Figure 2. AAV vectors efficiently transduce primary neurons and block eIF2α phosphorylation with minimal toxicity.

(A) A two-vector adeno-associated virus (AAV) system was used to decrease eIF2α phosphorylation specifically in excitatory forebrain neurons. The “activator” vector (CaMKII tTA-AAV) expresses the tetracycline transactivator (tTA) protein from a calmodulin kinase II (CaMKII) promoter, while the “responder” vector (GADD34 CA-AAV or GADD 34 cont–AAV) expresses GFP and either GADD34 constitutive active (CA) or GADD34 control (cont) from a bi-directional tetracycline operator-CMV promoter (tetO-CMV). All expressed transgenes contain a woodchuck post-trascriptional regulatory element (WRE) to enhance expression. ITR  =  inverted terminal repeat sequences. (B) Mixed cortical primary neurons were isolated from e15.5 mouse embryos and infected with 1×10⁷ viral genomes of CaMKII tTA-AAV and 1×10⁷ viral genomes of GADD34 CA-AAV or GADD 34 cont–AAV per well of a 12 well plate on the day of isolation. No virus was added to other wells as a negative control. Media was changed 48 hours later. After 8 days in culture, neurons were lysed for immunoblot analysis for total eIF2α, phosphorylated (p)-eIF2α, GFP, caspase 3 (full length and activated cleaved 17 kDa fragment), and β-actin as a loading control. (+) are HEK293 cells treated 30 minutes with 1 µM thapsigargin as a positive control for eIF2α phosphorylation, while (–) are untreated HEK293 cells as a negative control. Note that AAV transduction does not cause toxicity as indicated by minimal cleaved caspase 3 fragment. (C) Phosphorylated and total eIF2α immunosignal intensities were measured from the blot in (B) and phosphorylated:total eIF2α ratio (phospho/total eIF2α) was calculated and displayed as percentage of no virus control. Primary neurons transduced with GADD34 CA-AAV had dramatically less eIF2α phosphorylation than neurons transduced with GADD34 cont-AAV or no virus, as demonstrated by phospho/total eIF2α ratio. Note that GADD34 cont-AAV transduction caused a significant elevation of phospho/total eIF2α ratio compared to no virus control. Bars represent SEM, n = 3 samples per condition, p<0.01**.