TDP-43 causes neurotoxicity and cytoskeletal dysfunction in primary cortical neurons

Abstract

TDP-43-mediated proteinopathy is a key factor in the pathology of amyotrophic lateral sclerosis (ALS). A potential underlying mechanism is dysregulation of the cytoskeleton. Here we investigate the effects of expressing TDP-43 wild-type and M337V and Q331K mutant isoforms on cytoskeletal integrity and function, using rat cortical neurons in vitro. We find that TDP-43 protein becomes mislocalised in axons over 24-72 hours in culture, with protein aggregation occurring at later timepoints (144 hours). Quantitation of cell viability showed toxicity of both wild-type and mutant constructs which increased over time, especially of the Q331K mutant isoform. Analysis of the effects of TDP-43 on axonal integrity showed that TDP-43-transfected neurons had shorter axons than control cells, and that growth cone sizes were smaller. Axonal transport dynamics were also impaired by transfection with TDP-43 constructs. Taken together these data show that TDP-43 mislocalisation into axons precedes cell death in cortical neurons, and that cytoskeletal structure and function is impaired by expression of either TDP-43 wild-type or mutant constructs in vitro. These data suggest that dysregulation of cytoskeletal and neuronal integrity is an important mechanism for TDP-43-mediated proteinopathy.

Fig 1. TDP-43 is mislocalised in cortical neurons.

Rat cortical neurons were transfected with GFP control (A—D), and GFP-tagged TDP-43 wild-type and mutant isoforms (E—P) and analysed at 24–144 hours (H). Immunostained with anti-GFP antibodies. Scale bar = 10 µm. Insets (E’–P’)—higher power views of the corresponding panels above, converted to black and white pixels. Scale bar = 10 µm.

Fig 2. Cytoskeletal integrity is compromised over time in cortical neurons.

Rat cortical neurons were transfected with GFP control, or GFP-tagged TDP-43 wild-type and mutant isoforms as labelled and analysed at 24, 72 H and 144 H. Immunostaining with anti-GFP (green) and anti-tyrosinated tubulin (red) antibodies. Cellular fragmentation was observed at 144 H (bottom right panel). Scale bar = 10 µm.

Fig 3. TDP-43 mislocalisation visualised with anti-TDP-43 antibodies.

Rat cortical neurons were transfected with GFP control, and GFP-tagged TDP-43 wild-type and mutant isoforms as labelled and analysed at 72 H and 144 H. Immunostaining with anti-GFP (green) and anti-TDP-43 antibodies (red). Scale bar– 10 µm. Insets—higher power views of corresponding panels to the left, stained with anti-TDP-43 antibodies. Scale bar = 10 µm.

Fig 4. TDP-43 isoforms mediate toxicity in vitro.

Control and TDP-43 transfected neurons (as labelled) were stained using a TUNEL kit and imaged to show TUNEL-positive cells (black dots) and Hoechst staining (white dots) after 72H in culture (A—D). Scale bar = 10 µm. (E) Average number of apoptotic cells quantified from five 0.24mm² fields, at 3 time points as shown (3 experiments). Data is presented as means and standard errors. Significance was compared by ANOVA with GFP control at the respective timepoint and between constructs at various timepoints (no significance: p > 0.05; *** P < 0.0001, ** P < 0.001, * P < 0.01—compared with GFP control). (F) Semi-quantitative analysis of the viability of transfected cells assessed by Promega real-time glo viability assay at different time points from 3 experiments.

Fig 5. TDP-43 affects axonal growth in vitro.

(A) Quantitation of axon lengths of transfected neurons at time-points as labelled (n = 3 experiments; 100 neurons per experiment). Data is presented as means and standard errors. Significance were compared by ANOVA between constructs at same time points (no significance—p > 0.05; * P < 0.01; ** P < 0.001; *** P < 0.0001). (B)–(M) typical cortical neuron growth cones stained with anti-tyrosinated tubulin antibodies at timepoints and with constructs as labelled. Scale bar = 10 µm. (N) Quantitation of mean growth cone area at 3 different time points as labelled (70 neurons per construct per experiment, 3 experiments). Data is presented as mean and standard error. Significance denoted as for (A).

Fig 6. TDP-43 affects anterograde transport in cortical neurons.

(A) Example of a cortical neuron co-transfected with EB3-RFP, live imaged to show EB3 ‘comets’ (arrows) (A’) Higher power view. EB3 moves in an anterograde manner towards the growing ends of axon. (B)–(I) Example kymographs of neurons expressing GFP, or co-transfected with GFP and TDP isoforms, at 48 and 72H, as labelled. Distance along the X-axis and time along the Y-axis. Scale bar = 10 µm, except in A’ (J) Quantification of the velocity of EB3-RFP comets in the axons of transfected neurons. Graphs represent means and standard error of (n = 3). No significant differences were observed.