Fig 1: Exostosin-2 knockdown prevented internalization and accumulation of extracellular tau species in neurons.a, b Primary neurons (DIV2) were applied AccellTM SMARTpool siRNA for non-targeting (NT) or Ext2 gene (NM_001355075) at concentrations of 50–500 nM for 96 h. 18s rRNA was used for the reference gene and normalization in gene expression analysis. Ext2 siRNA (500 nM) significantly reduced the exostosin-2 mRNA (a) and protein (b) levels compared with NT siRNA. Results were from three independent experiments and shown as fold difference expression relative to control (mean ± SEM). Statistical analyses were measured by one-way ANOVA with Tukey’s test (a) or unpaired, two-tailed Student’s t test (b) from three biological independent experiment. Results showed as the value of mean ± SEM, a ****p < 0.0001 vs UT group, ##p < 0.01, ####p < 0.0001 vs NT group, b **p < 0.01 vs NT group. c–e Neurons (DIV2) were preincubated with NT or Ext2 siRNA for 96 h followed by AF568-tagged TauO from AD (c), PSP (d), or DLB (e) treatment for 18 h. Cells were immunolabeled with a mature neuronal marker (βIII-tubulin, blue), and an early endosomal marker (Rab5, green). Representative orthogonal images depicted AF568-tagged TauO co-localized to early endosomes (arrows). Scale bar: 2 and 10 μm. f–h Neurons were untreated (UT) or treated with 0.1 μM biotin-tagged TauO (+) from AD (f), PSP (g), or DLB (h) with similar experimental conditions and parameters as for (c–e). Internalized tau was detected using anti-Streptavidin antibody. Representative Western blot images depicted the appearance of exogenously applied TauO. Internal controls from the same blot were probed with anti-βIII-tubulin. Analysis of internalized tau levels was on the lower panel of each immunoblot showing as Streptavidin band intensity (HMW: 75–250 kDa) normalized to internal control and presented as the percentage of UT group. Statistical analyses were measured by one-way ANOVA with Tukey’s test from three biological independent experiments. Results showed as the value of mean ± SEM, **p < 0.01, ***p < 0.001 vs UT group. ##p < 0.01 vs TauO-treated group. Western blot analyses of TauO from AD, PSP, or DLB were performed on separate membranes. The same membranes were re-probed for marker proteins of autophagy–lysosomal pathway as shown in Fig. 5a–c. The immunoblots for internal controls shown in Fig. 4f–h were reused in Fig. 5a–c.
Fig 2: Mean fluorescence area of endosomal maturation markers (A) Rab5 and (B) Rab7. Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of dairy cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) (JD+ clinical n=7, JD+ subclinical n=7) or JD- controls (n=9). Cells were cultured 5-6 days to generate monocyte-derived macrophages (MDMs), pre-treated with vitamin D3 as detailed in methods, then incubated 24 hrs +/- 10:1 MOI live MAP +/- 25(OH)D3 or +/- 1,25(OH)2D3. Rab5 and Rab7 primary antibodies were both rabbit polyclonal so labeling for each marker was performed in a separate panel. Rab5 primary antibody was coupled with an AF594 secondary and an AF647 secondary antibody was used to detect Rab7. Intracellular labeling was measured by colocalization with macrophage marker CD68 detected with an AF488 secondary antibody. Data are presented as the mean fluorescence area ± SE and significance levels are as follows: * < 0.05, ** < 0.01, *** < 0.001. Comparisons between MAP treatment within JD infection status groups are c < 0.001 and intra-JD status comparisons are # < 0.001.
Fig 3: Colocalization of endosomal trafficking markers (A) Rab5 and (B) Rab7 with SYTO 9 labeled Mycobacterium smegmatis (M. smeg) and Mycobacterium avium subsp. paratuberculosis (MAP) K10-GFP within CD68+ monocyte derived macrophages (MDMs). Detection of fluorescent mycobacteria within acidic compartments was measured using (C) LysoTracker Red DND-99. A subset of cows naturally infected with MAP (JD+ clinical n=2, JD+ subclinical n=2) or JD- controls (n=2) were inoculated at 10:1 MOI with M. smeg or MAP K10-GFP. Data are presented as the mean fluorescence area ± SE and significance levels are as follows: * < 0.05, ** < 0.01, *** < 0.001.
Fig 4: Confocal microscopy image showing endosomal markers within monocyte derived macrophages (MDMs) cultured from a JD- control cow that were infected with Mycobacterium avium subsp. paratuberculosis (MAP) and treated with 25(OH)D3 in vitro. Separate protocols were performed for (A–D) early endosomal marker Rab5 and (E–H) late endosomal marker Rab7. All channels are overlayed and shown in the (A, E) composite panels. (B) Rab5 (magenta) was labeled with an AF594 secondary antibody and excited on the 561 nm laser, while (F) Rab7 (orange) was labeled with an AF647 secondary antibody and excited on the 640 nm laser. (C, G) CD68 (green) was labeled with an AF488 secondary antibody and excited on the 488 nm laser. (D, H) DAPI counterstain was used to detect nuclei (blue) and was excited on the 405 nm laser.
Fig 5: Co-staining of LCMV NP with markers of early endosomes and TLR-2. RAW 264.7 cells were infected with LCMV (ARM or WE) at an MOI of 5 and incubated for 30 or 60 min as indicated before being fixed, permeabilized, and stained with antibody against a conserved LCMV NP epitope (monoclonal M104, green), markers of early endosomes, (A) EEA1 (monoclonal F.43.1, red) or (B) Rab5 (polyclonal ab13253, red), and TLR2 (monoclonal CD282, grey). Nuclei of cells were stained with DAPI (blue).
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