Figure 5. HTT S421 phosphorylation affects presynaptic APP targeting.

(A) Effect of HTT S421 phosphorylation on exocytosis rate of APP was analyzed in COS cells co-transfected with APP-SEP (Super Ecliptic pHluorin) and with pARIS HTT or pARIS HTT_SA visualized by TIRF microscopy. Magnification represents a time lapse of events showing 2 events of APP vesicle exocytosis (green arrows). Histograms represent means +/- SEM of exocytosis event number per minute in 39 HTT and 40 HTT_SA cells from four independent experiments. Significance was determined using an unpaired t-test; *p<0.05. Scale bar = 20 µm. (see also Video 5). (B) Effect of HTT S421 phosphorylation on APP targeting at the synapse was assessed by anti-APP western blotting (22C11) analysis of extracts from synaptic chambers of a WT or HTT_SA corticocortical network. SNAP25 was used as a control for protein content in the synaptic compartment and nuclear marker Lamin B1 for the somatic compartment. Histograms represent means +/- SEM of APP signal per synaptophysin signal on five independent experiments. Significance was determined using a Mann-Whitney test; *p<0.05, ns = not significant. (C) Western blotting analysis of pre- and postsynaptic fractions obtained from synaptosome preparations. Fractionation gives the first pellet, P1, the first supernatant, S1, and the second supernatant, S2. Lamin B1, a nuclear marker is enriched in P1 fraction. The pre- (non-PSD) and the post-synaptic (PSD) fractions are respectively enriched in synaptophysin and PSD95. (D) APP from WT or HTT_SA cortices fractions was quantified by western blotting analyses. APP signal was quantified as the ratio of synaptophysin signal for non-PSD fraction and as the ratio of PSD95 signal for PSD fraction. One line represents one experiment. Significance was determined using Mann-Whitney test; *p<0.05, ns = not significant.