(A) Primary structure of full-length human Htt (3144 amino acids), including HEAT repeats (HR, gray regions) and the N-terminal variable-length polyQ region (green/red box), with the pathogenic threshold (~Q37) indicated by a white dotted line. C-termini of two N-terminal mHtt fragments used in this study (Httex1 and Httex1-12) are indicated. (B) Overall experimental approach. In the fly olfactory system, ORNs synapse with PNs in discrete regions of the antennal lobe known as glomeruli (gray circles). PNs send axons into higher brain centers (i.e., mushroom body and/or lateral horn). Draper-expressing glial cells project processes in the antennal lobe, where they ensheath individual glomeruli. To monitor spreading of mHtt aggregates between synaptically-connected ORNs and PNs, we generated transgenic flies that express mHttex1 or mHttex1-12 fragments in DA1 ORNs and wtHttex1 in DA1 PNs. Inset: Transfer of mHttex1 or mHttex1-12 aggregates between ORNs and PNs was assessed by monitoring the solubility and colocalization of mHtt and wtHtt fluorescent signals. (C and D) Maximum intensity z-projections of antennal lobes from 7 day-old adult males expressing either Httex1Q25-mCherry (C) or Httex1Q91-mCherry (D) in DA1 ORNs using Or67d-QF and Httex1Q25-GFP in GH146+ PNs using GH146-Gal4. Raw data are shown in grayscale for individual channels and pseudocolored in merged images. Merged images include Bruchpilot immunofluorescence in blue to mark neuropil, which was used to approximate the boundaries of the DA1 glomerulus (white dotted lines). Scale bars = 20 μm. (E–G) High-magnification confocal z-stacks of DA1 glomeruli from 1 day-old (E), 14 day-old (F), and 21 day-old (G) adult males expressing Httex1Q91-mCherry in DA1 ORNs and Httex1Q25-GFP in GH146+ PNs. Boxed regions in (F and G) are shown at higher magnification in insets. Raw data are shown in grayscale in individual channels (Httex1Q91: E1, F1, G1; Httex1Q25: E2, F2, G2) and pseudocolored in merged images (E3, F3, G3). mCherry+ ‘Httex1Q91 surfaces’ (E1’, F1’, G1’) and ‘Httex1Q91+Httex1Q25 surfaces’ (E2’, F2’, G2’) identified by semi-automated image segmentation are shown adjacent to raw data and pseudocolored red and yellow, respectively, in the ‘merged surfaces’ images (E3’, F3’, G3’). Arrows (yellow on grayscale images, white on merged images) indicate Httex1Q91+Httex1Q25 surfaces. Scale bars = 10 μm. (H and I) Confocal z-stacks from 1 day-old (H) and 21 day-old (I) adult females expressing RFP-Httex1-12Q138 in DA1 ORNs and Httex1Q25-GFP in GH146+ PNs. Boxed region in (I) is shown at higher magnification in insets. RFP+ surfaces identified by semi-automated image segmentation are shown in the last column, with Httex1-12Q138-only surfaces in red and Httex1-12Q138+Httex1Q25 surfaces in yellow. Scale bars = 10 μm. (J and K) Numbers of Httex1Q91 or Httex1-12Q138 (‘mHtt’) surfaces (J) and Httex1Q91+Httex1Q25 or Httex1-12Q138+Httex1Q25 (‘mHtt+wtHtt’) surfaces (K) identified in adult males (open bars) or females (solid bars) expressing Httex1Q91-mCherry in DA1 ORNs or adult females expressing RFP-Httex1-12Q138 in DA1 ORNs (striped bars) at the indicated ages. Data are shown as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, or ****p<0.0001 by two-way ANOVA followed by Tukey’s multiple comparisons tests. ‘*’s indicate statistical significance comparing flies of the same genotype and sex at different ages (black ‘*’s compare males or females expressing Httex1Q91, and gray ‘*’s compare females expressing Httex1-12Q138 over time). ‘#’s indicate statistical significance comparing different genotypes at the same age (black ‘#’s compare males vs females expressing Httex1Q91, and gray ‘#’s compare females expressing Httex1Q91 vs females expressing Httex1-12Q138).