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Open Access Highly Accessed Research article

Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation

Richard C Davis1, Ian T Marsden1, Michael T Maloney13, Laurie S Minamide1, Marcia Podlisny2, Dennis J Selkoe2 and James R Bamburg1*

Author Affiliations

1 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA

2 Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA

3 Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA

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Molecular Neurodegeneration 2011, 6:10  doi:10.1186/1750-1326-6-10

Published: 24 January 2011

Additional files

Additional file 1:

Preparation and quantification of Aβd/t. (A) Aβ Western blot (6E10 antibody) of gel filtration fractions from a single Superdex75 (10/30 HR) column run at a flow rate of 0.5 mL/min and loaded with 1 mL of a 10X concentrate of 7PA2 cell conditioned (16 h) DMEM medium [20,43]. (B, C) Dot blot standard curve for quantification of Aβ monomer equivalents in the 7PA2 culture medium and final Aβd/t fraction.

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Additional file 2:

Western blot showing time course of oxidative changes in synthetic human Aβ1-42 incubated under different conditions to generate SDS-stable dimers and higher oligomers. Synthetic human Aβ was dissolved to 5 μM directly into PBS alone or PBS containing 250 μM hydrogen peroxide, 25 μM CuCl2, or peroxide plus CuCl2, incubated at 37°C, and aliquots were removed at 1 day intervals for the times shown to examine the species present by Western blotting. After transfer, the blotting membrane was heated to boiling to expose the epitopes for detection with the 6E10 antibody.

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Additional file 3:

Western blot showing fractions of gel filtration column of Cu2+-peroxide-treated synthetic human Aβ1-42. Aβ Western blot (6E10 antibody) of gel filtration fractions from a single Superdex75 (10/30 HR) column run at a flow rate of 0.5 mL/min and loaded with 1 mL of the Cu2+-peroxide-treated synthetic human Aβ1-42 after 5 days of incubation. Fraction volumes are 0.5 mL and column void volume is about 6.5 mL. Two peaks of Aβ elute, one at the void volume and the second near the peak of the Aβd/t elution (about 12 ml). Upper plot shows the column calibration with points for chicken egg albumin (44 kDa), horse myoglobin (17 kDa) and vitamin B12 (1.35 kDa). Combined fractions of the high-n and low-n Aβ species used for the rod-induction assay in Figure 3B are underlined.

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Additional file 4:

The Aβd/t fraction remains stable for 48 h when incubated with neurons. Immunoprecipitates from 7PA2 medium (IP positive controls on left) and from neuronal culture medium 48 h after treatment with Aβd/t, the equivalent fraction from NC medium, or the monomer fraction. The load volume on the right is equivalent to 0.4 mL of starting 7PA2 medium and the dimer/trimer bands are slightly less than what is contained in the 0.5 mL of starting medium showing that the d/t fraction is stable over the 48 h of culture.

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Additional file 5:

Measurement of adenoviral infection efficiency of cells in organotypic slices. (A) Low magnification (4x objective) images of a hippocampal organotypic slice stained with DAPI, immunostained for cofilin, and imaged for GFP-expression after infection with adenovirus expressing GFP behind a CMV promoter. (B) Infection efficiency in organotypic slices, measured by the numbers of cells showing GFP fluorescence around a DAPI stained nucleus. Between 70-75% of the cells so examined were positive for GFP. (C) This panel was assembled to illustrate how we determined the percentage of infected cells. One region from a confocal plane of an image stack (60x objective) is shown. The areas surrounding nuclei within this plane were examined for GFP expression, which had to be above a threshold level to be counted as positive. Nuclei were often above or below the optical section containing the GFP and we used a Z stack to obtain the most accurate counts. Hundreds of nuclei across different areas were examined to obtain the infection efficiency.

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