Research ResourceAlzheimer’s Disease

Aβ and tau prion-like activities decline with longevity in the Alzheimer’s disease human brain

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Science Translational Medicine  01 May 2019:
Vol. 11, Issue 490, eaat8462
DOI: 10.1126/scitranslmed.aat8462
  • Fig. 1 Development of YFP-Aβ fusion proteins in HEK293T cell lines for measuring Aβ aggregates in brain samples.

    The indicated cell lines were developed to measure prion-like activity of preparations consisting of synthetic Aβ peptides and mouse brain–derived extracts based on their abilities to induce fluorescent aggregates (puncta). (A) Diagram illustrating the Aβ constructs used in this study (left). Stably transfected HEK293T cells expressing an Aβ-YFP fusion construct underwent Lipofectamine-based transduction with synthetic Aβ fibrils (right). (B) Representative confocal images of HEK293T cells expressing Aβ42 fused to YFP at the N terminus (clone #1), which were treated with phosphate-buffered saline (PBS) (left; control) or exposed to synthetic Aβ40 fibrils (initial monomeric concentration, 1 μM) (right, exposed). The aggregates of YFP-Aβ appear as fluorescent yellow puncta. To measure prion-like activity, we counted the number of puncta-positive cells and expressed this as a percent of the total number of cells in the field of view (% positive cells). Lower panels are higher-magnification images of white boxed areas in the upper panels. Scale bars, 20 μm (upper panels) or 5 μm (lower panels). (C) HEK293T cells transfected with YFP-Aβ42 were treated with two different types of Aβ ranging from 0.03 to 100 nM (initial monomeric concentration): synthetic Aβ40 (left) or Aβ purified from TgAPP23 mouse brains (right). Puncta-inducing activity in the HEK293T cells was quantified 2 days after the initial exposure to various Aβ preparations. Data shown are means ± SEM as determined from four images per well across four wells and are representative of three independent experiments. Statistical significance is indicated as **P < 0.01. (D) Cell lines stably expressing four different WT Aβ constructs [shown in (A)] were developed, and puncta formation was compared with synthetic Aβ40 and Aβ42 isoforms. Quantification of Aβ in 16 monoclonal cell lines (four randomly chosen clones from each construct; see fig. S2A) was performed 2 days after exposure to increasing concentrations of synthetic Aβ40 or Aβ42 isoforms (1 to 100 nM) (see fig. S1). Data shown are means ± SEM as determined from four images per well across four wells and are representative of two to three independent experiments. (E) Cell lines stably expressing four different Aβ40 constructs fused to YFP at the N terminus [shown in (A)] were developed, and puncta formation was compared after exposure to synthetic Aβ40 and Aβ42 isoforms. Quantification of Aβ in 16 monoclonal cell lines (four randomly chosen clones from each construct; see fig. S2B) was performed 2 days after exposure to increasing concentrations of synthetic Aβ40 or Aβ42 isoforms (1 to 100 nM) (see fig. S1). Data shown are means ± SEM as determined from four images per well across four wells and are representative of two independent experiments.

  • Fig. 2 Specificity of the Aβ cellular assays.

    Various transgenic mouse brain inocula were assayed for their Aβ, tau, and α-synuclein prion-like activities. (A) Representative confocal images of HEK293T cells stably expressing YFP-Aβ42 (top row), α-synuclein containing the A53T mutation fused to YFP (α-syn(A53T)–YFP, middle row), or tauK18(LM)-YFP (bottom row) (see Materials and Methods for construct details). The cells were treated with APP-derived peptides from the brains of TgAPP23 mice (0.1 × PTA sample, left column), or Tgα-Syn*A53T mice (0.1 × PTA sample, middle column), or homozygous Tg0N4Rtau*P301S mice (0.1 × PTA sample, right column). Scale bar, 20 μm. (B) Quantification of the responses of the YFP-Aβ42, α-syn(A53T)-YFP, and tauK18(LM)-YFP cell lines 2 days after exposure to increasing concentrations of transgenic mouse brain–derived Aβ (0.01× to 0.1× PTA sample; orange to dark red), α-synuclein carrying the A53T mutation (0.01× to 0.1× PTA sample; light blue to dark blue), and tau carrying the P301S mutation (0.01 × to 0.1× PTA sample; light gray to dark gray). Data shown are means ± SEM as determined from four images per well across four wells and are representative of four independent experiments.

