Fig. 3.

D-AKAP1/PKA protects dendrites and mitochondrial structure from Aβ₄₂-mediated toxicity via PKA-mediated phosphorylation of Drp1.

(a) Representative epifluorescence micrographs (60X) of primary cortical neurons expressing the indicated GFP-tagged DRP1-modulating plasmids in the presence of vehicle control (DMSO) or Aβ₄₂. Primary neurons were immunostained for MAP2B (red) to identify dendrites, for GFP (green) to identify transfected neurons, and for TOM20 (blue) to identify mitochondria. Mitochondria inside MAP2-positive dendrites are indicated by blue dashes. Transient expression of PKA phosphomimetic mutant of Drp1 (S656D) protects dendrites and the number of dendritic mitochondria from Aβ₄₂-treatment compared to primary neurons expressing OMM-GFP as a control. (b) Bar graph shows a compiled quantification of the mean dendrite length per neuron, as measured in MAP2B-positive neurites in primary cortical neurons treated with vehicle or with Aβ₄₂ (24 hr., 10μM). Means ± SEM, derived from 250–300 primary cortical neurons from at least 10 epifluorescence microscopic fields per experiment, were compiled from three independent experiments (**/***:p<0.01 vs. OMM-GFP or DRP1-S656D/Aβ42 in each series, *:<0.05 vs. OMM-GFP/Aβ4,, Two-Way ANOVA with Tukey’s post hoc test). (c) Bar graph shows a compiled quantification of the mean mitochondrial length of dendritic mitochondria, as identified by immunostaining with TOM20 within MAP2B-positive neurites, and quantified by Image J in primary cortical neurons treated with vehicle or with Aβ₄₂(24 hr., 10μM). Means ± SEM, derived from 250–300 primary cortical neurons from at least 10 epifluorescence microscopic fields per experiment, were compiled from three independent experiments (**/***:p<0.01 vs. OMM-GFP or for DRP1-S656D/Aβ42for each series, Two-Way ANOVA with Tukey’s post hoc test). (d) Bar graph shows a compiled quantification of the mean mitochondrial content in dendrites (% of dendrite length occupied by mitochondria), as identified by immunostaining with TOM20 within MAP2B-positive neurites, and quantified by Image J in primary cortical neurons treated with vehicle or with Aβ₄₂ (24 hr., 10μM). Means ± SEM, derived from 250–300 primary cortical neurons from at least 10 epifluorescence microscopic fields per experiment, were compiled from three independent experiments (Two-Way ANOVA with Tukey’s post hoc test).