Soluble phospho-tau from Alzheimer's disease hippocampus drives. Elisabeth Sanchez-Mejias1,4*, Victoria Navarro2,3,4*, Sebastian Jimenez2,3,4,

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1 Soluble phospho-tau from Alzheimer's disease hippocampus drives microglial degeneration Elisabeth Sanchez-Mejias,, Victoria Navarro,,, Sebastian Jimenez,,, Maria Sanchez-Mico,,, Raquel Sanchez-Varo,, Cristina Nuñez-Diaz,, Laura Trujillo-Estrada,, Jose Carlos Davila,, Marisa Vizuete,,, Antonia Gutierrez, and Javier Vitorica,, -Dept. Biologia Celular, Genetica y Fisiologia. Facultad de Ciencias, Universidad de Malaga, 97 Spain -Departamento Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Spain. -Instituto de Biomedicina de Sevilla (IBiS)-Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Spain. -Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain. These authors contributed equally in this work Co-Senior corresponding author Corresponding authors Javier Vitorica Dept. Bioquimica y Biologia Molecular Facultad de Farmacia. Universidad de Sevilla Instituto de Biomedicina de Sevilla (IBiS)-Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Spain C/ Prof. Garcia Gonzalez, Sevilla, Spain Telephone:

2 Antonia Gutierrez Dept. Biologia Celular, Genetica y Fisiologia. Facultad de Ciencias, Universidad de Malaga, Spain Telephone: agutierrez@uma.es

3 Supplemental Table. Human and murine Taqman probes used for qpcr Probes Murine Human c/ebpa Hs6997_m Pu. Hs7867_m Iba Mm7986_g Hs69_g CDb Mm55_m Hs55885_m CD5 Mm9577_m Hs897_m CD68 Mm7_m Hs8686_g TREM Mm9_g Hs9_m CD Hs768_m Prx Hs6_m CCR Mm87_m Hs77_s CxCr Mm6_s Hs958_s Pry Mm955_s Hs88698_m GAPDH Mm _g Hs9997_g Beta-actin Mm6958_g Hs999999_m

4 Supplemental Figures Supplemental Figure. Microglial activation and pathological characterization of, III-IV and V-VI hippocampus. a) The expression of microglial markers Pu. (a) and c/ebpa (a) was determined by qpcr. The expression of these two markers was increased significantly (Kruskal-Wallis and Dunn post-hoc test, see figure) only in Braak V- VI cases, as compared with Braak or samples. All affected microglial markers showed significant linear correlations using tau-kendall (a-6), corroborating the existence of microglial activation and the implication of a single cell population. b-c) Neuropathological characterization of phospho-tau and Abeta in human hippocampal samples using immunohistochemistry with AT8 (b-6) or G8 (c-6) antibodies. Phospho-tau accumulation was clearly observed in I-IV samples, mainly in CA field (b5) and in both CA (b6) and dentate gyrus (b) in stages. The Abeta plaques affected the CA region and molecular layer of dentate gyrus in I-IV cases (c and c5), however the hilar region was only affected in individuals (c and c6). Alv, alveus; g, granular layer; h, hilus; m, molecular layer; so, stratum oriens; slm, stratum lacunosum-moleculare; sp, stratum pyramidale; sr, stratum radiatum. Scale bars: b-b6 and c-c6, mm. Supplemental Figure. The expression of Pry was reduced in activated microglial cells. a) The co-localization (a and a6) between Iba (a and a) and Pry (a and a5) microglial specific markers was assessed by laser confocal microscopy in samples. Co-localization (a and a6, arrows) between both markers was clearly observed in microglial cells not in direct contact with Abeta plaques (visualized in blue colour). The boxed regions, at higher magnification in insets, showed activated Iba positive and Pry negative microglial cells, closely associated with Abeta plaques (in blue). Furthermore, ex vivo experiments, using isolated adult mice microglia, indicated that the Pry expression was slightly reduced after LPS activation (.5 ±. vs.65 ±.5 relative units for PBS or LPS respectively, n=, two-tailed t-test, t=.8,

