Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses
Figure Lengend Snippet: mHtt ex1 or mHtt ex1-12 aggregates formed in presynaptic ORNs induce the aggregation of wtHtt ex1 expressed in postsynaptic PNs. (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 (Htt ex1 and Htt ex1-12 ) 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 mHtt ex1 or mHtt ex1-12 fragments in DA1 ORNs and wtHtt ex1 in DA1 PNs. Inset: Transfer of mHtt ex1 or mHtt ex1-12 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 Htt ex1 Q25-mCherry (C) or Htt ex1 Q91-mCherry (D) in DA1 ORNs using Or67d-QF and Htt ex1 Q25-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 Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-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 (Htt ex1 Q91: E1, F1, G1; Htt ex1 Q25: E2, F2, G2) and pseudocolored in merged images (E3, F3, G3). mCherry+ “Htt ex1 Q91 surfaces” (E1’, F1’, G1’) and “Htt ex1 Q91+Htt ex1 Q25 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 Htt ex1 Q91+Htt ex1 Q25 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-Htt ex1-12 Q138 in DA1 ORNs and Htt ex1 Q25-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 Htt ex1-12 Q138-only surfaces in red and Htt ex1-12 Q138+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (J) and (K) Numbers of Htt ex1 Q91 or Htt ex1-12 Q138 (”mHtt”) surfaces (J) and Htt ex1 Q91+Htt ex1 Q25 or Htt ex1-12 Q138+Htt ex1 Q25 (“mHtt+wtHtt”) surfaces (K) identified in adult males ( open bars ) or females ( solid bars ) expressing Htt ex1 Q91-mCherry in DA1 ORNs or adult females expressing RFP-Htt ex1-12 Q138 in DA1 ORNs ( striped bars ) at the indicated ages. Data are shown as mean ± SEM; *p
Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).
Techniques: Expressing, Generated, Transgenic Assay, Solubility, Immunofluorescence