Fig. 5

Figure 5. Dendra-α-SYN clearance kinetics in vivo. (A) Confocal images of the green and red fluorescent signal of Dendra-α-SYN in isolated neurons in the spinal cord of fish with mosaic expression of Dendra-α-SYN-A53T. Neurons with transgene-expression can be identified by its dendra-derived green signal. After UV (405 nm) exposure targeting the soma of selected neurons (white arrowheads), part of Dendra protein is photoconverted from green to red. As a consequence, the pool of green protein is reduced and red Dendra-α-SYN can be visualised. Scale bar represents 50 μm. (B) Confocal images showing the reduction in the red signal of photoconverted Dendra-α-SYN-A53T over time. Newly synthesised Dendra-α-SYN protein is green, whereas red signal corresponds to the residual red photoconverted- protein after UV exposure. Red signal within neurons (delimited by dashed-lines) reduces over time as a consequence of its degradation and can be used as a readout for clearance kinetics. Scale bar represents 50 μm. (C) Graph representing the decrease in the red-Dendra intensity in single neuronal cells in the spinal cord of Dendra-α-SYN-wt and A53T fish with mosaic expression. Dendra-α-SYN-wt and A53T clear at the same rate (N ≧ 30 neurons per group). (D) Changes in the clearance rate of Dendra- α-SYN-A53T after inhibition of autophagic flux by ammonium chloride (NH₄Cl). NH₄Cl delays the clearance of Dendra-α-SYN-A53T (N = minimum 30 neurons per group; * p < 0.05, **** p < 0.0001 vs. untreated α-SYN-A53T).