Fig. 6

Expression of Rab4-mRFP rescues synaptic deformation and larval crawling defects caused by pathogenic HTT. a Representative images from larvae expressing HTT16Q or HTT128Q immunostained for the synaptic marker CSP and the neuronal membrane marker HRP-TxRED. Smooth staining is seen in HTT16Q larval nerves with the synaptic vesicle marker, CSP. Larval nerves expressing HTT128Q show axonal accumulations of CSP. Quantification of avg. CSP blocks per larvae (#) show a significant number of axonal blocks in larvae expressing HTT128Q compared to larvae expressing HTT16Q (p < 0.0001). n = 8. b Representative images of NMJs from muscle 6/7 segment A4–5 of WT larvae, larvae expressing HTT16Q or HTT128Q, or larvae co-expressing either HTT16Q or HTT128Q with Rab4-mRFP that have been stained with HRP-FITC. Note, NMJs of HTT16Q expressing larvae look similar to NMJs of WT larvae. c Quantification of the avg. number of boutons (#) per larvae revealed a significant decrease in the number of boutons at neuromuscular junctions of HTT128Q expressing larvae compared to WT (p < 0.001). Co-expression of Rab4-mRFP with HTT128Q rescued the number of boutons (ns) compared to WT, with a significant increase (p < 0.01) compared to HTT128Q larvae alone. n = 8. d Quantification of the avg. synapse length (μm) per larvae revealed a significant decrease in synapse length at neuromuscular junctions of HTT128Q expressing larvae compared to WT (p < 0.001). Co-expression of Rab4-mRFP with HTT128Q rescued the number of boutons (ns) compared to WT. n = 8. e Quantification of the avg. bouton area (μm²) per larvae revealed a significant increase in bouton area at neuromuscular junctions of HTT128Q expressing larvae compared to WT (p < 0.0001). Co-expression of Rab4-mRFP with HTT128Q rescued the number of boutons (ns) compared to WT, which were significant decreased in area compared to HTT128Q expressing larvae (p < 0.001). n = 8. f WT larvae, larvae expressing Rab4-mRFP or HTT128Q, and larvae co-expressing Rab4-mRFP with HTT128Q were subjected to larval crawling assays for 1.5 mins at 25C, 60% humidity. g Quantification of larval crawling behaviors (larval velocity = cm/min & larval contractions = contractions/minute) show significant decreases in both larval velocity (p < 0.0001) and contractions (p < 0.001) in HTT128Q larvae. Larvae co-expressing Rab4-mRFP and HTT128Q show no changes in crawling velocity (ns) or contractions (ns) compared to WT. n = 20. h Correlation scatterplots for ratios of avg. larval velocity normalized to WT (from panel g) compared to ratios of either bouton number (from panel c), synapse length (from panel d), or bouton area (from panel e) normalized to WT. Corresponding R-values from linear regressions indicate a positive correlation between crawling velocities and synaptic dysfunction. i Survival of HTT128Q and WT adult flies were measured for 70 days at 25 °C with 60% humidity. Quantification of survival percentage (%) reveals that HTT128Q flies do not survive beyond 40 days, with 50% not surviving beyond 20 days, while WT flies survived until day 63, with 50% of the WT flies surviving beyond 40 days. Adult flies co-expressing HTT128Q and Rab4-mRFP show a partial rescue of survival until day 53, with 50% flies surviving to day 40. n = 3 independent experiments, total of 60 adult flies per genotype. ns = p > 0.01, *p < 0.01, **p < 0.001, ***p < 0.0001. Statistical significance was determined using the two-sample two-tailed Student’s t-test