Exposure to ALS-FTD-CSF generates TDP-43 aggregates in glioblastoma cells through exosomes and TNTs-like structure

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent a continuum of devastating neurodegenerative diseases, characterized by transactive response DNA-binding protein of 43 kDa (TDP-43) aggregates accumulation throughout the nervous system. Despite rapidly emerging evidence suggesting the hypothesis of 'prion-like propagation' of TDP-43 positive inclusion in the regional spread of ALS symptoms, whether and how TDP-43 aggregates spread between cells is not clear. Herein, we established a cerebrospinal fluid (CSF)-cultured cell model to dissect mechanisms governing TDP-43 aggregates formation and propagation. Remarkably, intracellular TDP-43 mislocalization and aggregates were induced in the human glioma U251 cells following exposure to ALS-FTD-CSF but not ALS-CSF and normal control (NC) -CSF for 21 days. The exosomes derived from ALS-FTD-CSF were enriched in TDP-43 C-terminal fragments (CTFs). Incubation of ALS-FTD-CSF induced the increase of mislocated TDP-43 positive exosomes in U251 cells. We further demonstrated that exposure to ALS-FTD-CSF induced the generations of tunneling nanotubes (TNTs)-like structure and exosomes at different stages, which mediated the propagation of TDP-43 aggregates in the cultured U251 cells. Moreover, immunoblotting analyses revealed that abnormal activations of apoptosis and autophagy were induced in U251 cells, following incubation of ALS-CSF and ALS-FTD-CSF. Taken together, our data provide direct evidence that ALS-FTD-CSF has prion-like transmissible properties. TNTs-like structure and exosomes supply the routes for the transfer of TDP-43 aggregates, and selective inhibition of their over-generations may interrupt the progression of TDP-43 proteinopathy.

Keywords: ALS; FTD; TDP-43; exosomes; tunneling nanotubes.

Figure 1. ALS-FTD-CSF induces intracellular mislocalization and aggregation of endogenous TDP-43 in U251 cells

(A) Subcellular redistribution of TDP-43 in U251 cells following incubation of CSF. Immunofluorescent staining for endogenous TDP-43 labeled with rabbit anti-TDP-43 antibody (red), and the F-actin cytoskeleton labeled with phalloidin-Alexa Fluor 488 (green) were examined by fluorescent microscopy. The nucleus was stained with Hoechst 33258 (blue). Both mislocalization of TDP-43 (arrow) and TDP-43 aggregates (arrowhead) were assembled in ALS-FTD-CSF-cultured U251 cells, but the changes were not exhibited in ALS-CSF-cultured cells. Representative images of a field (n=8) are shown for one of three independent experiments from each culture. (B) High magnification microphotographs of TDP-43 mislocalization in cells following incubation of ALS-FTD-CSF. The cytoplasmic TDP-43 inclusions formed in cells and TDP-43 was diffusely distributed from the nucleus to the cytoplasm in the ALS-FTD-CSF-cultured cells. An arrowhead indicates the TDP-43 aggregates and an arrow indicates the TDP-43 distributes in the whole cells. (C) The ratio of the intensity for the fluorescence of TDP-43 located in nucleus (black) to TDP-43 in cytoplasm (red) in the CSF-cultured cells. Each bar represents values averaged from 200 cells, using student's t test with Bonferroni correction. (D) The percentage of cells containing TDP-43 aggregates following cultured with CSF. Values shown are the mean ± SD from three experiments. Level of statistical significance: **p < 0.01.

Figure 2. Full-length TDP-43 and TDP-43 CTFs are expressed in the exosomes derived from CSF

(A) Immunoblotting analysis of exosomal fractions isolated from NC-CSF and ALS-FTD-CSF. Flotillin-1 was used as internal standard. (B) Quantification showing the significant increase of full-length TDP-43 and TDP-43 CTFs in exosomes from ALS-FTD-CSF in comparison to NC-CSF. Values shown are the mean ± SD from three experiments. Level of statistical significance: **p < 0.01.

