Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: Implications for Alzheimer's disease

© 2016 The Author(s). Background: 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). Results: 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. Conclusions: 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

Shiga Toxin 1 Induces on Lipopolysaccharide-Treated Astrocytes the Release of Tumor Necrosis Factor-alpha that Alter Brain-Like Endothelium Integrity

The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS

Applying critical systems thinking to social prescribing: a relational model of stakeholder “buy-in”

Background Social prescribing (SP) allows health professionals to refer primary care patients toward health and wellbeing interventions and activities in the local community. Now widely implemented across the UK and adopted in other nations, questions arise concerning the modelling of present and future schemes, including challenges to full engagement encountered by stakeholders, which lie beyond the scope of traditional evaluations. Critical Systems Thinking (CST) allows for holistic analysis of fields where multiple stakeholders hold diverse interests and unequal power. Methods We use CST to (a) critically examine a developing rural social prescribing scheme from multiple stakeholder perspectives and (b) present a relational model for local social prescribing schemes. Our fieldwork included 24 in-depth interviews, regular planning meetings with key stakeholders, and discussions with those involved with national and international SP landscaping. A modified grounded theory approach was used for the analysis, and to consider the core elements of social prescribing sustainability. Results Our study confirms that local social prescribing schemes must operate with numerous stakeholder interests in mind, seeking to address real life social complexity and offer integrated solutions to multifaceted issues. Three main areas are discussed: holistic vision and boundary judgments; barriers and facilitators; relational issues and “emotional buy in”. Problems for staff include selecting suitable clients, feedback and technological issues and funding and evaluation pressures. Barriers for clients include health, transport and expense issues, also lack of prior information and GP involvement. Emotional “buy-in” emerged as essential for all stakeholders, but hard to sustain. Based on our findings we propose a positive relational model comprising shared vision, confidence and commitment; motivation and encouragement, support and wellbeing focus, collaborative relationships, communication and feedback, access to information /resources, learning in and from action, with emotional “buy-in” at its heart. Conclusion Those implementing social prescribing in different localities inevitably face hard choices about what and whom to include. Research on the sustainability of social prescribing remains limited, studies are required to ascertain which “holistic” models of social prescribing work best for which communities, who are the main beneficiaries of these approaches and how “buy-in” is best sustained