Enhanced Tau Aggregation in the Presence of Amyloid β

Abstract

Amyloid plaques and neurofibrillary tangles co-occur in Alzheimer disease, but with different topological and temporal patterns. Whether these two lesions are independent or pathobiologically related is uncertain. For example, amyloid deposition in the neocortex precedes the spread of tau neurofibrillary tangles from the limbic areas to the cortex. We examined the aggregation properties of tau isolated from human cases with early tau pathology (Braak II) with and without plaques. Using a well-established HEK cell biosensor assay, we show that tau from cases with plaques has an enhanced ability to induce tau aggregates compared to tau from cases without plaques. To further explore this effect, we combined mice carrying the APP/PS1 transgene array that develop plaques with rTg4510 mice carrying the P301L mutant human tau transgene that develop extensive tau pathology with age. The resulting APP/PS1-rTg4510 mice had a threefold increase in tau seeding activity over the rTg4510 strain, without change in tau production or extracellular release. Surprisingly, this effect was observed before overt amyloid deposition. The enhancement of tau aggregation was also apparent by an increase in histological measures of tau pathology in young APP/PS1-rTg4510 mice and an increase in high-molecular-weight tau. Overall, these data provide evidence that amyloid β acts to enhance tau pathology by increasing the formation of tau species capable of seeding new aggregates.

Figure 1

Plaques enhance tau aggregation in humans. A: Tissue homogenates were prepared from entorhinal cortex (EC) and hippocampus (HP) at the posterior end of the striatum. The posterior parahippocampal gyrus (PHG) was sampled at the level of the lateral geniculate nucleus approximately 2 to 3 cm posterior to the entorhinal cortex. Yellow highlighted areas indicate location of tissue sampling [1 (EC), 2 (HP), 3 (PHG)]. Boxed areas indicate locations where representative images were taken for B and E. B and E: Paraffin sections from contralateral entorhinal cortex (B) and hippocampal CA1 (E) were stained for Aβ plaques (red arrowheads) or tau (black arrowheads). Cases exhibiting no plaques have tau pathology in both the EC and CA1. Cases with plaques have similar tau pathology in addition to diffuse plaques in the EC and CA1. C: Aggregates in HEK cells are similar in brains with or without plaques in the EC, a region affected earliest in Braak staging, but are more prominent when cells are treated with plaque-containing brains from regions that develop tangle pathology with increasing Braak stage (HP and PHG). No aggregates are seen in nontreated cells (NT) or cells treated with homogenates from cerebellum (CBLM). D: SDD-AGE of representative cases to detect high-molecular-weight (HMW) and low-molecular-weight (LMW) tau using a polyclonal antibody against total tau. Comparisons of these Braak II cases are made versus a Braak VI sample (VI), which clearly contains both HMW and LMW tau species. HEK cell assay integrated FRET density values indicated at bottom. F: Total human tau protein measured by ELISA from prepared tissue homogenates. G: Quantification of HEK assay from C. Two-way analysis of variance. Data are expressed as means ± SD. ^∗P < 0.05. Scale bar = 100 μm (C). NP, no plaques; P, plaques.

Figure 2

Tau pathology is enhanced in APP/PS1-rTg4510 mice. A: AT8 (pS202/T205 Tau) labeling in somatosensory cortex of rTg4510 and APP/PS1-rTg4510 mice at 4, 6, and 12 months of age. No labeling is observed in wild-type or APP/PS1 mice. B: Quantification of AT8-positive neurons in cortex (t-test). C: Quantification of ThioS tangles in entorhinal cortex of the same mice [U-test (4 months), t-test (6 and 12 months)]. D: ThioS labeling shows neurofibrillary tangle pathology in rTg4510 and APP/PS1-rTg4510 mice at 4, 6, and 12 months. No ThioS labeling is observed in wild-type mice. Plaques are also apparent in APP/PS1 and APP/PS1-rTg4510 mice. Data are expressed as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01. Scale bars: 200 μm (A); 25 μm (D). EC, entorhinal cortex.

