Table 1.
Study Subject Demographics and Neuropathological Scoring for Young Adults.
Table 2.
Study Subject Demographics and Neuropathological Scoring for Middle-Aged and Oldest-Old.
Table 3.
Antibodies Used for Western Blot Analysis.
Figure 1.
Amyloid precursor protein and proteolytic-derived peptides assessed by Western blot or ELISA.
Sample numbers correspond to young adult (1–9), middle-aged (20–24) and oldest-old (30–35). Full length APP was detected in the precuneus (A) and posterior cingulate gyrus (B) by Western blot. C-terminal peptides (CT99 and CT83) of APP were determined by Western blot using an antibody against the last 20 amino acids of APP in the precuneus (C) and posterior cingulate gyrus (D). A total of 40 µg of total protein was loaded per lane. Data are reported in optical density units and were adjusted for actin. Actin loading probes are shown below each primary antibody blot. The molecular weight is shown on the left side of each blot. Brain tissue that was homogenized in GDFA/GHCl (see Materials and Methods) was used to quantify Aβ40 and Aβ42 in the precuneus (E, G) and posterior cingulate gyrus (F, H) by ELISA. The ELISA concentrations are reported in pg per mg of total protein. Abbreviations: Pc, precuneus; PCG, posterior cingulate gyrus; YA, young adult; MA, middle-aged; OO, oldest-old; APP, amyloid precursor protein; CT, C-terminal. The 3 age groups were statistically compared using the non-parametric Kruskall-Wallis test followed by the Dunn’s multiple comparison test (*p = 0.05–0.01; **p = 0.01–0.001; ***p≤0.001).
Figure 2.
Western blot analyses of BACE1 and APLP1 and APLP2.
Sample numbers correspond to young adult (1–9), middle-aged (20–24) and oldest-old (30–35). Western blotting was used to detect BACE1 in the precuneus (A) and posterior cingulate gyrus (B), APLP1 in the precuneus (C) and posterior cingulate gyrus (D) and APLP2 in the precuneus (E) and posterior cingulate gyrus (F). All Western blots were loaded with a total of 40 µg of protein per lane. APLP2 blots were performed under non-reducing conditions. Data are reported in optical density units and were adjusted for actin (BACE1, APLP1) or GAPDH (APLP2). Actin and GAPDH loading probes are shown below each primary antibody blot. Abbreviations: Pc, precuneus; PCG, posterior cingulate gyrus; YA, young adult; MA, middle-aged; OO, oldest-old; BACE1, β-site amyloid precursor protein-cleaving enzyme-1; APLP, amyloid precursor-like protein. For statistical treatment see legend to Figure 1.
Figure 3.
ELISA quantitative analyses of tau and α-synuclein.
Sample numbers correspond to young adult (1–9), middle-aged (20–24) and oldest-old (30–35). Tau results are shown in Figures A and B and α-synuclein are presented in Figures C and D for both precuneus and posterior cingulate gyrus as indicated. Concentrations are reported in ng per mg of total protein. Tau and α-synuclein were detected in GDFA/GHCl homogenates (for details see Materials and Methods section). Abbreviations: Pc, precuneus; PCG, posterior cingulate gyrus; YA, young adult; MA, middle-aged; OO, oldest-old; α-syn, α-synuclein. For statistical analyses see legend to Figure 1.
Figure 4.
ELISA quantitative analyses of total ApoE and Western blot analyses of ApoJ.
Sample numbers correspond to young adult (1–9), middle-aged (20–24) and oldest-old (30–35). ELISA analyses for ApoE were performed in RIPA homogenates from the precuneus (A) and posterior cingulate gyrus (B). ELISA concentrations are reported in ng per mg of total protein. Western blotting was used to visualize ApoJ-α and ApoJ-β chains in the precuneus and posterior cingulate gyrus (C, D). For Western blots, a total of 40 µg of total protein was loaded per lane. Data are reported in optical density units and were adjusted for actin. Actin loading probes are shown below each primary antibody blot. The molecular weight is shown on the left side of each blot. Abbreviations: Pc, precuneus; PCG, posterior cingulate gyrus; YA, young adult; MA, middle-aged; OO, oldest-old; ApoE, apolipoprotein E; ApoJ, apolipoprotein J. For statistical treatment see legend to Figure 1.
Figure 5.
ELISA quantitative analyses of TNF-α and CD200 and Western blot analyses of PEDF.
As indicated in the Figure both the precuneus (A, C and E) and posterior cingulate gyrus (B, D and F) were investigated. Sample numbers, shown above each blot, correspond to young adult (1–9), middle-aged (20–24) and oldest-old (30–35). ELISA concentrations are reported in ng per mg of total protein. For Western blot analyses a total of 40 µg of total protein was loaded per lane. Data are reported in optical density units and were adjusted for actin. The actin loading probes is shown below the primary antibody blot. The molecular weight is shown on the left side of each blot. Abbreviations: Pc, precuneus; PCG, posterior cingulate gyrus; YA, young adult; MA, middle-aged; OO, oldest-old; TNF-α, tumor necrosis factor-α; PEDF, pigment epithelium-derived factor. For statistical treatment see legend to Figure 1.
Figure 6.
ELISA quantitative analyses of BDNF and GFAP and Western blot analyses of S100B.
As indicated in the figure both the precuneus (A, C and E) and posterior cingulate gyrus (B, D and F) were investigated. Sample numbers, shown above each Western blot correspond to young adult (1–9), middle-aged (20–24) and oldest-old (30–35). ELISA concentrations are reported in ng per mg of total protein. For the S100B Western blot, a total of 25 µg of total protein was loaded per lane. Data are reported in optical density units and were adjusted for GAPDH. The GAPDH loading probe is shown below the primary antibody blot. The molecular weights are shown on the left side of each blot. Abbreviations: Pc, precuneus; PCG, posterior cingulate gyrus; YA, young adult; MA, middle-aged; OO, oldest-old; BDNF, brain-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; S100B, S100 calcium binding protein-B. For statistical analyses see legend to Figure 1.