Display Settings:

Items per page

Results: 12

1.
Figure 9

Figure 9. Binding of tau to microtubules is not altered by the P301L mutation. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

QBI293 cells transfected with expression plasmids for WT or P301L tau were harvested 48 hours after transfection for microtubule (MT) binding assays, as described in “Materials and Methods.” Representative immunoblots with anti-tau (17025) or anti-β-tubulin antibodies of unbound fraction (supernatant; S) or microtubule-bound fraction (pellet; P) and concurrent experiment on cells with or without fibril treatment and harvested by biochemical cellular fraction (Triton-soluble and Triton-insoluble fractions). Representative immunoblots are from the same Western blot per antibody, but rearranged for presentation. 41±6% [SD] of WT and 40±14% [SD] of P301L tau were bound to microtubules (n=3).

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
2.
Figure 6

Figure 6. Thioflavin S reactivity assessed by confocal microscopy on tau expressing cells treated with recombinant, pre-fibrillized α-syn. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

Representative images of Thioflavin S (ThioS; green) immnuoreactivity of QBI293 cells that were transfected with expression plasmids for WT (A) or P301L (B-C) tau and treated with recombinant, pre-fibrillized full-length α-syn. Representative images are of cells fixed 72 hours after transfection. Intracellular, ThioS-positive aggregates were double-labeled with AT8 (A) or 17025 (B) (red). (C) Recombinant α-syn (SNL4 immunoreactivity; red) was observed sticking to the outside of cells, and only larger extracellular aggregates displayed robust ThioS reactivity. Anti-α-syn antibody SNL4 did not immunolabel large, intracellular ThioS aggregates (arrow). Bar scale = 20 μm.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
3.
Figure 2

Figure 2. Representative confocal microscopy of double-immunofluorescence with phospho-specific antibodies on α-syn and tau show mingling of α-syn and tau aggregates. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

(A) Cells that were co-transfected with expression plasmids for α-syn and tau display a paucity of immunostaining with pSer129 or PHF1. (B-D) Transfected cells treated with recombinant, pre-fibrillized 21–140 α-syn protein presented pSer129 and PHF1 (B,C) or AT8 (D) positive aggregates. Aggregates sometimes appeared intertwined or with phosphorylated α-syn circling phosphorylated tau. Representative images are of cells fixed 72 hours after transfection. (E) Double-immunofluorescence between pSer129 and AT8 on the amygdala of a patient diagnosed with LBVAD shows similar morphology in vivo. Equivalent exposures were provided for all representative samples. Bar scale = 60 μm for A,C-D; 40 μm for B; 100 μm for E; 20 μm for C, inset.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
4.
Figure 5

Figure 5. Polymer subpopulations of fibrillized, recombinant α-syn can promote cellular tau aggregation. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

Recombinant full-length or 21–140 α-syn was pre-fibrillized. Prior to sonication, polymers of full-length α-syn were separated by centrifugation, as described in “Materials and Methods,” and all populations were subjected to sonication. (A-D) Ultrastructural EM analyses were performed on full-length α-syn that had not been separated by centrifugation (mix) (A), small α-syn oligomers (B), large α-syn fibrils (C), or 21–140 α-syn mix (D). (E) Cells expressing WT or P301L tau were treated with each of these polymer types at a final concentration of 1 μM. Cells were harvested 72 hrs after transfection and biochemical cellular fractionation was performed, separating the Triton X-100 (TX) soluble from the TX-insoluble fraction. No differences were noted in aggregation propensity between these polymer types, while soluble, recombinant, non-fibrillized α-syn did not promote cellular tau aggregation.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
5.
Figure 1

Figure 1. Tau co-localized with α-syn in cells that form cellular aggregates. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

Representative double-immunofluorescence between SNL4 (anti-α-syn antibody) and an anti-tau antibody was performed on QBI293 cells co-transfected with expression plasmids for human wild-type α-syn and tau. (A,B) In the absence of fibril treatment, α-syn appeared diffuse and tau appeared diffuse and bundled. (C-E) For cultures treated with recombinant, pre-fibrillized 21–140 α-syn protein, intracellular, endogenously generated α-syn formed large cellular aggregates that sometimes contained tau. Co-localization of α-syn and tau was assessed by confocal microscopy (B,D,E). Overlay between a portion of SNL4 and anti-tau immunoreactivity was observed in aggregate-containing cells. Many of these cells appeared rounded in morphology, and occasional tau “tails” were observed attached to aggregated α-syn proteins. Representative images are of cells fixed 72 hours after transfection. Equivalent exposures were provided for all representative samples. Bar scale = 100 μm for A,C; 30 μm for B,D,E.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
6.
Figure 4

