Article Figures & Data
Figures
- Figure 1.
Selectively overexpressing hAPP in either mature or immature OSNs. a, Schematic for generating transgenic lines that selectively overexpress hAPP in either mature (OMP promoter, OMP-hAPP line) or immature (Gγ8 promoter, Gγ8-hAPP line) OSNs (see Materials and Methods). b, Colocalization of hAPP protein (red) with OMP protein (green) in OMP-hAPP line (left), and with GAP43 protein (green) in Gγ8-hAPP line (right). Scale bar, 40 μm. c, Overexpression of human Aβ42, Aβ40 in OMP-hAPP line (Aβ42, 0.016 ± 0.002; Aβ40, 0.037 ± 0.005 μg/g; ratio, 0.44 ± 0.05; n = 5), and Gγ8-hAPP line (Aβ42, 0.015 ± 0.006; Aβ40, 0.033 ± 0.010 μg/g; ratio, 0.46 ± 0.07; n = 5), compared with control, tetO-hAPP line (Aβ42, 0; Aβ40, 0 μg/g; n = 3). Values are mean ± SD. Animals: 3–4 weeks old.
- Figure 2.
Rapid and widespread apoptosis in OSNs expressing hAPP. a, Mature OSN numbers (OMP-positive; green) were significantly smaller in both OMP-hAPP (230 ± 26, n = 5) and Gγ8-hAPP (177 ± 41, n = 4) than tetO-hAPP (505 ± 77, n = 6), while immature OSNs (GAP43-positive; red) remained similar: OMP-hAPP (209 ± 42, n = 4), Gγ8-hAPP (219 ± 31, n = 4), tetO-hAPP (212 ± 31, n = 4). b, Increased cleaved-caspase3 expression in mutant OE (white dots, collapsed confocal stack). c, Colocalization of cleaved-caspase3 (red) and hAPP (green) in both mature (OMP-hAPP line, top) and immature (Gγ8-hAPP line, bottom) OSNs (arrowheads). d, Significant increase in caspase3-positive OSNs in both OMP-hAPP (8 ± 2, n = 8) and Gγ8-hAPP (9 ± 2, n = 4), compared with tetO-hAPP (2 ± 1, n = 7). However, there were fewer hAPP-expressing cells and a greater ratio of caspase3:hAPP-positive cells in OMP-hAPP (hAPP, 184 ± 58; ratio, 4.9 ± 1.4%) than in Gγ8-hAPP (hAPP, 294 ± 62; ratio, 3.0 ± 0.3%). Scale bars, 40 μm. Values are mean ± SD per mm OE, single optical slice. Significance: *p < 0.05; **p < 0.001. Animals: 3–4 weeks old.
- Figure 3.
Cell-autonomous hAPP-induced ultrastructural disruption of the OE. a, hAPP immunostaining showed intracellular cytoplasmic signal (arrowheads). b, Immunoelectron microscopic images of an OSN in OMP-hAPP OE. Black dots indicate hAPP signal (see Materials and Methods). Right panels correspond to boxed regions indicating hAPP protein in the nuclear (pink) envelope and ER (blue) lumen. c, d, EM images of control tetO-hAPP OE. c, Well-formed OSN dendritic knobs (left, arrowheads), each containing many cut cilia (right, arrows) and some intact (arrowheads). d, Spatially organized OSN cell bodies. e, f, EM images of OMP-hAPP OE. e, Very few smaller dendritic knobs (left, arrowhead) with less cilia (right, arrowhead). f, Disorganized OSN cell bodies around an apoptotic nucleus (red arrowhead). Scale bars: a, 10 μm; b, 200 nm; c–f, 1 μm. Animals: 3–4 weeks old.
- Figure 4.
Both mature and immature hAPP-expressing OSNs showed reduced survival but were differentially rescued by turning off hAPP transgene. a, Experimental timeline shows all BrdU injections in 2-week-old animals and olfactory tissue harvested 2 h, 7 d, or 14 d later. Images show OE immunostained for BrdU (white dots) corresponding to time points above. b, Quantification of BrdU-positive cells at 2 week (+2 h) point showed a significantly greater number in Gγ8-hAPP line (107 ± 18, n = 5) than either OMP-hAPP (59 ± 6, n = 3) or tetO-hAPP controls (52 ± 3, n = 4) (black asterisk), revealing increased proliferation in Gγ8-hAPP line. A significant drop in BrdU-positive cells in 3-week-old Gγ8-hAPP mice (22 ± 7, n = 4) compared with that in 2-week-old Gγ8-hAPP mice (red asterisks), as well as 3-week-old OMP-hAPP (69 ± 15, n = 4) and tetO-hAPP (65 ± 21, n = 5) (black asterisk) identified a susceptibility period of apoptosis in immature OSNs. In OMP-hAPP mice, a similar significant drop occurred between 3 and 4 weeks of age (4w: 9 ± 4, n = 4; green asterisk), revealing a later susceptibility period in mature neurons. Both mutant lines had significantly fewer BrdU-positive neurons at 4 weeks of age (Gγ8-hAPP: 8 ± 1, n = 3) than tetO-hAPP (43 ± 16, n = 4) (black asterisk). c, Timeline of recovery experiments shows BrdU injections in 2-week-old mice, followed by Dox-chow (which turns off hAPP expression) given to Gγ8-hAPP mice from 2 to 3 weeks of age, and OMP-hAPP mice from 3 to 4 weeks of age (see Materials and Methods), based upon the susceptibility periods for each line determined above. d, OE from Dox-treated mice described in c, showing rapid recovery in Gγ8-hAPP line and delayed recovery in OMP-hAPP line. Graphs show significant changes in Dox-treated compared with nontreated Gγ8-hAPP OE, including increased BrdU-positive cells (Dox, 67 ± 14, n = 3; no-Dox, 22 ± 7, n = 4); decreased caspase3-positive cells (Dox, 4 ± 1, n = 3; no-Dox, 9 ± 2, n = 4) and increased OMP-positive cells (Dox, 383 ± 62, n = 3; no-Dox, 177 ± 41, n = 4). Dox-treated OMP-hAPP mice showed smaller changes compared with nontreated in BrdU-positive cells (Dox, 13 ± 5, n = 3; no-Dox, 9 ± 4, n = 4), caspase3-positive cells (Dox, 7 ± 3, n = 3; no-Dox, 8 ± 2, n = 8) and OMP-positive cells (Dox, 296 ± 22, n = 3; no-Dox, 230 ± 26, n = 5). Significance: *p < 0.05; **p < 0.001. Scale bar, 50 μm.
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