Figure 3. TDP-43 aggregates are propagated through exosomes

(A) Localization of flotillin-1 with mislocated TDP-43 observed by fluorescence microscopy in the U251 cells following ALS-FTD-CSF incubation. Double immunofluorescence staining images shown are one representative experiment of five independent experiments performed. (B) Immunoblotting analysis of flotillin-1 expression in CSF-cultured U251 cells. The upper panel shows the increased expression of flotillin-1 in the cells exposed to ALS-FTD-CSF compared to the control groups. The lower panel shows GAPDH band used as a loading control. (C) Quantification showing significant increase of flotillin-1 in ALS-FTD-CSF group in comparison to NC-CSF. Values shown are the mean ± SD from three experiments. Level of statistical significance: *p < 0.05.

Figure 4. TDP-43 aggregates are transmitted through TNTs-like structure

(A) Alexa Fluor 488 conjugated-WGA (top image) and Alexa Fluor 488 conjugated–phalloidin (bottom image) stained TNTs-like structures were analyzed by fluorescence microscope following ALS-FTD-CSF incubation of U251 cells. Fixed cells are connected to surrounding cells by numerous ultrafine membrane extensions, namely TNTs-like structure (arrows). (B) Percentage of TNT-connected U251 cells at various time points during exposure of NC-CSF or ALS-FTD-CSF. Values shown are the mean ± SD from three experiments. (C) Transfer of TDP-43 aggregates occurs through TNTs-like structure in ALS-FTD-CSF-cultured U251 cells. Cells were stained with Alexa Fluor 488 conjugated–phalloidin (green) to label TNTs-like structure and rabbit polyclonal antibody (red) to label TDP-43 aggregates. TDP-43 aggregates were found inside TNTs-like structure adjacent cells. The amplified-field image is shown in the right panel. The staining merge indicates that TDP-43 aggregates are present within the lumen of the TNTs-like structure (enlarged views of the boxed areas, aggregates are clearly present within the lumen of the TNTs-like structure).

Figure 5. Exposure to ALS-FTD-CSF actives apoptosis and autophagy, and increases the expression of TDP-43 CTFs in U251 cells

(A) Western blotting was performed using lysates of U251 cells following CSF incubation for 21 days. Cell lysates were examined by immunoblotting with the indicated antibodies. (B) Quantification showing significant increase in TDP-43 CTFs, cleaved caspase-3, p53, LC3II/LC3I, Beclin-1 levels in ALS-FTD-CSF-cultured cells compared to NC-CSF, whereas expression level of Bcl-2 was significantly decreased. Compared ALS-FTD-CSF group with ALS-CSF group, expression levels of TDP-43 CTFs, cleaved caspase-3, p53, LC3II/LC3I, Beclin-1 show significant increase, whereas Bcl-2 level decreases. Values shown are the mean ± SD from three experiments. Level of statistical significance, compared NC-CSF with ALS-CSF: ^& p< 0.05, ^&& p< 0.01; compared NC-CSF with ALS-FTD-CSF: *p< 0.05, **p< 0.01; compared ALS-CSF with ALS-FTD-CSF: ^# p< 0.05, ^## p< 0.01.

Figure 6. Overexpression of TDP-43 CTFs induces apoptosis and autophagy

(A) Western blotting was performed using lysates of 293A cells following transfection of pEGFP, pEGFP-TDP-43, pEGFP-TDP-35, pEGFP-TDP-25 for 72 h. Each sample was probed with the indicated antibodies. (B) Quantification showing significant increase in cleaved caspase-3, p53, LC3II/LC3I, Beclin-1 levels in pEGFP-TDP35-transfected cells and pEGFP-TDP-25-transfected cells in comparison to pEGFP-transfected cells, whereas expression level of Bcl-2 was significantly decreased. Compared pEGFP-TDP-35-transfected cells or pEGFP-TDP-25-transfected cells with pEGFP-TDP-43-transfected cells, expression levels of cleaved caspase-3, p53, LC3II/LC3I, Beclin-1 significantly increase, whereas Bcl-2 level decrease. Values shown are the mean ± SD from three experiments. Level of statistical significance, compared pEGFP-transfected cells with pEGFP-TDP-35-transfected cells or pEGFP-TDP-25-transfected cells: *p< 0.05, **p< 0.01; compared pEGFP-transfected cells with pEGFP-TDP-43-transfected: ^& p< 0.05, ^&& p< 0.01; compared pEGFP-TDP-43-transfected cells with pEGFP-TDP-35-transfected cells or pEGFP-TDP-25-transfected cells: ^# p< 0.05, ^## p< 0.01. (C) TDP-43 aggregates spread among glioblastoma cells through exosomes and TNTs-like structure.