Figure 3

Increased aggregation and uptake of tau in homogenates from APP/PS1-rTg4510 mice. A: Aggregate formation markedly increases in HEK cells in the presence of APP/PS1-rTg4510 homogenates (with lipofectamine, 12 hours after application). No aggregates are seen in cells treated with wild-type (WT) or APP/PS1 homogenates (insets). B: Quantification of aggregate formation in cells treated with 0.1 μg protein homogenates in 1% lipofectamine OPTIMEM [t-test (4 and 12 months), U-test]. C: Quantification of cell uptake and aggregate formation in cells treated without lipofectamine and 1 μg protein homogenates [U-test (6 months), t-test (6 and 12 months)]. Data are expressed as means ± SD. ^∗P < 0.05, ^∗∗P < 0.01. Scale bar = 50 μm (A, main images and insets).

Figure 4

No change in tau release or production in APP/PS1-rTg4510 mice. Cerebrospinal fluid (CSF) levels of extracellular total tau (A) and total tau in cortical homogenates (B) are unchanged between rTg4510 mice and APP/PS1-rTg4510 mice, as measured by ELISA. Data are expressed as means ± SD. P = 0.054 (U-test).

Figure 5

Tau-containing high-molecular-weight (HMW) complexes and phosphorylation at specific epitopes in APP/PS1-rTg4510 mice. A: Cortical homogenates (15 μg total protein) from 4-month-old mice of four different genotypes [wild type (WT)] were loaded on an agarose gel under semidenaturing (SDD-AGE) conditions, and the resulting protein blot was probed with a polyclonal total tau antibody (AbCam). The approximate locations of HMW and low-molecular-weight (LMW) are noted. Dashed lines indicate divisions between genotypes. B: Western blot of sarkosyl-soluble and sarkosyl-insoluble extracts was also performed and probed using a polyclonal total tau antibody (Dako) in a separate cohort. C: Western blots of seven unique phospho-tau epitopes performed using cortical homogenates from 4-month-old rTg4510 and APP/PS1-rTg4510 mice. Separate blots were used for each phospho-site, with actin shown for loading control. n = 2 (A and C).

Figure 6

Tau phosphorylation in human Alzheimer disease cases with and without plaques. A–C, G–I, and M–O: Western blots of parahippocampal gyrus homogenates from three representative no plaque and three plaque cases were assessed using antibodies to detect phosphorylated tau species. D–F, J–L, and P–R: Bands were quantified by densitometry and normalized to actin loading controls. Phospho-tau species that were assessed include pT181 (A and D), pS199 (B and E), pT205 (C and F), pT231 (G and J), pS262/pS356 (H and K), pS396 (I and L), pS400 (M and P), and pS422 (N and Q). E: Actin shown for the pS396 blot is from a second pS396 blot and is a sample processing control. O and R: A total tau antibody (HT7) that detects the midregion of tau was also used. Separate blots were used for each tau antibody, with actin shown for loading control below. Data are expressed as means ± SD. ^∗P < 0.05 (t-test). AU, arbitrary unit.

Supplemental Figure S1

Uncropped Western blots (APP/PS1-rTg4510 mice). A and B: Blot of sarkosyl soluble (A) and insoluble (B) fractions probed with a mid-domain polyclonal anti-tau antibody (Dako) or an N-terminal total tau antibody (Tau13; Biolegend). C: Phospho-tau blots and total tau (Dako) blot. D: Blots were reprobed with an anti–β-actin antibody and are shown below their corresponding blots from C. All blots were used in Figure 5. L, ladder; T, rTg4510; W, wild type; X, APP/PS1-rTg4510. Molecular weight ladders were visible at 680 nm (Tau13; actin blots).

