Mitochondrial PKA and AKAP1 promote neuronal survival and oppose mitochondrial fragmentation in vitro and in vivo.

(A) Hippocampal neurons were transfected with the indicated cDNA and shRNA plasmids (AKAP1ΔPKA = I310P,L316P [PKA binding defective]; Figure S2C). After 3 d, cells were treated ± 400 nM rotenone for 2 d, fixed, and analyzed by counting apoptotic nuclei in the transfected neuron population (means ± s.e.m. of n = 3–7 experiments). The inset shows two transfected neurons (green) with apoptotic nuclei and an untransfected neuron with normal nucleus (asterisk). (B, C) Representative confocal sections of TMRM-stained (mito) hippocampal neurons (B) and their mitochondrial length scores (C) 3 d after transfection with the indicated cDNA and shRNA constructs are shown (means ± s.e.m. of n = 3–5 experiments; Student's t test comparisons between GFP fusion proteins and omGFP and between AKAP1 and NS shRNAs). (D–F) Rats injected with lentivirus expressing mitochondrial (m)GFP and AKAP1-GFP into the hippocampus and striatum of left and right hemispheres, respectively, were analyzed 7–14 d later for mitochondrial shape. Perfusion-fixed cryostat sections immunolabeled for GFP (representative confocal image in (D), counterstained for nuclei with TOPRO-3 [blue]) were subjected to ImageJ software-based morphometry. Scatter plots (E, F) correlate form factor (inverse of circularity of individual mitochondria) with cumulative area:perimeter ratio (a measure of network connectivity). Each open symbol represents average shape metrics from 10–22 z-sections of one neuron; filled symbols are population averages (± s.d., 29–42 neurons per condition from 2 (E) and 3 (F) rats).