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

Unraveling the cellular uptake of bioreducible poly(amido amine) — Gene complexes in cells of the retinal pigment epithelium

In vitro endocytosis of gene complexes composed of a bioreducible polyamidoamine CBA ABOL and plasmid DNA, in cells of the retinal pigment epithelium (RPE) was studied, the latter being an interesting target for ocular gene therapy. We found that cationic CBA ABOL DNA polyplexes attach to cell surface proteoglycans of these RPE cells and get subsequently internalized via a phagocytosis-like mechanism, as well as Flotillin dependent endocytosis.\ud \ud Introduction\ud \ud Proper delivery of therapeutic genes to designated cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. To this end, typically sub-micron sized particles are made by combining the therapeutic genes with a carrier material, such as cationic polymers, that aid in delivering the genes to the target site. In this work, for the first time, we have evaluated the ability of the highly promising bioreducible polymer carrier cystamin bisacrylamid aminobutanol (CBA ABOL) [1] (Fig. 1) to deliver plasmid DNA in cultured cells of the retinal pigment epithelium (ARPE-19) and characterized in vitro the cellular interactions with these target cells. These studies are of crucial importance since the further design and functionalization of polymeric gene carriers depend strongly on our understanding of the mechanisms involved in cellular adhesion, intracellular uptake and intracellular processing of the polyplexes.\ud \ud Experimental methods\ud \ud ARPE-19 cells (retinal pigment epithelial cell line; ATCC number CRL-2302) were cultured in DMEM:F12 supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 2% penicillin-streptomycin. All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. CBA ABOL gene complexes with an average hydrodynamic diameter of 130 nm and an average zeta potential of + 45 mV in 20 mM HEPES buffer were obtained by adding the polymer in a mass ratio of 48/1 in 20 mM HEPES to the plasmid and vortexing the mixture for 10 min. For every transfection, fresh polyplexes were prepared and applied to the cells within 30 min after complexation. For all uptake studies, YOYO-1™ (λex = 491 nm, λem = 509 nm, Molecular Probes, Merelbeke, Belgium) labeled pGL4.13 plasmid (Promega, Leiden, The Netherlands) was used. For all transfection studies, gWiz™GFP plasmid (Aldevron, Freiburg, Germany) was used. siRNAs were all purchased from Dharmacon and transfected in cells with the help of LipofectaminRNAiMAX (Invitrogen, Merelbeke, Belgium). Protein knockdown was assessed on Western Blot. Uptake of polyplexes or endocytic markers or GFP expression was measured on a FACS Calibur Flow Cytometer (Beckton Dickinson, Erembodegem, Belgium). For genetic labeling of endosomes, cells were transfected with GFP-fusion proteins.\ud \ud For fluorescence colocalization studies with GFP labeled cellular structures, cells were transfected with GFP-fusion proteins using Lipofectamin2000 (Invitrogen, Merelbeke, Belgium) and 24 h later exposed to red fluorescent labeled polyplexes. For this, CBA ABOL was complexed with pGL4.13 plasmid, covalently labeled with Cy5 (Label IT Nucleic Acid Labeling Kit, Mirus Bio Corporation, WI, USA). Live cell fluorescence colocalization was then performed on a custom built laser epi-fluorescence microscope set-up. A Nikon Plan Apo VC 100× 1.4 NA oil immersion objective lens (Nikon Belux, Brussels, Belgium) was used for imaging. GFP and Cy5 were excited with 491 nm and 636 nm laser light and emission was detected on an EMCCD camera (Roper Scientific, Nieuwegein, The Netherlands). For live cell imaging the cells were placed in a stage top incubation chamber (Tokai Hit, Shizuoka, Japan), set at 37 °C, 5% CO2 and 100% humidity.\ud \ud Result and discussion\ud \ud First, we found evidence that these net positively charged CBA ABOL polyplexes adhere to the negatively charged heparan sulfate proteoglycans (HSPGs) at the cell surface and that polyplex internalization is blocked by antibodies against Toll-like receptor 9\u

siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors

Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates

The increased need for macromolecular therapeutics, such as peptides, proteins and nucleotides, to reach intracellular targets necessitates more effective delivery vectors and a higher level of understanding of their mechanism of action. Cell penetrating peptides (CPPs) can transport a range of macromolecules into cells, either through direct plasma membrane translocation or endocytosis. All known endocytic pathways involve cell-cortex remodelling, a process shown to be regulated by reorganisation of the actin cytoskeleton. Here using flow cytometry, confocal microscopy and a variety of actin inhibitors we identify how actin disorganisation in different cell types differentially influences the cellular entry of three probes: the CPP octaarginine – Alexa488 conjugate (R8-Alexa488), octaarginine conjugated Enhanced Green Fluorescent Protein (EGFP-R8), and the fluid phase probe dextran. Disrupting actin organisation in A431 skin epithelial cells dramatically increases the uptake of EGFP-R8 and dextran, and contrasts strongly to inhibitory effects observed with transferrin and R8 attached to the fluorophore Alexa488. This demonstrates that uptake of the same CPP can occur via different endocytic processes depending on the conjugated fluorescent entity. Overall this study highlights how cargo influences cell uptake of this peptide and that the actin cytoskeleton may act as a gateway or barrier to endocytosis of drug delivery vectors

Fluorescence labelling of extracellular vesicles using a novel thiol-based strategy for quantitative analysis of cellular delivery and intracellular traffic

Extracellular vesicles, including exosomes, are naturally derived nanovesicles generated in and released by numerous cell types. As extracellular entities they have the capacity to interact with neighbouring cells and distant tissues and affect physiological processes as well as being implicated in numerous diseases including tumorigenesis and neurodegeneration. They are also under intense investigation as delivery vectors for biotherapeutics. The ways in which EVs interact with recipient cells to influence cell physiology and deliver a macromolecular payload are at the early stages of exploration. A significant challenge within these studies is the ability to label EVs directly or indirectly with fluorescent probes to allow visualization without compromising functionality. Here, we present a thiol-based fluorescence labelling method allowing comprehensive analysis of the cellular uptake of prostate cancer derived EVs in live cells using confocal microscopy. Labelling of the EVs in this way did not influence their size and had no effect on their ability to induce differentiation of lung fibroblasts to myofibroblasts. For endocytosis analyses, depletion of key endocytic proteins and the use of chemical inhibitors (Dynasore, EIPA, Rottlerin and IPA-3) indicated that fluid-phase endocytosis and/or macropinocytosis was involved in EV internalisation. Over a period of six hours EVs were observed to increasingly co-localise with lysosomes, indicating a possible termination point following internalisation. Overall this method provides new opportunities for analysing the cellular dynamics of EVs as biological entities affecting cell and whole body physiology as well as investigating their potential as drug delivery vectors