Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

Abstract

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.

Supplemental Figure S1

Controls for oAβ ELISA for soluble synaptic Aβ oligomers. A: Standard curve for cross-linked oligomeric Aβ 40 standards and stability of cross-linked oligomeric Aβ standards in 0.1% HFIP. B: Cross-linked oAβ standard (15 ng/mL) measured by ELISA in the presence of excess monomeric Aβ40. Aβ, amyloid-β; ELSIA, enzyme-linked immunosorbent assay; HFIP, 1,1,1,3,3,3-hexafluoro-2-proponol; oAβ, soluble Aβ oligomers.

Supplemental Figure S2

Total in situ synaptic Aβ is not removed by incubation with trypsin and heparinase. Aβ immunolabeling with the 10G4 antibody was quantified by flow cytometry in an AD sample with (A) and without (B) incubation with 0.01% trypsin (2 minutes room temperature) and 10 u/mL heparinase (5 minutes, 32°C) before the fixation step. C–E: Comparison of flow cytometric immunolabeling of synaptosomes with 10G4 (C), 6E10 (D), and MOAB2 (E) antibodies. Aβ, amyloid-β; AD, Alzheimer disease.

Figure 1

Flow cytometric analysis of Aβ immunolabeling across disease stage in Alzheimer disease parietal cortex synaptosomes. Representative dot plots show background in the presence of isotype-specific control antibody (A), SNAP-25 as a positive control and indicator of synaptosomal purity (B). Representative Aβ labeling in an aged cognitively normal control case (C), a case with Braak (synaptosomal-associated protein, 25 kDa NFT) stage IV and plaque stage 0 (D), and a late-stage case with extensive plaques and tangles (E). Ten thousand synaptosomes are plotted for each sample. The FSC variable is proportional to size. Percentages are positive fraction in analysis gate. RFU is mean relative fluorescence in analysis gate. Aβ, amyloid-β; FSC, forward scatter; NFT, neurofibrillary tangle; POS, positive; RFU, relative fluorescence unit; SNAP-25, synaptosomal-associated protein, 25 kDa.

Figure 2

Synaptic Aβ pathology across disease stage. A: Group data show total in situ synaptic Aβ, measured by flow cytometry, plotted by Braak NFT stage. For Braak staging cases were divided into four groups: Con, HPC, AD ≤ IV, and AD ≥ V. B: The assigned NFT stage was plotted against plaque stage for each of the three groups' Cons, HPCs, and AD; HPCs are distributed across plaque stages. C and D: Total in situ Aβ level measured by flow cytometry significantly correlates with the local plaque level in the parietal cortex, taken from the neuropathology report, when all samples are included (C) and for the HPC group considered alone (D). E: ELISA measurements of total soluble Aβ in synaptosome-enriched P-2 samples across disease stage. F: Western blots with 6E10 antibody across disease stage in P-2 samples from parietal cortex. G: Soluble oligomeric Aβ levels in parietal cortex P-2 samples plotted across Braak stages. Data are expressed as means ± SEM. n = 6 Cons, n = 12 HPCs, n = 9 AD ≤ IV, and n = 7 AD ≥ V (A); n = 6 Cons, n = 12 HPCs, and n = 16 AD (B); n = 34 samples (C); n = 12 HPC samples (D); n = 5 Cons, n = 7 HPCs, n = 4 AD ≤ IV, and n = 6 AD ≥ V (E); controls n = 6 Cons, n = 12 HPCs, n = 7 AD ≤ IV, and n = 9 AD ≥ V (G). *P < 0.05, **P < 0.01. Aβ, amyloid-β; AD, Alzheimer disease; AD ≤ IV, Braak stage I to IV; AD ≥ V, Braak stage V to VI; B, Braak stage; Con, control; ELISA, enzyme-linked immunosorbent assay; HPC, high pathology control; NFT, neurofibrillary tangle.

Figure 3

Conformation-dependent antibodies label Aβ in synaptic terminals. A–D: Synaptosomes were dual-labeled with synaptotophysin and the monoclonal antibody M55 directed against pre-fibrillar oligomers: M55 (A); SYP (B); overlay image with arrows indicate colocalization (C); DIC image (D). E–H: Synaptosomes dual-labeled with SYP and the monoclonal antibody M116 directed against fibrillar oligomers: M116 (E); SYP (F); overlay image with arrows indicate co-localization (G); DIC image (H). Scale bar. Aβ, amyloid-β; DIC, differential interference contrast; SYP, synaptophysin. Scale bar = 5 μm.

