1. Unconventional Secretion Mediates the Trans-cellular Spreading of Tau
Taxiarchis Katsinelos, Marcel Zeitler, Eleni Dimou, Andromachi Karakatsani, Hans-Michael Müller, Eliana Nachman, Julia P. Steringer, Carmen Ruiz de Almodovar, Walter Nickel, Thomas R. Jahn 
Cell Reports 
Volume 23, Issue 7, Pages (May 2018)
DOI: /j.celrep
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2. Cell Reports 2018 23, 2039-2055DOI: (10.1016/j.celrep.2018.04.056)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Tau Is Unconventionally Secreted from Neurons
(A) WB analysis of the tau variants expressed in SH-SY5Y cells. Lysates were blotted against HA for tau detection, whereas GAPDH was employed as loading control.
(B) The tau variants co-localize with MTs in SH-SY5Y cells. HA and tubulin antibodies were used against tau and tubulin, respectively, whereas the nucleus was stained with Hoechst. The scale bar represents 15 μm.
(C) Equal amounts of lysed SH-SY5Y cells were dephosphorylated. Treated and untreated samples were immunoblotted against HA for detecting tau and GAPDH as loading control.
(D) Conditioned medium from neuroblastoma cells expressing the tau variants was immunoprecipitated against HA and subjected to WB analysis. GAPDH was used as assay quality control.
(E) Exosomes (E) from conditioned medium were isolated through ultracentrifugation (see also Figure S3A), and the supernatant containing free tau (F) was subjected to IP against HA. Endogenous flotillin 2 was used as exosomal marker. See also Figure S1E.
(F) SH-SY5Y cells expressing tau E14 were treated with increasing concentrations of NaClO3. Lysates and IP medium were analyzed by WB. GAPDH was employed as loading and assay quality control. See also Figures S1F and S1G.
(G) Mouse primary neurons were stained against tau and tubulin. The scale bars represent 50 μm and, in magnified image, 30 μm.
(H) Lysates from mouse primary hippocampal neurons and Sf9-derived recombinant 0N4R tau WT were dephosphorylated. Treated and untreated samples were blotted against tau and GAPDH.
(I) Primary hippocampal neurons were treated with increasing concentrations of NaClO3, and the conditioned medium was subjected to IP against tau. Lysate and medium fractions were immunoblotted against tau, whereas GAPDH and MAP2 were employed as loading and assay quality controls.
(J) Densitometric quantification from WB replicates described in (I) (n = 9; from 4 independent mouse preparations).
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4. Figure 2 Tau Phosphorylation Promotes Its Secretion
(A) HA and tubulin antibodies were used against tau and tubulin, respectively, whereas nucleus was stained with Hoechst. The scale bar represents 30 μm.
(B) Increasing concentrations of recombinant tau variants were incubated with Taxol-stabilized MTs. The % of bound fractions relative to total tau was determined by densitometric quantification, and non-linear regression is displayed (n ≥ 3). See also Figure S2D.
(C) The dissociation constant (KD) was calculated from experimental replicates described in (B).
(D) Sarkosyl-insoluble fractions were isolated from lysates, and medium samples were subjected to IP against HA. GAPDH was used as loading, fractionation, and assay quality control.
(E) Lysates and IP medium from tau WT cells transfected with HA-GSK3β or empty vector were blotted against HA for total tau and GSK3β detection (longer exposure), AT8 for phosphorylated tau, and GAPDH as loading and assay quality control.
(F) Western blot replicates as described in (E) were densitometrically quantified. The secreted tau WT levels were normalized to intracellular expression and compared to the cells transfected with the empty vector (n = 12).
(G) Sarkosyl-insoluble secreted proteins (P) were separated by centrifugation, whereas the soluble tau (S) was immunoprecipitated against HA.
(H) Densitometric quantification of WB replicates described in (G) (n = 4).
(I) Exosomal (E) and free supernatant (F) fractions from conditioned medium were analyzed by WB. Endogenous Flotillin 2 was employed as exosomal marker. See also Figures S3A and S3B.
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5. Figure 3 Interaction of Tau with Components of the Plasma Membrane
(A and B) Cells expressing tau E14 (A) or FGF4-GFP (B) were treated with brefeldin A or monensin. Secreted proteins were immunoprecipitated against HA and GFP, respectively. GAPDH was employed as intracellular loading control. See also Figures S3D–S3G.
(C) Recombinant tau variants and GFP were incubated with immobilized phospholipids. The tau-incubated membranes were probed with the KJ9A pan-tau antibody, whereas the GFP was detected by a GFP antibody.
(D) Binding experiments of tau E14-GFP to liposomes analyzed by FACS-based assay. PC- or PM-like LUVs, supplemented with or without PI(4,5)P2, were incubated with increasing concentrations of recombinant protein (n = 3). See also Figure S4C.
(E and F) CF-sequestered PM-like LUVs supplemented with PI(4,5)P2 (E) or Ni-NTA lipid (F) were incubated with 2 μM recombinant proteins or vehicle only. The disruption of the membrane integrity was measured by fluorescence dequenching. See also Figures S4D–S4G.
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6. Figure 4
Tau Secretion Is Dependent on PI(4,5)P2 and Sulfated Proteoglycans
(A) Cell surface biotinylation was performed on living cells, and samples from cell lysate (C) and surface fraction (S) were analyzed by WB. The tau variants were detected by immunoblotting against HA, whereas the other samples against GFP. GAPDH was employed as loading and assay quality control.
