50 years of age a nonsignificant preventive effect was seen. Glutathione peroxidase in erythrocytes did not correlate well with serum selenium and was not a marker for the risk of breast cancer.     

Effect of pH on the association behavior in aqueous solutions of pig gastric mucin

In this study, dynamic light scattering (DLS), turbidity, and rheo-small angle light scattering (rheo-SALS) methods have been utilized to examine the impact of pH (1 < or = pH < or = 7) on aqueous solutions of noncommercial purified pig gastric mucin. The asymmetric flow field-flow fractionation (AFFFF) measurements established that the mucin sample has a high molecular weight and is polydisperse. DLS measurements on dilute solutions of mucin disclosed large interchain aggregates at pH 2, where the polymer has a low charge density or is uncharged. At lower or higher values of pH, mucin is charged and the tendency of forming interpolymer complexes is affected. In the semidilute concentration regime, pronounced junction zones ('lumps' of polymer) are evolved and a heterogeneous connected network is formed at pH 2, whereas the association structures are disintegrated (smaller 'lumps') at lower or higher pH values due to electrostatic repulsive interactions, and a more homogeneous network is evolved. The DLS and viscosity results at pH 1 indicate the development of a fragmented network, composed of contracted chains that are decorated by some positive charges. The effect of shear flow on the structure of semidilute solutions of mucin was investigated with the aid of rheo-SALS methods. The scattered intensity revealed a strong upturn at low values of the wave vector (q) for mucin solutions at pH 2 and pH 4, which suggests the evolution of large association domains. At these pH values, a flow-induced anisotropy in the 2D SALS patterns in the form of elliptical shapes was observed at high shear rates.     

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin-dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. Here, we report age-dependent constitutive activation of glycogen synthase kinase 3β, protein kinase B, mitogen-activated protein kinase, and c- Jun -N-terminal kinase in adult p62^−/− mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62^−/− brain led to recovery of aggregated K63-ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain-derived neurotrophic factor levels. Our findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer-like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid-beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity.     

Effects of beta-cyclodextrin addition and temperature on the modulation of hydrophobic interactions in aqueous solutions of an associative alginate

Novel information about the effects of beta-cyclodextrin (beta-CD) addition and temperature on structural and rheological features of semidilute solutions of alginate and its hydrophobically modified analogue (HM-alginate) is given. Enhanced turbidity is observed for the HM-alginate solutions at high levels of beta-CD addition and low temperatures. The viscosity results revealed cross-linking of the alginate chains at high beta-CD concentrations and low temperatures. Rheological results for the HM-alginate solutions demonstrated that high levels of beta-CD addition and elevated temperatures promoted decoupling of the hydrophobic polymer-polymer associations via inclusion complex formation between beta-CD cavities and the hydrophobic side chains of the polymer. Analysis of small-angle neutron scattering (SANS) results from HM-alginate solutions in the presence of beta-CD suggested that the polymer chains are locally stretched at all of the considered levels of beta-CD and temperatures. The SANS results revealed association structures. The general picture that emerges is that beta-CD addition and temperature can be combined to tune the intensity of the hydrophobic interactions and to cross-link the unmodified alginate.