Overlapping profiles of Aβ peptides in the Alzheimer's disease and pathological aging brains

Abstract

Introduction: A hallmark of Alzheimer's disease (AD) is the presence of senile plaques composed of aggregated amyloid β (Aβ) peptides. Pathological aging (PA) is a postmortem classification that has been used to describe brains with plaque pathology similar in extent to AD, minimal cortical tau pathology, and no accompanying history of cognitive decline in the brain donor prior to death. PA may represent either a prodromal phase of AD, a benign form of Aβ accumulation, or inherent individual resistance to the toxic effects of Aβ accumulation. To attempt to distinguish between these possibilities we have systematically characterized Aβ peptides in a postmortem series of PA, AD and non-demented control (NDC) brains.

Methods: Aβ was sequentially extracted with tris buffered saline (TBS), radioimmunoprecipitation buffer (RIPA), 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA) from the pre-frontal cortex of 16 AD, eight PA, and six NDC patients. These extracts were analyzed by 1) a panel of Aβ sandwich ELISAs, 2) immunoprecipitation followed by mass spectrometry (IP/MS) and 3) western blotting. These studies enabled us to asses Aβ levels and solubility, peptide profiles and oligomeric assemblies.

Results: In almost all extracts (TBS, RIPA, 2% SDS and 70% FA) the average levels of Aβ1-40, Aβ1-42, Aβ total, and Aβx-42 were greatest in AD. On average, levels were slightly lower in PA, and there was extensive overlap between Aβ levels in individual PA and AD cases. The profiles of Aβ peptides detected using IP/MS techniques also showed extensive similarity between the PA and AD brain extracts. In select AD brain extracts, we detected more amino-terminally truncated Aβ peptides compared to PA patients, but these peptides represented a minor portion of the Aβ observed. No consistent differences in the Aβ assemblies were observed by western blotting in the PA and AD groups.

Conclusions: We found extensive overlap with only subtle quantitative differences between Aβ levels, peptide profiles, solubility, and SDS-stable oligomeric assemblies in the PA and AD brains. These cross-sectional data indicate that Aβ accumulation in PA and AD is remarkably similar. Such data would be consistent with PA representing a prodromal stage of AD or a resistance to the toxic effects of Aβ.

Figure 1

Immunohistochemical characterization of amyloid and tau pathology in Alzheimer's disease (AD), pathological aging (PA) and normal non-demented controls (NDC). A-E. Representative paraffin embedded formalin fixed hippocampi from human AD (A, B), PA (C, D) and control (NDC; E) subjects were stained with 33.1.1 antibody (pan Aβ1-16). Insets show higher magnification with special emphasis on cored Aβ plaques associated with blood vessels in subjects with a post-mortem clinical pathological diagnosis of cerebral amyloid angiopathy (CAA+). Scale Bar, 600 μm (A-E), insets 60 μm (A-E). F-J. Representative paraffin embedded formalin fixed hippocampi from human AD (F, G), PA (H, I) and NDC (J) subjects were stained with anti-phosphorylated tau (CP13). Insets highlight phorphorylated tau containing tangles and neuritic structures. Scale Bar, 600 μm (A-E), insets 60 μm (A-E). Aβ, amyloid β.

Figure 2

Immunohistochemical characterization of Aβ1-40 and Aβ1-42 in Alzheimer's disease (AD), pathological aging (PA) and normal non-demented controls (NDC). A-E. Representative paraffin embedded formalin fixed hippocampi from human AD (A, B), PA (C, D) and control (NDC; E) subjects were stained with 13.1.1 antibody (Aβ35-40). Insets show higher magnification with special emphasis on cored Aβ plaques associated with blood vessels in subjects with a post-mortem clinical pathological diagnosis of cerebral amyloid angiopathy (CAA+). Scale Bar, 600 μm (A-E), insets 60 μm (A-E). F-J. Representative paraffin embedded formalin fixed hippocampi from human AD (A, B), PA (C, D) and control (NDC; E) subjects were stained with 2.1.3 antibody (Aβ35-42). Insets show higher magnification with special emphasis on cored Aβ plaques associated with blood vessels in subjects with a post-mortem clinical pathological diagnosis of cerebral amyloid angiopathy (CAA+). Scale Bar, 600 μm (A-E), insets 60 μm (A-E). Aβ, amyloid β.

