title: Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: implications for Alzheimer's disease.
abstract: 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.
authors.affiliation: School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, UK.
authors.forename: Rhian S
authors.initials: RS
authors.lastname: Thomas
authors.affiliation: School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, UK.
authors.forename: Alex
authors.initials: A
authors.lastname: Henson
authors.affiliation: MRC Centre for Neuropsychiatric Genetics and Genomics, School of Medicine, Cardiff University, Hadyn Ellis Building, Maindy Road, Cardiff, CF24 4HQ, UK.|West Midlands Regional Genetics Laboratory, Birmingham Women's NHS Foundation Trust, Mindelsohn Way, Edgbaston, Birmingham, B15 2TG, UK.
authors.forename: Amy
authors.initials: A
authors.lastname: Gerrish
authors.affiliation: MRC Centre for Neuropsychiatric Genetics and Genomics, School of Medicine, Cardiff University, Hadyn Ellis Building, Maindy Road, Cardiff, CF24 4HQ, UK.
authors.forename: Lesley
authors.initials: L
authors.lastname: Jones
authors.affiliation: MRC Centre for Neuropsychiatric Genetics and Genomics, School of Medicine, Cardiff University, Hadyn Ellis Building, Maindy Road, Cardiff, CF24 4HQ, UK.
authors.forename: Julie
authors.initials: J
authors.lastname: Williams
authors.affiliation: School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, UK. KiddEJ@cf.ac.uk.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Kidd
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D034041:Monomeric Clathrin Assembly Proteins / Q000172:deficiency* / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D020033:Protein Isoforms / Q000172:deficiency / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D014168:Transferrin / Q000378:metabolism
chemical_list: C545487:APP protein, human; D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D034041:Monomeric Clathrin Assembly Proteins; C439003:PICALM protein, human; D010446:Peptide Fragments; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014168:Transferrin; C078592:amyloid beta-protein (1-40); D033922:Clathrin Heavy Chains; D053829:Amyloid Precursor Protein Secretases
keywords: Alzheimer’s disease; Amyloid precursor protein; Clathrin-mediated endocytosis; PICALM; β-C-terminal fragment; β-Secretase