Figure 4

A–C: Flow cytometric analysis of Aβ in a transgenic rat model of Alzheimer disease (APP/PS1). Representative Aβ labeling in transgenic rat mixed cortex samples (Tg) is shown at 3 (A), 9 (B), and 20 (C) months; 10,000 synaptosomes are plotted for each sample; the FSC variable is proportional to size; percentage, positive fraction in analysis gate, RFU, mean relative fluorescence in analysis gate. D and E: Group data show total in situ synaptic Aβ at 3, 9, and 20 months measured by flow cytometry in WT and Tg rat mixed cortex (D) and frontal cortex (E). F: Soluble oligomeric Aβ level in mixed cortex P-2 samples is plotted for WT and Tg at 3, 9, and 20 months. Data are expressed as means ± SEM. n = 6 to 9 in each group. ***P < 0.001. Aβ, amyloid-β; APP, Aβ (A4) precursor protein; FSC, forward scatter; PS1, presenilin-1; RFU, relative fluorescence unit; Tg, transgenic; WT, wild-type.

Figure 5

Synaptic p-tau is not elevated until late-stage Alzheimer disease in parietal cortex. A, C, and E: Representative dot plots from the flow cytometric analysis are shown for p-tau labeling with the PHF-1 antibody; 10,000 synaptosomes are plotted for each sample; the FSC variable is proportional to size; percentage, positive fraction in analysis gate, RFU, mean relative fluorescence in analysis gate. B, D, and F: Aggregate plots of flow cytometric data from synaptosome-enriched P-2 samples from parietal cortex for total tau with HT-7 (B), and p-tau with PHF-1 (D), and pS⁴²² (F) across disease stages. Cases were divided into four groups: Cons, HPCs, AD ≤ IV, and AD ≥ V. G: Representative dot plot from flow cytometric analysis for an early Braak stage case with elevated p-tau signal in parietal cortex P-2. Data are expressed as means ± SEM. n = 5 to 6 Cons, n = 11 to 12 HPCs, n = 9 AD ≤ IV, and n = 8 to 9 AD ≥ V (B, D, and F). ***P < 0.001. AD, Alzheimer disease; AD ≤ IV, Braak I to IV; AD ≥ V, Braak V to VI; Con, control; FSC, forward scatter; HPC, high pathology control; p-tau, hyperphosphorylated tau; RFU, relative fluorescence unit.

Figure 6

Synaptic p-tau follows amyloid pathology in a transgenic rat model of Alzheimer disease. A–C: Representative dot plots from the flow cytometric analysis are shown for background staining in the presence of isotype-specific control antibody (A), and p-tau immunolabeling with the PHF-1 antibody in a 20-month-old WT (B)] and Tg (C) P-2 samples. Ten thousand synaptosomes are plotted for each sample; the FSC variable is proportional to size; percentage, positive fraction in analysis gate, RFU, mean relative fluorescence in analysis gate. D: Group data for 3-, 9-, and 20-month-old animals; p-tau immunolabeling is shown for the PHF-1 in frontal cortex P-2 samples. Data are expressed as means ± SEM. n = 8 animals per group. **P = 0.01. FSC, forward scatter; p-tau, hyperphosphorylated tau; RFU, relative fluorescence unit; Tg, transgenic; WT, wild-type.

Figure 7

Elevation of p-tau immunolabeling in Aβ-positive synaptosomes. A–C: Flow cytometric analysis of a single AD parietal cortex synaptosome-enriched P-2 sample dual-labeled for Aβ (10G4 antibody) and p-tau (PHF-1 antibody). Representative plots of the same dual-labeled sample show the size of the total p-tau positive fraction (8.82% (A); 10,000 particles], the collection of Aβ-positive synaptosomes inside the analysis gate (B), and increased p-tau in the Aβ-positive synaptosomes (21.5%; C, upper right quadrant). Five thousand particles were collected for each sample; the FSC variable is proportional to size; percentage, positive fraction in analysis gate, RFU, mean relative fluorescence in analysis gate. D: Group data for dual-labeling analysis in PAR, HIPP, and EC. E and F: Correlation analysis of Aβ and p-tau immunolabeling in early-stage AD (F) and Aβ and p-tau correlation in early-stage AD HIPP and EC cortex. Data are expressed as means ± SEM. n = 28 PAR, n = 7 HIPP, and n = 6 EC (D); n = 20 ≤Braak IV (E); n = 20 ≤Braak IV (F). ***P < 0.001. Ab, antibody; Aβ, amyloid-β; AD, Alzheimer disease; EC, entorhinal cortex; FSC, forward scatter; HIPP, hippocampus; PAR, parietal cortex; p-tau, hyperphosphorylated tau; RFU, relative fluorescence unit.