Axonal uptake and transmission of TDP-43. (A) Experimental design of the anterograde transmission experiment. (B) Primary cortical mouse neurons in microfluidic devices (5 DIV) were transduced with rAAV6.2 TDP-Luc. After 24 h, H4 cells were coplated in the axonal terminal compartment. Excess culture medium was added to the axonal compartment during the entire experiment to maintain a positive hydraulic pressure and prevent diffusion of TDP-43 or virus particles. After 5 d of co-culture, H4 cells were harvested, and luciferase activity was detected. (C) The same setup without primary neurons was used as a diffusion control. (D) Experimental design of the axon terminal uptake experiment. (E) CM from HEK-293 cells transfected with mock, Luc, or TDP-Luc were added to the axonal compartment of microfluidic devices containing primary cortical neurons (5 DIV). Excess culture medium was added to the cell body compartment to maintain a positive hydraulic pressure against protein diffusion during the entire experiment. After 5 d, neuron bodies were harvested, and luciferase activity was measured. (F) The same setup without primary neurons was used as a diffusion control. For all experiments, n = 9. Mean ± SEM. *, P < 0.05; **, P < 0.01. Yellow rectangles in A and D indicate compartments from which cell bodies were harvested for luciferase activity measurement in the respective experiment. n.s., not significant.