Supplemental Figure S2

Uncropped Western blots (human samples). A–H: Uncropped phospho-tau and actin blots from Figure 6. Actin and pS396 shown are from the same blot (F). I: Uncropped total tau blot (Dako) from Figure 6. J–L: Total tau was also detected using an additional rabbit polyclonal antibody (AbCam; J), a monoclonal N-terminal tau antibody (Tau13; Biolegend; K), or a mid-domain monoclonal antibody (HT7; Invitrogen; L). No differences in truncated forms of tau were revealed between groups. Samples were loaded the same for all blots from left to right: ladder, no plaques 1 to 3, and plaques 1 to 3.

Supplemental Figure S3

Aβ pathology in APP/PS1-rTg4510 mice. A pan-Aβ antibody was used to label plaques in mice at 4 (A and E), 6 (B and F), or 12 (C and G) months of age. No Aβ labeling was observed in wild-type (D) or rTg4510 (H) mice but was observed in the brains of APP/PS1 (A–C) and APP/PS1-rTg4510 (E–G). The total number of plaques per mm² did not differ between APP/PS1 and APP/PS1-rTg4510 mice at 4 (I) or 6 (J) months but was significantly reduced at 12 months of age (K). ThioS labeling of dense core plaques reveals that the total plaque area is also reduced at 12 months (L), but the size of ThioS-positive plaques is greater (M). Images are taken in somatosensory cortex, but plaques were quantified throughout a single hemisphere (t-test). Total human/rodent Aβ1-42 was measured in CSF from a separate cohort of mice at 12 months of age (Kruskal-Wallis analysis of variance, P = 0.0024; N), and total human Aβ1-42 was measured in cortical homogenates (Kruskal-Wallis analysis of variance, P < 0.001; O). Data are expressed as means ± SD (I–O). ^∗∗P < 0.01, ^∗∗∗P < 0.001 (Dunn's post hoc comparisons). Scale bar = 200 μm (A–H).

Supplemental Figure S4

Astrocyte pathology in APP/PS1-rTg4510 mice. To assess gliosis in 4-, 6-, and 12-month-old mice, sections were labeled with glial fibrillary acidic protein (GFAP; A–H) and wild-type mice (4-month-old mouse; D) were compared to APP/PS1 (4-month-old mouse; H), rTg4510 (A–C), and APP/PS1-rTg4510 mice (E–G). Stereological quantification reveals an increase in the number of GFAP-positive astrocytes seen in APP/PS1-rTg4510 hemispheres at 6 months (one-way analysis of variance; J) and 12 months (Kruskal-Wallis analysis of variance, P = 0.002; K), but not at 4 months (Kruskal-Wallis analysis of variance, P = 0.01; I) of age. Data are expressed as means ± SD (I–K). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 (Dunn's or Bonferroni post hoc comparisons). Scale bar = 50 μm (A–H).

Supplemental Figure S5

Microglial pathology in APP/PS1-rTg4510 mice. Iba-1 was used to label microglia in 4-month-old mice (A, D, E, and H), 6-month-old mice (B and F), and 12-month-old mice (C and G). Compared to rTg4510 mice (A–C), APP/PS1-rTg4510 mice (E–G) exhibit active phenotypes and clustering near plaques. A representative 4-month-old wild-type and APP/PS1 mouse is shown in D and H, respectively. Total numbers of Iba-1–positive cells are unchanged between genotypes at 4 months (one-way analysis of variance, P = 0.117; I), 6 months (P = 0.580; J), or 12 months (P = 0.099; K) of age. Scale bar = 50 μm (A–H).

Supplemental Figure S6

Cortical volume measurements in APP/PS1-rTg4510 mice. Estimates of the size of cortical volumes are given for each genotype at 4 (A), 6 (B), and 12 (C) months of age based on tracing the area of the cortex from serial sections (40 μm thick) sampled every 400 μm from the frontal cortex to the posterior edge containing entorhinal cortex (4 months, Kruskal-Wallis analysis of variance P = 0.0006; 6 months, one-way analysis of variance P = 0.0003; 12 months, P < 0.0001). Data are expressed as means ± SD (A–C). ^∗P < 0.05, ^∗∗∗∗P < 0.0001 (Dunn's or Bonferroni post hoc comparisons).