Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1 (original) (raw)
Figure 7
AKAP1 promotes mitochondrial elongation and neuronal survival by enhancing phosphorylation of Drp1 at SerPKA.
(A, B) COS cells co-expressing GFP-Drp1 and either empty vector, wild-type, or ΔPKA-mutant GFP-AKAP11–524 were stimulated for 45 min with increasing concentrations of forskolin/rolipram (rolipram at 1/30th of the indicated forskolin concentrations), and total cell lysates were probed for phospho-SerPKA Drp1 and GFP (detecting Drp1 and AKAP1) on the same blot using a dual-channel infrared imager. Drp1 phosphorylation was quantified as the ratio of phospho- to total Drp1 signals normalized to the highest value and is shown as part of the representative blots (A) and the summary graph (B, means ± s.e.m. of 6 independent experiments). (C, D) HeLa cells co-expressing the indicated constructs (ΔPKA = I310P, L316P-mutant) were treated with forskolin/rolipram (25/2 µM, 3 h) and processed for immunofluorescence for mitochondrial cytochrome oxidase II (mito, red) and V5-tagged AKAP1 (green). Shown are representative images (C) and mitochondrial morphology analysis (D, mean ± s.e.m. of ∼200–300 cells per condition from a representative experiment). (E, F) PC12 cells were cotransfected with GFP-Drp1/shRNA expression plasmids and either AKAP1 cDNAs or shRNAs. Cultures were treated 48 h posttransfection with 0.5 µM staurosporine for 24 h, fixed, and stained with Hoechst 33342. Representative images in (E) show overlays of cell contours (bright field), GFP-Drp1 (green), and Hoechst-stained nuclei (blue), with transfected cells displaying normal nuclear morphology and many untransfected cells having condensed, apoptotic nuclei (arrows). Apoptosis was quantified as the percentage of GFP-positive cells with condensed or fragmented nuclei (E, means ± s.e.m. of 6–13 image fields with ∼300 transfected cells per condition from one experiment representative of three).