Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis.

저자: Xiao Q, Gil SC, Yan P, Wang Y, Han S, Gonzales E, Perez R, Cirrito JR, Lee JM.

저널: J Biol Chem. 2012 Jun 15;287(25):21279-89

사용한 제품: Dynabeads Streptavidin MyOne T1 (Cat# 65601, 65602)

Abstract

One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism.

제품 사용법

Cell Surface Biotinylation and Internalization Assays

 Cell surface biotinylation and internalization assays were performed as described (22) with modifications. Briefly, cells in 6-well plates were washed with cold PBS, and surface proteins were labeled with a non-membrane-permeant, cleavable biotin derivative, sulfo-NHS-SS-biotin (1 mg/ml in PBS). Cells were kept at 4 °C for 30 min in the dark and gently rocked during the incubation period. The biotin reagent was quenched by treating the cells with two 15-min washes of 0.1 M glycine in PBS. Cells were rinsed with PBS and lysed in radioimmune precipitation assay buffer containing 50mM Tris-HCl, pH 7.5, 150mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) deoxycholate, and 1 protease inhibitor mixture. Lysates were incubated with Dynabeads MyOne Streptavidin T1 (Invitrogen) for 2 h in a rotary mixer to isolate biotin-labeled proteins. For the internalization assay, surface biotin-labeled cells were incubated for varying times at 37 °C to allow internalization. Internalization was stopped by rapid cooling on ice. To cleave biotin exposed at the cell surface, cells were incubated three times for 20 min at 4 °C with 50 mM 2-mercaptoethanesulfonic acid (Sigma) in 50mM Tris-HCl, pH 8.7, 100 mM NaCl, and 2.5 mM CaCl2. After thorough rinsing with PBS containing 20 mM Hepes, cells were lysed in radioimmune precipitation assay buffer, and internalized biotinylated proteins were immunoprecipitated with streptavidin and subjected to immunoblotting for APP (CT695; Invitrogen).

 To analyze transferrin uptake, cells were rinsed with DMEM twice and cultured in serum-free medium for 2 h. Cells were incubated with biotin-labeled transferrin (20  g/ml) in serumfree medium for various time periods at 37 °C. Cells were then placed on ice, washed with ice-cold PBS twice, and then subsequently incubated three times for 10 min on ice in 25 mM MesNa, pH 5.0 containing 150mM NaCl and 50 M deferoxamine mesylate and for 10 min in PBS. Cells were lysed with radioimmune precipitation assay buffer, and the lysates were fractionated by SDS-PAGE followed by immunoblotting with anti-biotin antibody.