Fig 1.
Representative amyloid-beta (Aβ) immunohistochemistry demonstrates similar levels of pathology between elderly high-pathology controls and elderly subjects with mild ADD.
Scale bar = 0.5 cm, applies to panels A-F. (A-C) Aβ plaque pathology in frontal cortex sections from nondemented elderly subjects (CDR 0 + plaques). (D-F) Aβ plaque pathology in frontal cortex sections from elderly subjects with mild ADD (CDR 1). (G) Gray matter coverage by Aβ plaque pathology was not different in the nondemented elderly subjects with plaques (CDR 0 + plaques) versus subjects with mild ADD (CDR 1) (not significant [n.s.] by Mann–Whitney U test).
Table 1.
Characteristics of human brain frontal cortex samples.
Fig 2.
Exemplar fluorescent confocal microscopy of Aβ1-42-biotin binding to unfixed, frozen frontal cortex sections.
(A, D) Representative fluorescent confocal images of labelled (streptavidin-Alexa594, red channel) Aβ1-42-biotin binding in elderly high-pathology controls (CDR0 + plaques) and elderly subjects with mild ADD (CDR1). Scale bar = 200 μm. (B, E) Higher magnification confocal images reveal distinct plaque morphology and minimal background fluorescence in both in elderly high-pathology controls (CDR0 + plaques) and elderly subjects with mild ADD (CDR1). Scale bar = 20 μm. (C) Fluorescent images of labelled (streptavidin-Alexa594, red channel) Aβ1-42-biotin binding display an absence of plaque structure morphology with minimal background signal in cognitively normal elderly subjects without plaque pathology. Nuclei stained with DAPI (blue channel). Scale bar = 50 μm. (F) Fluorescent images of labelled (streptavidin-Alexa594, red channel) Aβ1-42-biotin binding display punctate staining of a central core with peripheral decoration in elderly subjects with mild ADD (CDR1). Nuclei stained with DAPI (blue channel). Scale bar = 50 μm. Orthogonal XZ and YZ views, centered on the yellow crosshairs, demonstrate the labelling extent through the tissue section. Scale bar = 10 μm.
Fig 3.
Assessments based on confocal fluorescence microscopy of Aβ1-42-biotin binding does not distinguish between elderly high-pathology controls and elderly subjects with mild ADD.
(A) Gray matter coverage of fluorescently labelled (streptavidin-Alexa594) Aβ1-42-biotin binding in CDR 0 + plaques group versus CDR 1 group (not significant [n.s.] by Mann–Whitney U test). (B) Correlations between fluorescently labelled Aβ1-42-biotin gray matter coverage and overall Aβ plaque coverage. (C) Ratio of the normalized fluorescently labelled Aβ1-42-biotin signal to the percentage fluorescent positive coverage (not significant [n.s.] by Mann–Whitney U test).
Fig 4.
Assessments based on enzyme-linked immunosorbent assay of bound Aβ1-42-biotin does not distinguish between elderly high-pathology controls and elderly subjects with mild ADD.
(A) There was no difference between groups in the levels of overall Aβ1-42-biotin recovered from dissociated tissue, as measured using an indirect ELISA which only detects biotinylated Aβ (not significant [n.s.] by Mann–Whitney U test). Data expressed as picograms Aβ per nanogram of total measurable protein. (B) The ratio of the amount of Aβ1-42-biotin to the amount of Aβ1-x as measured by sandwich ELISA was not different between groups (not significant [n.s.] by Mann–Whitney U test). (C) The ratio of Aβ Aβ1-42-biotin as measured by sandwich ELISA to the percent gray matter plaque coverage did not differ between groups (not significant [n.s.] by Mann–Whitney U test). (D) There was no difference between groups in the levels of overall Aβ1-x recovered from dissociated tissue, as measured using an sandwich ELISA (not significant [n.s.] by Mann–Whitney U test).
Fig 5.
Size exclusion chromatography reveals that the Aβ1-42-biotin monomer remained predominantly low molecular weight during the experiment.
(A) Globular protein molecular weight standards as run on a size exclusion Superdex 200 10/300 GL column. mAu = milli-absorbance units, kDa = kilodaltons. (B) The predominance of the pre-incubation (black closed circles) Aβ1-42-biotin elutes as a low molecular weight peak in fractions 18 to 21 likely corresponding to monomer, and a minority of the Aβ1-42-biotin eluted as a high molecular weight peak in fractions 8 and 9 corresponding to aggregated Aβ. The post overnight incubation sample (red open circles) has a minor shift to include a shoulder of fractions 16 and 17 in the low molecular weight peak suggesting the accumulation of oligomeric species while most the sample remained monomeric.