Technical Reference #1629

1629.

Low Molecular Weight Alpha Beta Induces Collapse of Endoplasmic Reticulum Cora Lai; Julie Preisler; Larry Baum; Daniel Lee; Ho-Keung Ng; Jacques Hugon; Kwok-Fai So; Raymond Chang, The University of Hong Kon, Molecular and Cellular Neuroscience, 41(1629), (2009)

Abstract:
The endoplasmic reticulum (ER) is a dynamic multifunction organelle that is responsible for Ca2+homeostasis protein folding post-translational modification protein degradation and transportation ofnascent proteins. Disruption of ER architecture mi

Keywords:
Alzheimer's disease; beta-amyloid; endoplasmic reticulum; autophagy; cytoskeleton

Materials & Methods:
Antibodies and chemicals PDI antibody (1:500) EEA1 (1:400) were from BD Laboratories (San Jose CA USA). LC3 antibody (1:1000) was from Medical and Biological Laboratories (Naka-ku Nagoya Japan). LC3 antibody (1:400) for human sections staining was from Abgent (CA USA). Acetylated-α-tubulin (1:500) and α-tubulin (1:500) antibodies were from Sigma-Aldrich Inc. (Saint Louis USA). Calreticulin antibody (1:200) was from Stressgen Bioreagent (MI USA). Thasagargin Nocodazole and Trichostatin A were purchased from Calbiochem (CA USA). Taxol was purchased from Sigma. Preparation of oligomeric Aβ Aβ1–40 peptides were purchased from Biosource (CA USA). Aβ1–42 was purchased from Yale University. The peptide powder was dissolved in 111333-hexafluoro-2-propanol (Sigma) by vortexing. The peptide solution was then dried down by gentle stream of nitrogen gas. The peptide was resuspended by anhydrous DMSO (Sigma) to a final concentration of 2 mM. The peptide was incubated Fig. 8 (continued). in bath sonicator for 30 min at room temperature. Then the peptide was aliquoted and snap frozen in liquid nitrogen. The aliquoted peptidewas dissolved in culture medium to working concentration for treatment. The structure of Aβ peptides were examined under electron microscope. Both Aβ1–40 and Aβ1–42 peptides contained rod and globular shape structure with about 100 nm in diameter. Primary hippocampal culture and transfection Primary cultures for hippocampal neurons were prepared form day 18 embryonic Sprague–Dawley rats (Laboratory Animal Unit The University of Hong Kong accredited by Association for Assessment and Accreditation of Laboratory Animal Care International). Briefly the meninges were removed and the hippocampi were mechanically dissociated in Tyrode's solution. Neurons were then plated onto poly- L-lysine (25 μg/ml) coated 6- or 12-well plates with cover slips or MatTek (MatTek Ashland MA USA) culture dish at 6×105 7×104 and 1×105 cells per well respectively. Neurons were cultured with Neural Basal medium (GIBCO-BRL Rockville MO USA) supplemented with B-27 supplement L-glutamine (2 mM) penicillin/streptomycin (100 U and 100 μg/ml) and β-mercaptoethanol (10 μM) at 37 °C in a humidified 5% CO2 atmosphere. Deoxyfluorouridine (2 μM) was added 1 day after of plating to inhibit the growth of non-neuronal cells. Neurons were cultured for 12 days prior to treatment. Hippocampal neurons were transfected at DIV 7 for 4 h with Lipofectamine 2000 (Invitrogen Carlsbad CA USA). CLIMP63-GFP construct was a gift from Dr. D.R. Klopfenstein (University of Basel Switzerland). LC3-DsRedwas a gift from Dr. N.S.Wong (The University of Hong Kong Hong Kong). KDEL-DsRed was a gift from Dr. S.K. Kong (Chinese University of Hong Kong). Neurons were treated 48 h after transfection. KDEL-GFP (pShooter-ER-GFP) plasmid was purchased from Invitrogen. Live cell imaging and immunocytochemistry For live cell imaging neurons were cultured on poly-L-lysine coated MetTek glass-bottom dish. Transfection was performed at DIV 7. Experiments were performed on DIV 12. Lysosomes were visualized with live cell stain neurons were incubated with 200 nM LysoTracker Green® (Molecular Probes Invitrogen) 15 min before imaging. Confocal Z-stack images were taken at 1 μm intervals using Carl Zeiss LSM510-Meta system (Göttingen Germany). For immunocytochemical staining neuron were rinsed with TBS fixed in 4% formaldehyde for 20 min permeabilized with 0.1% Triton X-100 in TBS for 5 min at room temperature and washed with TBS twice. Cells were blocked by 10% BSA for 1 h incubated with primary antibodies for 2 h then rinsed with TBS and incubated with secondary antibodies (Anti-mouse Alexa-fluor 488 or 568 1:500 Molecular Probes Eugene OR USA) for 1 h at RT. Confocal images were captured by using Zeiss LSM510-Meta. For the ratio of acetylated-α-tubulin/α-tubulin the signal of gain and offset of acetylated-α-tubulin signal was adjusted to be about the same as α- tubulin in control and then the gain and offset setting was fixed and captured other treated group for comparison. Electron microscopy After treatment hippocampal neurons cultured on coverslip were fixed with 2% glutaldehyde in 0.1 M Na cacodylate buffer for 20 min. After 1 h postfixation with 1% osmium tetroxide neurons were dehydrated in series of ethanol and finally embedded in Embed 812. Glass coverslip was removed. Ultra-thin sections (50–90 nm thick sections) were cut and mounted on copper grids. The sections were further stained with lead citrate and uranyl acetate. The sections were viewed with an 80-kV transmission electron microscope (Philips EM208s) equipped with a Gatan camera for digital imaging. Immunohistochemical staining on human and transgenic mice hippocampal sections Human brain sections containing hippocampal region from agematched control individuals and AD patients (five from each group) were provided by Dr. H.K. Ng (Department of Anatomical and Cellular Pathology at the Chinese University of Hong Kong). The clinical information are as follows (Chang et al. 2002) (Table 1). Brains from transgenic mice expressing human APP with the Swedish mutation (Tg2576) and age-matched control (13 month-old) were fixed embedded sectioned (10 μm) deparaffinized by incubating the sections in xylene for 10 min twice and rehydrated with descending concentrations of ethanol (100% 95% 70% for 3–5 min each). Subsequently the sections were treated heated in 0.01Mcitrate buffer at 80 °C for 15 min for antigen retrieval. The brain sections were then washed with TBS and were blocked with 5% goat serum in 0.5% Triton X-100-containing TBS. For the staining of LC3 and calreticulin the sections were incubated with antibody (1:200 dilutions in background reducing agent) at 4 °C for 2 h. The sections were then stained with secondary antibody anti-mouse or anti-rabbit Alexa fluor-488 or -568 (1:400) for 2 h at room temperature.

Microscopic Technique
Electron Microscopy, Transmission Electron Microscopy

Cell Type(s)
Neurons