Figure 3.

PA drives FOXO3a into the neuronal nucleus, promotes FOXO-dependent transcription, and protects against the proteotoxicity of mutant SOD and mutant p150^glued. A, The chemical structure of PA. B, Subcellular fractionation of 14 DIV spinal cord culture lysates treated with PA or vehicle. Nuclear fraction (enriched for the nuclear marker lamin) contains more FOXO3a in PA- versus vehicle-treated cultures. The abundance of total FOXO3a, lamin, or actin is the same under both conditions. Quantification of band intensities show that PA treatment leads to a statistically significant increase in nuclear FOXO3a/nuclear lamin compared with vehicle-treated cultures. C, PA promotes transcription from a 3xFHRE-luciferase reporter in a dose-dependent manner. PA affected neither transcription from the parent vector (pGL3 that lacks the FHREs) nor a 3xRARE-luciferase reporter. There was a statistically significant difference between vehicle treatment of 3xFHRE versus PA concentrations 10 nm (*p < 0.05), 50 nm (**p < 0.01), and 100 nm (***p < 0.001). D, Pretreatment of mixed spinal cord cultures with PA (+PA) protected motor neurons from excitotoxic insult (left bar graph) and DHT-dependent death of polyQ expanded AR toxicity (right bar graph). E, One set of 14 DIV mixed spinal cord cultures was infected with HSV–LacZ, HSV–WT-SOD, or HSV-mutant SOD, and a second set of cultures was infected with HSV–LacZ, HSV–WT-p150^glued, or HSV-mutant p150^glued. Survival of SMI32-stained motor neurons was determined 4 d later. Infected cultures were either treated with vehicle (−PA) or PA (+PA). Mutant SOD led to a statistically significant reduction in motor neuron number (by ANOVA; see Results for details), and this was prevented in PA-treated cultures. Similarly, mutant p150^glued led to a statistically significant reduction in motor neuron number, and this was prevented in PA-treated cultures. *p < 0.05.