Figure 4. Biochemical cellular fractionation performed on tau expressing cells after treatment with recombinant, pre-fibrillized α-syn protein. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

QBI293 cells were co-transfected with expression plasmids for WT or P301L tau and α-syn or pcDNA3.1 (pcDNA) empty vector. Parallel samples either received no treatment or were treated with recombinant pre-fibrillized 21–140 α-syn protein. Cells were harvested 72 hours after transfection, and biochemical cellular fractionation was performed, as described in “Materials and Methods.” (A) Representative immunoblots of biochemical fractionation with anti-tau antibody 17025, with phospho-specific antibodies PHF1, AT8, 12E8, AT180, and AT270, and with anti-α-syn antibody SNL4. Triton-insoluble tau and α-syn were only observed after treatment with recombinant, pre-fibrillized α-syn treatment, and robust increases in Triton-insoluble tau were observed with cells expressing P301L tau. Representative immunoblots are from the same experiment. (B) Quantitative summary of time-course of the formation of Triton-insoluble tau, quantified by densitometry of anti-tau (17025) immunoreactivity. Indicated time is hours after transfection. Percent of Triton-insoluble tau was calculated as Triton-insoluble tau/(Triton-soluble + Triton-insoluble tau) *100. Data represent average ± SD (n=4).

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
7.
Figure 8

Figure 8. Confocal microscopy analyses of cytoskeletal markers on cells containing tau aggregates. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

QBI293 cells were transfected with expression plasmids for WT (A,C) or P301L (B) tau and treated with recombinant, pre-fibrillized full-length α-syn. Cells containing tau aggregates were identified by the characteristic increased anti-tau immunoreactivity and displacement of the nuclear membrane. Representative images are cells fixed 72 hours after transfection. Double-immunofluorescence was performed between anti-tau antibodies (green) and anti-β-tubulin (A), anti-γ-tubulin (B), or anti-vimentin (C) (red). 3-Dimensional (3D) projections of cells were composed by stacking 10–12 confocal images and rotating images 15–30 degrees on a central axis (first three columns). A single, merged Z-plane (<0.7 μm) of each representative image is provided at the far right column. (A) Anti-β-tubulin and (C) anti-vimentin immunoreactivity was observed around and inside some tau aggregates. (B) Anti-γ-tubulin immunoreactivity was observed outside tau aggregates, often displayed outside of the center of the cellular plane. Similar morphology of tau aggregates were observed for wild-type and P301L expressing cells. Bar scale = 40 μm.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
8.
Figure 3

Figure 3. Recombinant, pre-fibrillized α-syn promotes the formation of tau aggregates in cells only overexpressing tau. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

QBI293 cells were transfected with expression plasmids for WT tau (A-C) or tau containing the P301L mutation (D), and double-immunofluorescence was performed between an anti-tau antibody (green) and AT8 (A-B,D) or PHF1 (C) (red). Representative images are of cells fixed 72 hours after transfection. In the absence of recombinant, pre-fibrillized α-syn treatment, rare AT8 positive cells were observed, and tau appeared mostly bundled (A). After recombinant, pre-fibrillized α-syn treatment, AT8 immunoreactivity increased for both wild-type (B) and P301L (D) tau expressing cells. These aggregates displayed greater anti-tau immunoreactivity, and a greater percentage of transfected cells were AT8-immunopositive in the presence of the P301L mutation. (C) Cellular tau aggregates were also positive for PHF1, and were often observed as encompassing the entire cell body, as observed by confocal microscopy. Equivalent exposures were provided for all representative samples. Bar scale = 100 μm for A-B,D; 20 μm for C. (E,F) Summary of the percent of tau-positive cells that displayed AT8 (E) or PHF1 (F) immunoreactivity in the absence (-) or presence (+) of fibril treatment (for AT8 - *, p = 0.0005 by two-way, parametric t-test; **, p = 0.005; #, p = 0.007 by non-parametric Welsh t-test; for PHF1 - †, p = 0.04; ‡, p = 0.008; §, p = 0.001 by non-parametric Welsh t-test; n = 4 for all sets). Data represent average ± SD.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
9.
Figure 11

