Polymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer’s disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME). Following depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression. The depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD.

Introduction: Articular cartilage functions in withstanding mechanical loads and provides a lubricating surface for frictionless movement of joints. Osteoarthritis, characterised by cartilage degeneration, develops due to the progressive erosion of structural integrity and eventual loss of functional performance. Osteoarthritis is a multi-factorial disorder; two important risk factors are abnormal mechanical load and genetic predisposition. A single nucleotide polymorphism analysis demonstrated an association of hip osteoarthritis with an Arg324Gly substitution mutation in FrzB, a Wnt antagonist. The purpose of this study was two-fold: to assess whether mechanical stimulation modulates β-catenin signalling and catabolic gene expression in articular chondrocytes, and further to investigate whether there is an interplay of mechanical load and Wnt signalling in mediating a catabolic response. Methods: Chondrocytes were pre-stimulated with recombinant Wnt3A for 24 hours prior to the application of tensile strain (7.5%, 1 Hz) for 30 minutes. Activation of Wnt signalling, via β-catenin nuclear translocation and downstream effects including the transcriptional activation of c-jun, c-fos and Lef1, markers of chondrocyte phenotype (type II collagen (col2a1), aggrecan (acan), SOX9) and catabolic genes (MMP3, MMP13, ADAMTS-4, ADAMTS-5) were assessed. Results: Physiological tensile strain induced col2a1, acan and SOX9 transcription. Load-induced acan and SOX9 expression were repressed in the presence of Wnt3A. Load induced partial β-catenin nuclear translocation; there was an additive effect of load and Wnt3A on β-catenin distribution, with both extensive localisation in the nucleus and cytoplasm. Immediate early response (c-jun) and catabolic genes (MMP3, ADAMTS-4) were up-regulated in Wnt3A stimulated chondrocytes. With load and Wnt3A there was an additive up-regulation of c-fos, MMP3 and ADAMTS-4 transcription, whereas there was a synergistic interplay on c-jun, Lef1 and ADAMTS-5 transcription. Conclusion: Our data suggest that load and Wnt, in combination, can repress transcription of chondrocyte matrix genes, whilst enhancing expression of catabolic mediators. Future studies will investigate the respective roles of abnormal loading and genetic predisposition in mediating cartilage degeneration.

Background and Purpose: Asthma exacerbations contribute to corticosteroid insensitivity. LPS is ubiquitous in the environment. It causes bronchoconstriction and airway inflammation and may therefore exacerbate allergen responses. This study examined whether LPS and ovalbumin co‐administration could exacerbate the airway inflammatory and functional responses to ovalbumin in conscious guinea pigs and whether these exacerbated responses were insensitive to inhaled corticosteroid treatment with fluticasone propionate (FP).Experimental Approach: Guinea pigs were sensitized and challenged with ovalbumin and airway function recorded as specific airway conductance by whole body plethysmography. Airway inflammation was measured from lung histology and bronchoalveolar lavage. Airway hyper‐reactivity (AHR) to inhaled histamine was examined 24 h after ovalbumin. LPS was inhaled alone or 24 or 48 h before ovalbumin and combined with ovalbumin. FP (0.05–1 mg·mL⁻¹) or vehicle was nebulized for 15 min twice daily for 6 days before ovalbumin or LPS exposure.Key Results: Ovalbumin inhalation caused early (EAR) and late asthmatic response (LAR), airway hyper‐reactivity to histamine and influx of inflammatory cells into the lungs. LPS 48 h before and co‐administered with ovalbumin exacerbated the response with increased length of the EAR, prolonged response to histamine and elevated inflammatory cells. FP 0.5 and 1 mg·mL⁻¹ reduced the LAR, AHR and cell influx with ovalbumin alone, but was ineffective when guinea pigs were exposed to LPS before and with ovalbumin.Conclusions and Implications: LPS exposure exacerbates airway inflammatory and functional responses to allergen inhalation and decreases corticosteroid sensitivity. Its widespread presence in the environment could contribute to asthma exacerbations and corticosteroid insensitivity in humans.