Participation of Presenilin 2 in apoptosis: Enhanced basal activity conferred by an Alzheimer mutation

Science

Volumn 274, Issue 5293, 1996, Pages 1710-1712

  Wolozin, Benjamin ^a,f   Iwasaki, Katsunori ^a   Vito, Pasquale ^b   Ganjei, J Kelly ^b   Lacanà, Emanuela ^b   Sunderland, Trey ^a   Zhao, Boyu ^c   Kusiak, John W ^c   Wasco, Wilma ^d   D'Adamio, Luciano ^e  

^a NATIONAL INSTITUTE OF MENTAL HEALTH   (United States)

^b NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES   (United States)

^c NATIONAL INSTITUTE ON AGING   (United States)

^d HARVARD MEDICAL SCHOOL   (United States)

^e NATIONAL INSTITUTES OF HEALTH   (United States)

^f LOYOLA UNIVERSITY MEDICAL CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AMYLOID BETA PROTEIN; CELL PROTEIN; GUANINE NUCLEOTIDE BINDING PROTEIN; NERVE GROWTH FACTOR; PERTUSSIS TOXIN; PRESENILIN 2;

ALZHEIMER DISEASE; ANIMAL CELL; ARTICLE; BRAIN PROTECTION; CONTROLLED STUDY; GENE EXPRESSION REGULATION; GENETIC SUSCEPTIBILITY; HUMAN; HUMAN CELL; MOUSE; NERVE CELL DIFFERENTIATION; NERVE DEGENERATION; NONHUMAN; ONSET AGE; PRIORITY JOURNAL;

ALZHEIMER DISEASE; AMYLOID BETA-PROTEIN; AMYLOID BETA-PROTEIN PRECURSOR; ANIMALS; APOPTOSIS; DNA, ANTISENSE; GTP-BINDING PROTEIN ALPHA SUBUNITS, GI-GO; GTP-BINDING PROTEIN ALPHA SUBUNITS, GS; HUMANS; MEMBRANE PROTEINS; MUTATION; NERVE GROWTH FACTORS; NEURONS; PC12 CELLS; PEPTIDE FRAGMENTS; PERTUSSIS TOXIN; PRESENILIN-2; RATS; TRANSFECTION; VIRULENCE FACTORS, BORDETELLA;

ANIMALIA;

EID: 10544224542     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5293.1710     Document Type: Article

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30. PC12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing NGF (100 ng/ ml), 10% fetal bovine serum (FBS). 5% horse serum, and gentamycin sulfate (50 ng/ml). The cells were plated into 60-mm dishes and transfected the following day with lipofectamine (12 μl/ml) (Gibco-BRL) and plasmid (2 μg/ml). Trophic deprivation was accomplished by washing the cells twice with DMEM and then maintaining the cells in DMEM plus gentamycin sulfate (50 ng/ml). For extraction of apoptotic DNA fragmented in the internucleosomal spaces, cells were pelleted and suspended in 100 μl of 10 mM tris, 0.1 mM EDTA (pH 8.0) (TE). A volume of 200 μl of 2% NP-40 in TE was added, and the cells were agitated for 30 min at 4°C and spun for 15 min in a microfuge at 4°C. Supernatants were extracted with phenol and chloroform and precipitated in 300 mM sodium acetate (pH 5.1) and two volumes of ethanol. DNA was separated on a 1.5% agarose gel by electrophoresis.
31. For TUNEL analysis, the day after transfection cells were treated with trypsin and transferred to chamber slides. A peroxidase-based TACS-TdT kit (Trevigen) was used according to the manufacturer's directions. After staining for apoptosis, three to six fields were chosen at random, and the numbers of total nuclei and apoptotic nuclei were counted. The experiments were repeated four to eight times with similar results.
32. For protein immunoblot analysis, the cells were harvested 3 days after transfection, lysed in radioimmunoprecipitation assay (RIPA) buffer containing the protease inhibitors aprotinin, pepstatin, and leupeptin (100 μg/ml each, Sigma). Cell lysates (10 μg) were denatured in modified 5X loading buffer (320 mM tris, pH 6.8, 50% glycerol, 0.5% bromophenol blue, 10% SDS, 100 mM dithiothreitol, and 8M urea) to reduce aggregation that typically occurs with proteins having seven transmembrane domains. After heating at 37°C for 45 min and then 75°C for 5 min, proteins were separated on a 4 to 20% polyacrylamide-SDS gradient gel, blotted onto nitrocellulose membranes (Gelman Science), and probed with either affinity-purified anti-PS2n (3), anti-APP (22C11, Boehringer Mannheim), or anti-β-tubulin (Boehringer Mannheim). Immunoblots were developed with the ECL System (Amersham).
33. For immunocytochemistry, 1 day after transfection cells were treated with trypsin and plated onto cover slips in a 24-well plate. The following day the cells were fixed for 20 min in 3% paraformaldehyde at 4°C, washed once with 1.5% glycine in PBS, twice with PBS, and blocked for 1 hour at 37°C with 10% bovine serum albumin in PBS. Samples were washed three times with 0.1% saponin in PBS (Sigma), incubated for 1 hour at 4°C with anti-FLAG (M2, Eastman Kodak), diluted to 5 μg/ml in PBS-0.1% saponin, and washed three more times with PBS-0.1% saponin. Cells were then stained for 1 hour at 4°C with fluorescein isothiocyanate-conjugated donkey antibody to mouse immunoglobulin G diluted 1:200 in PBS-0.1% saponin. After three washes, the cover slips were mounted onto a slide with fluoromount-g. Cells were viewed on a confocal Bio-Rad MRC1024 microscope at a ×20 magnification.
34. TUNEL studies were performed as described (17). PTX (Sigma, St. Louis, MO) was added once to achieve a final concentration of 100 ng/ml on day 2 and maintained in the medium until day 4.
35. For Aβ treatments the cells were maintained in DMEM containing 10% dialyzed FBS, 5% dialyzed horse serum, and gentamycin (50 ng/ml). The cells were treated with 10 μM Aβ( 1 - 42) (generated from a 1 mM Aβ stock solution that had been aged 7 days at 37°C) starting on day 2 after the transfection. For all studies, the cells were fixed on day 4 and analyzed by TUNEL.
36. We would like to thank R. Schwartz and D. LeRoith for their helpful review and useful comments and J. Yewdell, J. Bennick, and P. Day for their time and expertise in using the confocal microscope.