5 p=.75). Thus, the expression of Pry receptor was down-regulated after microglial activation g, granular layer. Scale bars: a-a6, 5 µm (insets in a and a6, µm) Supplemental Fig. Microglial response in Abeta (APP/PS) and tau (Thytau) transgenic models. a) APP/PS model displayed a strong age-dependent accumulation of Abetaplaques (a; -month-old mice) and total Abeta (a, representative westernblot using 6E+8E) in the hippocampal formation. This Abeta accumulation was associated with a clear microglial activation. Representative images showing Iba (a, panoramic image; a, high magnification) or CD5 (a5, panoramic image; a6, high magnification) immunostained microglial cells from -month-old mice. The microglial activation was also assessed by determining (qpcr) the expression (a7) of microglial markers (ANOVA and Tukey post hoc test; p<.5). Data (n=5 animals per age) were normalized using 9-month-old WT mice (n=5; not shown) and are mean ± S.D. Quantitative Iba-load determinations at the hilar region (a8), from 6- or -month-old mice, also demonstrated the existence of a significant (ANOVA and Tukey post hoc test; p<.5; n=5/age and genotype) increase in the area covered by Iba- positive cells, as compared with age-matched WT mice. b) Thy-tau transgenic model presented an age-dependent phospho-tau accumulation principally in the CA region, determined by AT8 immunohistochemistry (b) or western-blots (b). This phospho-tau accumulation produced an attenuated microglial response as probed by Iba immunostaining, from -6-month-old WT (b) and Thy-tau (b) mice. Microglial activation was also assessed by qpcr (b5). Only the expression of CD5, CD68 and TREM was slightly but significantly increased (ANOVA and Tukey post hoc test; p<.5). Data (n=5 animals per age) were normalized using 9-month-old WT mice (n=5; not shown) and are mean ± S.D. Quantitative analysis of the microglial domain (b6) demonstrated the existence of a significant decrease (Mann-Whitney U test; p=.; WT n= 75 or Thytau n= cells from or different animals, respectively) in the area covered by the individual microglia cells in aged (-6-month-old) Thy-tau. DG, dentate gyrus; CA- hippocampal subfields. Asterisks indicate amyloid

6 plaques. Scale bars: a, a and a5, 5μm; a and a6, μm; b, μm; b and b, μm Supplemental Figure. Preparation and characterization of the S soluble fractions from, III-IV and V-VI hippocampus. a) Hippocampal samples were fractionated into: S, soluble fraction containing extracellular/cytosolic proteins; S, intracellular vesiculated fraction; S, aggregated proteins, releasable by SDS treatment and P, highly-aggregated proteins, only releasable by SDS-Urea. The presence of phospho-tau species was analyzed by western blots using AT (a) or AT8 (not shown). As expected, I-IV and samples showed a prominent phosphotau accumulation (AT positive tau in the SDS releasable pool (S). This is consistent with the presence of aggregated and phosphorylated tau protein, forming intracellular tangles. We also observed a clear accumulation of AT positive tau protein in the S soluble fraction (extracellular and/or cytosolic) from samples. Total tau in the soluble S fractions was assessed by sandwich ELISA (a). b) Soluble Abeta present in S fractions was quantitatively analyzed by ELISA (b) or dot-blots (b-) using the OC antibody. Supplemental Figure 5. Sarkosyl-insoluble phospho-tau was not toxic for microglial cells and astrocytes were resistant to soluble phospho-tau. a) Representative AT8 western blot of sarkosyl-soluble and sarkosyl-insoluble tau, isolated from two different and samples (a). The toxicity on BV cells of the sarkosyl-insoluble phospho-tau was then tested by Flow Cytometry (a). Although we have assessed three different doses (., and μl of sarkosyl-insluble tau/ μl culture media; equivalent to ten to a hundred times higher dose than S), we did not detect any toxic effect on BV cells (a). Data are mean ± SD of three independent experiments. b) Toxicity (using flow cytometry, b) of S fractions isolated from Braak,, Braak III-IV and samples was also assessed using the astrocytic cell line WJE. As shown (b), no toxicity was observed in any case. Data are mean ± S.D. of three independent experiments.

7 Supplemental Figure 6. Characterization and toxicity of the S soluble fractions prepared from APP/PS and Thy-tau tansgenic models and SH-SY5Y-tau transfected cells. a) S fractions, isolated from APP/PS mice, accumulated oligomeric soluble Abeta (detected by dot-blots using OC, a). S fractions isolated from agematched WT or Thy-tau mice (see below) were used as negative controls. a) Quantitative analysis of BV cell toxicity after treatment with 6- or 8-monthold APP/PS-derived S fractions showing percentage of viable, early apoptotic cell (Annexin V positive) or late apoptotic cells (Annexin V and PI positive cells). Data are mean ± S.D. of three different experiments. b) S fractions isolated from Thy-tau models. The phospho-tau (b, upper panel) or total tau content (b, middle panel) in these fractions was assessed by western blots using AT8 or Tau6 antibodies. As shown, there was an age-dependent increase in the soluble AT8-positive phosho-tau in the S fractions. (b) Quantitative analysis of BV cell viability after treatment with -, 9- or -monthold Thy-tau-derived S fractions. Percentage of viable, early apoptotic cell (Annexin V positive) or late apoptotic cells (Annexin V and PI positive cells) was indicated in the figure. Only S fractions derived from -month-old Thy-tau presented toxicity at the dose used (.μl/μl culture media). Data are mean ± S.D. of three different experiments. c) The presence of soluble phospho-tau in transfected SH-tau cells was also assessed. For these experiments, the cells were disrupted by sonication and the soluble and insoluble fractions isolated by centrifugation at,xg ( min at ºC). Total proteins were isolated using Laemmli-dissociation buffer. As shown, most tau forms (either phosphorylated, AT8-positive, or total, Tau6-positive) were isolated in the soluble fraction. This experiment was repeated twice with similar results.