(B) Densitometric quantification from WB replicates described in (A) (n ≥ 6). For the matter of clearness, not all statistical comparisons are indicated.
(C) CHOK1 tau WT cells were treated with forskolin and subjected to cell surface biotinylation. Cell lysate (C) and surface (S) samples were blotted against HA for detection of tau, whereas GAPDH was used as loading and cellular integrity control. The fractions of tau retained on the cell surface were subsequently compared to the untreated control (n = 9).
(D) Cell surface biotinylation was employed on neomycin-treated CHOK1 tau E14 cells. Cell lysate (C) and surface (S) samples were analyzed by WB. Tau E14 was detected by blotting against HA, whereas GAPDH was employed as loading and cellular integrity control. All samples were compared to the untreated control (n = 14). See also Figure S5D.
(E) CHOK1 tau E14 cells were treated with increasing concentrations of NaClO3. After surface biotinylation, samples from cell lysate (C) and surface biotinylated fractions (S) were analyzed by immunoblotting against HA. GAPDH was employed as loading and cellular integrity control. The WBs were subsequently quantified densitometrically (n = 9).
(F) CHOK1 tau E14 cells were treated as described in (C) and probed for tau E14 secretion. Cell lysates and IP-medium fractions were analyzed through WB. GAPDH was employed as loading and assay quality control. The secreted tau E14 levels were normalized to intracellular levels, and all samples were compared to the untreated control (n = 9).
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7. Figure 5
Impaired Tau Secretion from sGAG-Deficient Cells Is Restored by Co-culturing with Wild-Type Cells
(A) Phosphomimetic tau E14 expression and secretion from CHOK1 and CHO745 cells. Cell lysates and IP medium fractions were probed with the HA antibody, whereas GAPDH was used as loading and assay quality control. Three representative experimental repeats are displayed. See also Figures S6A–S6C.
(B) Densitometric quantification from WBs shown in (A). Tau E14-secreted levels were normalized to intracellular expression and subsequently compared to CHOK1 tau E14 secretion (n = 12).
(C) Immunofluorescence images from single or co-culture combinations. FGF2-GFP and tau E14 were detected using GFP and HA antibodies, respectively, whereas Hoechst was used for nuclear staining. The scale bar represents 30 μm.
(D) WB analysis of cell lysate and IP medium from co-cultured cells. Both blots were probed using the HA and GFP antibody for detection of tau E14 and FGF2, respectively. GAPDH was employed as loading and assay quality control. Three representative experimental replicates are displayed. See also Figures S6D and S6E.
(E) Densitometric quantification from experiments described in (D). Secreted tau E14 levels were normalized to intracellular expression and compared to the sGAG-deficient combination (n = 12).
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8. Figure 6
Tau E14 Transfer between Cells Depends on sGAG-Mediated Secretion
(A) FACS analysis of GFP-positive cells from different combinations: tau E14 and FGF2-GFP cells cultured separately, co-cultured, or mixed right before sorting. Box labeled as “P3” indicates the sorted populations.
(B) The GFP-sorted populations were lysed and immunoprecipitated against HA. Samples from each step were probed with HA and the GFP antibodies for detection of tau E14 and FGF2, respectively. GAPDH was used as loading control.
(C) Tau E14 spreads to neighboring FGF2-GFP cells. HA and GFP antibodies were employed for tau E14 and FGF2 detection, respectively, whereas Hoechst was used for nuclear staining. The scale bars represent 20 μm and, in magnified image, 5 μm. See also Figure S5F.
(D) Image-based analysis of different co-culture combinations. Tau E14-positive signal inside FGF2-GFP cells was quantified through image analysis (3 independent experimental replicates with total n ≥ 300 cells processed). For the matter of clearness, not all statistical comparisons are indicated.
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9. Figure 7 Secreted Tau E14 Induces Tau RD-GFP Aggregation
(A) CHOK1 RD-GFP cells were transduced with monomeric or fibrillar tau E14 and stained with GFP and His antibody for RD-GFP and recombinant tau, respectively, whereas Hoechst was used for nuclear staining. Intracellular aggregates (arrowhead) were detected only upon fibrillar tau transduction. The scale bar represents 7 μm. For more examples, see also Figure S7C.
(B) Quantification of GFP-positive intracellular aggregates upon transduction. The GFP-positive inclusions were counted through image analysis of z-projected scans (3 independent experimental replicates with total n ≥ 700 cells processed).
(C) CHOK1 tau RD-GFP cultured alone or with CHOK1 or CHO745 tau E14-expressing cells. Cells were stained with tau10 and the GFP antibodies full-length tau E14 and RD-GFP detection, respectively, whereas Hoechst was used for nuclear staining. GFP-positive inclusions were detected in significantly higher occurrence upon co-culturing with CHOK1 tau E14 cells (magnifications in e and f) compared to culturing alone (a and b) or with CHO745 (c and d). The scale bars represent 20 μm and, in magnification, 5 μm. See also Figure S7D.
(D) Quantification of GFP-positive intracellular aggregates from fixed cells as described in (C). The GFP-positive inclusions were counted through a semi-automated analysis of randomly selected z-projected scans (3 independent experimental replicates with total n ≥ 700 cells).
Cell Reports  , DOI: ( /j.celrep )
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