Figure 11. Overexpression of GSK3β or MARK2 each inhibit the propensity of tau to form cellular aggregates. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

QBI293 cells were co-transfected with plasmids for the expression of WT or P301L tau and pcDNA (empty vector), GSK3β (A, B) or MARK2 (C). Cellular experimentation was performed in the absence (- fibrils) or the presence (+ fibrils) of recombinant, pre-fibrillized α-syn. Cells were harvested 72 hrs after transfection and biochemical cellular fractionation was performed, separating the Triton X-100 (TX) soluble from the TX-insoluble fraction. (A) Representative immunoblots of biochemical fractionation with anti-tau antibody 17025 on WT or P301L tau expressing cells in the presence or absence of GSK3β overexpression. (B,C) Quantitative summary of the percent of tau present in the Triton-insoluble fraction for each transfection and fibril treatment condition, as determined by densitometry of anti-tau (17025) immunoreactivity. (B) With WT tau and GSK3β co-expression, no significant increase in Triton-insoluble tau was observed after the addition of α-syn fibrils, but a significant increase in Triton-insoluble tau was observed above samples with WT tau and pcDNA in the absence of fibrils (p < 0.002 by one-way ANOVA; *, p < 0.01 by Bonferroni post-test analyses; n = 4). With P301L tau, GSK3β co-expression resulted in a significant increase in Triton-insoluble in the absence of fibril treatment and a reduction of Triton-insoluble tau in the presence of treatment (p < 0.0001 by one-way ANOVA; **, p < 0.001; #; p < 0.05 by Bonferroni post-test analyses; n = 4). (C) MARK2 co-expression significantly reduced the propensity of WT and P301L tau to aggregate after α-syn fibril treatment (for WT, p = 0.0006 by one-way ANOVA; for P301L, p < 0.0001 by one-way ANOVA; **, p < 0.001; #; p < 0.05 by Bonferroni post-test analyses; n = 5). ns = not significant. Data represent average ± SD.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
10.
Figure 7

Figure 7. Ultrastructural analyses of cells forming tau aggregates. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

Electron microscopy was performed on transfected cells expressing P301L tau and treated with recombinant, prefibrillized α-syn protein. Representative images are cells fixed 72 hours after transfection. (A-C) Progressively increasing magnification on the center of same cell to show cellular morphology and the cytoplasmic localization of fibrous protein aggregates. Examples of damaged mitochondria were observed (arrowheads). (D) Higher magnification of cytoplasmic fibrils isolating organelles. (E) High magnification of intracellular tau fibril morphology. (F) Electron microscopy of a normal cell is provided for comparison. (G-H) Immunolabeling with anti-tau antibody 17025 of P301L tau transfected cells in the absence (G) or presence (H) of recombinant, pre-fibrillized α-syn. (I) Immunolabeling with anti-α-syn antibody SNL4 on P301L transfected cells treated with recombinant, pre-fibrillized α-syn. Clusters of SNL4-positive fibrils were noted (arrows). H-I Insets provide higher magnification of cellular fibrils within each frame. N = nucleus; I = inclusion. Bar scale = 1.5 μm for A; 1 μm for B, F; 500 nm for C; 416 nm for G; 333 nm for H, I; 300 nm for D; 185 nm for E; 166 nm for inset H; 100 nm for inset I.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
11.
Figure 12

Figure 12. Site-specific mutations of the 12E8 and AT8 epitopes alter the propensities of tau to form cellular aggregates. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