Figure 3

Biochemical analysis of Aβ from human brain lysates. Human prefrontal cortical tissue from Alzheimer's disease (AD), pathological aging (PA) and normal controls (NDC) was sequentially extracted with TBS (A), RIPA (B), 2% SDS (C) and 70% FA (D). End-specific sandwich ELISAs measuring Aβ1-40, Aβ1-42, Aβtotal and Aβx-42 are presented for each of these fractions. N = 16 (AD), 8 (PA) and 6 (NDC). (***P < 0.001, **P < 0.01, *P < 0.05 by ANOVA with tukey post-hoc analysis raw data analyzed (A, B) and log-transformed data analyzed (C, D)). Aβ, amyloid β; ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; FA, formic acid; RIPA, radioimmunoprecipitation buffer; SDS, sodium dodecyl sulfate; TBS, Tris buffered saline.

Figure 4

Mass spectrometric (MS) analysis following immunoprecipiation of Aβ from 2% SDS extracted lysates of human subjects. 2% SDS lysates of Alzheimer's disease (AD; A, D), pathological aging (PA; B, E) and control (NDC; C, F) subjects were subjected to immunoprecipitation with pull-down by Ab9 (A-C), sequential pull-down with Ab9 and 4 G8 (D-F). Representative MS spectra are shown (A-F). Peaks corresponding to Aβ peptides have been labeled according to m/z. Aβ1-28 was spiked in as an experimental control. The 1-42* peak denotes a possible modified Aβ1-42 species (Aβ1-42 + 16 Da, A-E). Aβ, amyloid β; SDS, sodium dodecyl sulfate.

Figure 5

Mass spectrometric (MS) analysis following immunoprecipiation of Aβ peptides from 70% formic acid extracted lysates of human subjects. 70% formic acid extracted lysates of Alzheimer's disease (AD; A, D), pathological aging (PA; B, E) and control (NDC; C, F) cohorts were subjected to immunoprecipitation with pull-down by Ab9 (A-C), sequential pull-down with Ab9 and 4G8 (D-F). Representative MS spectra are shown. Peaks corresponding to Aβ peptides have been labeled according to m/z. Aβ1-28 was spiked in as an experimental control. Different peaks corresponding to modified Aβ are shown (4-42*, Aβ4-42 + 16 Da; 1-40*, Aβ1-40 +16 Da; 1-42**, Aβ1-42 + 22 Da). Aβ, amyloid β.

Figure 6

Summary of SDS soluble Aβ peptides in test subjects. 2% SDS extracted lysates from human prefrontal cortical tissues were subjected to immunoprecipitation with pull-down by Ab9 (A) or sequential pull-down with Ab9 and 4G8 (B), followed by MS. Data are graphically presented as a percentage of subjects in each cohort (Alzheimer's disease (AD), Pathological aging (PA) or controls (NDC)) containing Aβ peptides (x-axis). N = 16 (AD), 8 (PA) and 6 (NDC). Aβ, amyloid β; MS, mass spectrometry; N, number; SDS, sodium dodecyl sulfate.

Figure 7

Summary of formic acid soluble Aβ peptides in test subjects. 70% FA extracted lysates from human prefrontal cortical tissue were subjected to immunoprecipitation with pull-down by Ab9 (A) or sequential pull-down with Ab9 and 4G8 (B), followed by MS. Data are graphically presented as a percentage of subjects in each cohort (Alzheimer's disease (AD), Pathological aging (PA) or controls (NDC)) containing Aβ peptides (x-axis). N = 16 (AD), 8 (PA) and 6 (NDC). Aβ, amyloid β; FA, formic acid; MS, mass spectrometry; N, number.

Figure 8

Immunoblot analysis of Aβ species from sequentially extracted human prefrontal cortical tissue lysates. Human hippocampi from Alzheimer's disease (AD), pathological aging (PA) and normal (N) cohorts were sequentially extracted with TBS, 2% SDS extracted lysates from human subjects. Representative immunoblots probed with 82E1 antibody are shown. Control lanes includes cell lysates expressing C99 (CTFβ) and recombinant Aβ1-42 (43 pmol). Aβ, amyloid β; SDS, sodium dodecyl sulfate; TBS, Tris buffered saline.

Figure 9

Immunoblot analysis of Aβ species from RIPA soluble lysates from human subjects. Human hippocampi from Alzheimer's disease (AD), pathological aging (PA) and normal (N) cohorts were extracted with RIPA. Representative anti-82E1 immunoblot of RIPA extracted lysates of Alzheimer's disease (AD), pathologic aging (PA) and control (N) subjects with (A) and without (B) a post-mortem clinical pathological diagnosis of cerebral amyloid angiopathy (+CAA). Longer exposure panels below highlight no significant differences between the three cohorts in the dimer/trimer molecular weight range. Aβ, amyloid β; RIPA, radioimmunoprecipitation buffer.