8 a Relative Units Relative Units a Pu Kruscal-Wallis = p=. Braak I-IV a Cebpa Kruscal-Wallis =.77 p =.7 p<.5 p<.5 CD68 Cebpa 6 5 CD5 vs CD68 Braak I-IV τ =.6 p=. 5 6 CD5 a a CD5 vs Cebpa Braak I-IV PU. Cebpa a5 a5 Braak I-IV CD5 vs Pu. Braak τ=.9 I-IV p=, 5 6 CD5 a6 Pu.-Cebpa Braak I-IV τ =.67 p=. 5 6 CD5 b I-IV τ=.88 p=. Pu DG CA C b b h gm alv so sp sr slm b b5 h g m I-IV alv so sp sr slm b b6 h gm alv so sp sr slm DG CA c h gm al so v sp sr c c c5 c6 slm h g m alv so sp sr c h gm alv so sp sr Supplemental Fig.

9 a Iba- Pry Merge a a a Iba- Pry Merge a a5 a6 Supplemental Fig.

10 a Abeta a Iba a CD5 CA DG CA DG CA DG a Iba a CD5 M APP/PS 9M M a7 mrna expression (relative units) Iba-positive Iba-positive area (%) 8 6 a CDb M 9 M M WT APP/PS Tukey p<.5 PS-APP APP/PS Hippocampus Iba CD5 Iba-load Iba-load Hilus Hilus ANOVA F(,)=6.57 p=. CD68 CXCR Pry TREM Tukey p<.5 b a5 a6 6 Age (months) AT8 Iba Iba CA DG b b b b Tau WT M 9M M AT8 Tau6 b5 mrna expression (relative units) 8 6 CDb M 9 M M Thy-Tau Hippocampus Iba CD5 CD68 CXCR Pry TREM b6 Microglial domain (μm/cell) Microglial domain p=. WT Thy-Tau Supplemental Fig.

11 a AT a I-IV S S S P S S S P S S S P a 5 a ELISA TAU total 78 5 pg Tau / μg protein S 5 Braak I-IV b pg Aβ / μg protein S b,5,,5,,5 b ANOVA; F(,5)=.78 p=. ELISA Aβ total p<.5 b OC I-IV Relative units (vs ) b b ANOVA, F (,5) =7. p=. Aβ (OC), Braak I-IV I-IV Supplemental Fig.

12 a a Braak Sarkosyl Soluble Sarkosyl Insoluble II II VI VI II II VI VI a P.I Braak VI Braak VI a Viable cells Annexin V Viable cells (%) 8 6 b b... μl Sarkosyl-insoluble tau / μl b Braak WJE-toxicity Viable cells Annexin positive cells Annexin-PI positive cells P.I I-IV Number of cells (%) 8 6 Annexin V Braak I-IV S (.μg/μl) Supplemental Fig.5

13 a a WT APP/PS ThyTau OC 6M 6M a S from PSxAPP S from APP/PS a Viable cells Annexin positive cells Annexin-PI positive cells M 9M BV cells (%) 8 6 8M M b b AT8 S-ThyTau M 9M M b PBS WT APP/PS PSxAPP 6M 6M PSxAPP APP/PS 8M S (.μg/μl) S from Thy-Tau Viable cells Annexin positive cells ANOVA F(,)=5. Annexin-PI positive cells p= BV cells (%) 8 6 Tukey p<.5 Tukey p<.5 Tukey p<.5 Tau 6 PBS Thy-Tau M Thy-Tau 9M Thy-Tau M S (.μg/μl) GAPDH c 9 - Total AT8 Soluble Insoluble Tau6 Total Soluble Insoluble Supplemental Fig.6