QBI293 cells were co-transfected with the indicated tau plasmids and pcDNA or MARK2. Cellular experimentation was performed in the absence (-) or presence (+) of recombinant, pre-fibrillized α-syn. (A) Representative immunoblots of biochemical fractionation with antibodies 17025, 12E8, AT8, PHF1, or anti-myc (to identify MARK2 overexpression) performed on cells expressing WT tau or tau containing S262A and/or S356A mutations in the presence or absence of MARK2 overexpression. (B) Quantitative summary of the percent of tau present in the Triton-insoluble fraction after treatment with recombinant α-syn fibrils. MARK2 co-expression significantly reduced the amount of Triton-insoluble tau in all conditions that did not contain the P301L mutation (p < 0.0001 by one-way ANOVA; *, p < 0.05; **, p < 0.001 by Bonferroni post-test analyses; n = 4). Further, tau S356A mutants co-expressed with MARK2 and treated with recombinant fibrils further reduced the propensity of tau to form cellular aggregates (#, p < 0.01 by Bonferroni post-test analyses; n = 4). No significant changes were observed between conditions expressing the P301L mutation of tau and treated with recombinant α-syn fibrils when multiple comparisons were factored into analyses. (C) Quantitative summary of the percent of tau present in the Triton-insoluble fraction after treatment with recombinant α-syn fibrils. Tau was mutated at the AT8 epitope residues S202 and T205 to either Ala or Glu. Both the S202A/T205A and the S202E/T205E mutations inhibited the propensity of tau to aggregate (p = 0.0004 by repeated measures ANOVA; *, p < 0.05; **, p < 0.001, by Bonferroni post-test analyses; n = 3). In the presence of the P301L mutation, only the additional S202E/T205E mutation significantly inhibited the propensity of cellular tau to aggregate (p = 0.02 by repeated measures ANOVA; *, p < 0.05 by Bonferroni post-test analyses; n = 3). All data represent average ± SD.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.
12.
Figure 10

Figure 10. The P301L mutation in tau enhances the ability of pre-fibrillized α-syn to promote tau fibrillization in vitro. From: Induction of intracellular tau aggregation is promoted by ?-synuclein seeds, and provides novel insights into the hyperphosphorylation of tau.

(A – left panel) Representative Commassie Blue R-250 stained SDS-polyacrylaminde gel of sedimentation analyses of pre-formed, sonicated recombinant α-syn fibrils (0.25 mg/ml) and tau (1 mg/ml) after 3 days under assembly conditions in vitro, as described in “Materials and Methods.” (A – right panel) Representative Commassie Blue R250 stained gel of soluble, recombinant α-syn (0.3 mg/ml) and tau (1 mg/ml) after 7 days of incubation in the absence of recombinant α-syn fibril seeds show an inability of tau to sediment. (B) Quantitative summary of the ability of recombinant α-syn fibril seeds to promote tau aggregation at 3 days of incubation. P301L tau sedimented to a greater extent than WT tau (*, p<0.0001 by Mann-Whitney non-parametric U-test). (C) K114 fluorometry of assembled α-syn and tau under the above indicated conditions provided in arbitrary fluorescence units (AFU). α-Syn promoted increased K114 fluorometry in the presence of P301L tau (*, p<0.0001 by one-way ANOVA; p<0.001 for P301L tau when directly compared to other conditions by Bonferroni post-test analyses). All data represent average ± SD (n=9 in 3 independent experiments). (D) The ability of recombinant, pre-fibrillized, sonicated α-syn to promote sedimentation of soluble α-syn (0.3 mg/ml), WT tau, or P301L tau was compared at 1, 3, and 7 days of incubation. Maximum sedimentation of WT tau and soluble α-syn was observed after 1 day of incubation, while sedimentation of P301L tau increased over the course of 7 days. Significant differences between all three protein types were observed at 1 and 3 days (at 1 day, p< 0.0001 by one-way ANOVA, with p<0.05 between WT and P301L tau, and p<0.001 between α-syn and WT or P301L tau by Bonferroni post-test analyses; at 3 days, p<0.0001 by one-way ANOVA, with p<0.001 between all conditions by Bonferroni post-test analyses; at 7 days, p<0.0001 by one-way ANOVA, with p<0.001 between WT tau and P301L or α-syn by Bonferroni post-test analyses; n = 5 to 13, depending on condition, each performed in a minimum of 3 independent experiments). Data represent average ± SD.

Elisa A. Waxman, et al. J Neurosci. ;31(21):7604-7618.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk