In vitro prion-like behaviour of TDP-43 in ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and >95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded 'prion-like' protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.

Keywords: ALS; Prion-like disease; Propagation; Protein misfolding; Seeding; TDP-43.

Graphical abstract

Fig. S1

Morphological diversity of pTDP-43 inclusions seeded into HEK cells. A) and B) are skein-like pTDP-43 and FLAG positive inclusions. C) Mixtures of cytoplasmic pTDP-43 and FLAG positive dot and skein-like dash inclusions. D) Mixtures of dash, dense compact and ring inclusions positive for pTDP-43 and FLAG. E) Round pTDP-43 and FLAG positive inclusions. F) Round inclusion with radiating spiculae (racket shaped) positive for pTDP-43 and FLAG. G) Round pTDP-43 and FLAG positive inclusions with radiating spiculae. H) Circular pTDP-43 and FLAG positive inclusion. I) Dot like inclusions positive for FLAG and pTDP-43. J) Numerous dot inclusions positive for pTDP-43 and FLAG. K) Widespread dot inclusions throughout the cell positive for pTDP-43 and FLAG. L) Widespread dot inclusions positive for pTDP-43 and FLAG. M) Large dot inclusions positive for pTDP-43 and FLAG. N) Dot inclusions positive for TDP-43 and FLAG. O) Pre-inclusion with a mix of wispy straight and wavy filaments positive for pTDP-43 and FLAG. P) Diffuse granular pre-inclusion positive for pTDP-43 and FLAG with early coalescence into round inclusions. All images are representative images for the types of inclusions observed in each ALS sample examined. Cells were stained for DAPI (DNA/nuclear marker) in blue, pS409/410 (pTDP-43) in green, FLAG in red and all are images merged.

Table S1

Control and ALS CNS tissue samples used in this study.

Fig. 1

Seeded aggregation of pTDP-43 from ALS brain and spinal cord.A) Western blotting of Sarkosyl insoluble, urea soluble control and ALS patient samples which were used as pTDP-43 enriched inocula, labelled using polyclonal anti-pTDP-43 (pS409/410) antibody. B) Western blotting of Sarkosyl insoluble (SI) fractions of HEK cells either without plasmid or expressing the FL WT TDP-43 plasmid. The cells received either no inocula or 5 μg of normal control (NC), parkinson's disease control (PDC) or ALS inocula from motor cortex (MC), temporal cortex (TCX), or spinal cord (SC). Western blots labelled using anti-FLAG and anti-pTDP-43 antibodies. The graph shows the densitometry of pTDP-43 46 kDa band from SI fractions of cells expressing WT TDP-43 treated with control or ALS inocula (n = 3 and **p < 0.01) C) Sarkosyl insoluble (SI) fractions of HEK cells transfected with the FL WT TDP-43 plus 5 μg of ALS inocula from ALS MC at 1, 2 and 3 days post inoculation and western blotted using monoclonal anti-FLAG and polyclonal anti-pTDP-43 antibodies. The pTDP-43 bands are indicated with arrows, and quantification was performed on the 46 kDa band using densitometry from n = 5. D) Quantification of endogenous TDP-43 levels from SS and SI fractions of HEK cells expressing no construct (blue) and expressing the FL WT construct (red). Blots are labelled with polyclonal TDP-43 and β-actin of cells either without TDP-43 plasmid (−) or expressing the TDP-43 plasmid (+), and are representative of n = 3. E) Immunofluoresence (IF) staining of HEK cells transfected with 5 μg control or ALS pTDP-43 MC and SC enriched inocula with the FL WT plasmid. Scale bars = 44 μm, NC = normal control, MC = motor cortex, TCX = temporal cortex, PDC = Parkinson's disease control, SS = sarkosyl soluble, SI = sarkosyl insoluble, FL WT = full length wild type TDP-43 plasmid. All blots and IF images are representative images of n = 3. All western blots were loaded with equal amounts of protein in each well per gel. Error bars represent SEM. Statistical significance was calculated using an unpaired two tailed students t-test where **p < 0.01 and ***p < 0.001.

Fig. 2

Morphological diversity of pTDP-43 inclusions seeded into HEK cells.A) Diagrammatic illustration of different morphological types of neuronal cytoplasmic inclusions seen in motor neuron cell bodies from patients with ALS. B) Different morphological types of inclusions observed upon seeding of pathological pTDP-43 (green) from ALS patient CNS tissue onto cells expressing FLAG tagged FL WT TDP-43 (red).

Fig. 3

Serial passage of pTDP-43 aggregates to naïve cells expressing TDP-43. HEK cells containing aggregates that were co-transfected with full length wild type TDP-43 plasmid (FL WT) and ALS TCX were extracted in sarkosyl and the insoluble fraction was inoculated into naïve cells expressing FL WT TDP-43 in order to passage the seeding reaction. Western blot (A) and immunofluorescence (B) demonstrates the formation of pTDP-43 bands and aggregates with the FL WT + ALS TCX, and then stronger bands and aggregates in the FL WT + SK (insoluble fraction of HEK cells containing aggregates) after 3 days. Diagram (C) shows the increased amount of pTDP-43 46 kDa bands quantified by densitometry in passaged cells compared to cells exposed to initial ALS TCX extract, and corrected for FLAG expression. D) Densitometry of pTDP-43 46 and 25 kDa bands corrected for using β-actin in the initial inocula and the first and second passage of the cells. Error bars represent mean ± SEM where n = 3 and significance was determined using an unpaired two tailed students t-test (*p < 0.05, **p < 0.01,***p < 0.001).

Fig. 4

pTDP-43 aggregate spread to naïve cells expressing GFP. Co-culture of cells containing pTDP-43 aggregates and cells expressing GFP in a 1:1 ratio. After 3 days of incubation the cells were stained with pS409/410 (pTDP-43) (red) and DAPI (blue). Images (A) and (B) are 3D reconstructions of cells with pTDP-43 aggregates (Red). The red lines represent the X-Y axis, the green line represents the X-Z axes and blue line represents the Y-Z axes. Images (C) and (D) are intensity distribution profiles of images (A) and (B) respectively, with blue lines representing DAPI, green lines representing GFP and red line representing pTDP-43 in a merged image. All images are representative of 3 independent experiments.

Fig. 5

TDP-43 seeding, toxicity and oligomer formation in a motor neuron like (NSC-34) cell line. A) Western blot showing the formation of pTDP-43 46 and 25 kDa bands only when both the FL WT and ALS TCX and SC samples are transfected and not present in cells transfected with the ALS TCX or SC samples alone. B) MTT assay measuring NSC-34 cell viability after inoculation with control and ALS samples ± the FL WT TDP-43 plasmid showing significantly reduced cell viability only in cells inoculated with the plasmid and ALS samples compared to all other treatments. Error bars represent mean ± SEM with three repeats, and analysis was conducted by a one way ANOVA with a post hoc Tukey test to compare all groups. **p < 0.01 and ***p < 0.001. C) Representative images of the formation of pTDP-43 aggregates (green) in the NSC-34 cell line treated with ALS TCX and SC samples which are not present in control treated cells. All scale bars = 35 μm. D) Representative images of TDP-43 oligomers (TDP-O) (green) present in cells seeded with ALS samples and not controls.