  • Fig. 3 Quantitation of postmortem human brain Aβ and tau in parallel cellular assays.

    (A) The prion-like activities of Aβ and tau in postmortem human brain samples from patients with FTLD-tau, AD, CAA, and MSA (multiple system atrophy) were added to three different HEK293T cell lines expressing YFP-Aβ42, tauK18(LM)-YFP, or α-syn(A53T)-YFP. (B to D) The prion-like activities of Aβ and tau in human brain tissue samples were quantified using the YFP-Aβ42 and tauK18(LM)-YFP cell lines. The cell lines were exposed to a 0.03× dilution of PTA-precipitated brain homogenates derived from 86 patients with sporadic or familial AD or CAA, 10 aged healthy controls, and 10 patients with FTLD-tau (7 with PSP and 3 with CBD). Data shown are means ± SEM as determined from four images per well across four wells per sample. Statistical significance is indicated as ***P < 0.0001 for Aβ puncta–inducing prion-like activity compared to control or ###P < 0.0001 for tau puncta–inducing prion-like activity compared to control. (E) α-Synuclein abundance in brain homogenates was quantified as a percentage of α-syn(A53T)–YFP–expressing cells positive for α-synuclein puncta. The α-syn(A53T)–YFP cell line was exposed to a 0.03× dilution of PTA-precipitated brain homogenates from all postmortem brain samples. Data shown are means ± SEM as determined from four images per well across four wells per sample. (F) Correlation analyses between Aβ load (x axis) and tau load (y axis). Shown is a summary plot for brain tissue from 75 AD cases (37 sporadic AD, red; 25 familial AD with the PSEN mutation, blue; 13 familial AD with the APP mutation, orange) compared to brain tissue from aged healthy controls (gray) and from patients with CAA (sporadic CAA, purple; familial CAA, pink) or FTLD-tau (PSP/CBD, cyan). Brain tissue from one patient with sporadic AD lacked a Braak stage score and had comorbid Lewy body dementia; brain tissue samples from two patients with sporadic AD were Braak stage III/IV; all three data points fell well within the range of other close-lying points and, thus, were not removed. Data are representative of three independent experiments.

  • Fig. 4 Inverse correlation between longevity and self-propagating Aβ and tau activities.

    The Aβ and tau prion-like activity in brain tissue from sporadic and familial AD samples (Braak stages V and VI) was plotted as a function of patient age at death (A): familial AD with the PSEN mutation, blue; familial AD with the APP mutation, orange; sporadic AD, red. Statistical values for correlation, linear regression, and 95% confidence intervals are shown. (B) Histogram of Aβ load for the same dataset binned into three age groups: <60 years old (y.o., top); 60 to 80 years old (middle); and >80 years old (bottom). (C) Tau load in brain tissue from patients with sporadic AD and patients with familial AD plotted as a function of patient age at death: familial AD with the PSEN mutation, blue; familial AD with the APP mutation, orange; sporadic AD, red. Statistical values for correlation, linear regression, and 95% confidence intervals are shown. (D) Histogram of tau abundance for the same dataset binned into three age groups: <60 years old (top); 60 to 80 years old (middle); and >80 years old (bottom). Fraction of total sample number in each age bin with 0 to 5% tau-positive cells. The SDs of individual data points are similar to those in Fig. 1 and are much smaller than the deviation from the regression line, indicating that measurement error did not contribute significantly to the deviations from the trend line. Data are representative of three independent experiments.

