Figure 13.

Secreted ASPD Induced Apoptotic Degeneration of Surrounding Neurons

(A) APP- or GFP-transduced or untransduced control neuronal cultures were treated with 75 nM MG132 for the indicated time. Viability was determined with a Cell Counting Kit-8 (see Transparent Methods; mean ± SD, n = 3; Scheffé post hoc test, ***p < 0.0001, **p = 0.0268). The viability of GFP-transduced cultures was 93.5% ± 7.0% of that of the untransduced control cultures (n = 3, p = 0.1854 by Scheffé posy hoc test), indicating that AAV transduction itself does not affect cell viability.

(B) APP-transduced or untransduced control cultures were treated with 75 nM MG132 for the indicated time and stained with the anti-ASPD rpASD1 antibody, DAPI, and TUNEL (arrowhead) or with the anti-MAP2 antibody (Transparent Methods). Solid and hatched scale bars, 50 and 100 μm, respectively.

(C) APP-transduced or untransduced control cultures were treated with or without 75 nM MG132 for 24 h and quadruple-stained with rpASD1, DAPI, TUNEL, and MAP2/GFAP. The number of MAP2-labeled neurons or GFAP-labeled astrocytes in APPswe-transduced cultures was counted and is shown (n = 7, Scheffé post hoc test, ***p < 0.0001). Representative images are shown in Figure S14.

(D) APPswe-transduced primary rat hippocampal neuronal cultures were treated with 75 nM MG132 for the indicated time; triple-stained with propidium iodide (PI), calcein-AM, and anti-ASPD mASD3 antibody (see Transparent Methods); and photographed (see Figure S15). Representative images of the corresponding PI/mASD3 staining are shown with 50-μm scale bars. PI-detectable non-apoptotic death was detected in ASPD-producing neurons at 48-h MG132 treatment.

See Figures S14 and S15.