  • Fig. 5 Measurement of APP expression and the amounts of soluble and insoluble Aβ and tau species as a function of the age at death of patients with AD.

    ELISA was used to measure different proteins in brain samples (Braak stages V and VI) from patients with familial or sporadic AD. The following proteins were measured: (A) APP in the clarified brain homogenate (PBS soluble), (B and C) Aβ40 and Aβ42 (formic acid soluble), respectively, (D) total tau in the clarified brain homogenate (PBS soluble), (E) total tau (formic acid soluble), and (F) p-tau (phosphorylated epitope Ser396, formic acid soluble). All data are plotted as a function of patient age at death. Statistical values for correlation, linear regression, and 95% confidence interval are shown. The measurements were made in duplicate and are much smaller than the deviation from the regression line, indicating that measurement error did not contribute significantly to the deviations from the trend line. Data are representative of one to two independent experiments.

  • Fig. 6 The abundance of self-propagating and p-tau decreased exponentially over six decades in patients with AD.

    The specific activity of prion-like tau in AD brain homogenates decreased in parallel with a reduction in hyperphosphorylated tau. (A) Prion-like tau abundance measured in AD brain tissue samples (Braak stages V and VI) was normalized to the adjusted value of insoluble tau as measured by ELISA (see Fig. 5E). Shown are normalized data plotted as a function of AD patient age at death and fitted using an exponential decay model equation (one-phase decay). (B) p-tau (phosphorylated on Thr231; p-tauT231) was measured in AD brain samples normalized to the adjusted value of insoluble tau as measured by ELISA (see Fig. 5E). Normalized data were plotted as a function of AD patient age at death and fitted using an exponential decay model equation (one-phase decay). To normalize the data, the prion-like tau values from Fig. 4 or the p-tau concentration from Fig. 5 was divided by the concentration of total insoluble tau obtained from the regression line for total tau versus age at death for patients with AD shown in Fig. 5E. (C) p-tau (phosphorylated on Ser396; p-tauS396) was measured in AD brain samples normalized to the adjusted value of insoluble tau as measured by ELISA. (D) p-tau (phosphorylated on Ser199; p-tauS199) was measured in AD samples normalized to the adjusted value of insoluble tau as measured by ELISA. (E) Statistical values for correlation, decay constant (k), half-life (T), and their respective 95% confidence intervals (CIs) are shown.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/490/eaat8462/DC1

    Fig. S1. Development of YFP-Aβ fusion HEK293T cell lines: Synthetic Aβ fibril inocula- and puncta-inducing kinetics.

    Fig. S2. Development of YFP-Aβ fusion HEK293T cell lines: Individual clones.

    Fig. S3. Amyloid plaque pathology, astrogliosis, and prion-like Aβ abundance during disease progression in TgAPP23 and TgCRND8 mice.

    Fig. S4. APOE ε4 status, gender, and brain region influence the extent of prion-like Aβ and tau abundance in AD postmortem brain tissue.

    Fig. S5. Correlation of different Aβ and tau species with age at death of patients with AD.

    Table S1. Source of postmortem human brain tissue samples.

    Data file S1. Source data.

  • The PDF file includes:

    • Fig. S1. Development of YFP-Aβ fusion HEK293T cell lines: Synthetic Aβ fibril inocula- and puncta-inducing kinetics.
    • Fig. S2. Development of YFP-Aβ fusion HEK293T cell lines: Individual clones.
    • Fig. S3. Amyloid plaque pathology, astrogliosis, and prion-like Aβ abundance during disease progression in TgAPP23 and TgCRND8 mice.
    • Fig. S4. APOE ε4 status, gender, and brain region influence the extent of prion-like Aβ and tau abundance in AD postmortem brain tissue.
    • Fig. S5. Correlation of different Aβ and tau species with age at death of patients with AD.
    • Table S1. Source of postmortem human brain tissue samples.

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    Other Supplementary Material for this manuscript includes the following:

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