Fig. 1

Co-expression of AMPKα subunits and α-syn in mouse cortical neurons. a AMPKα constructs used in the study, overexpressed using AAV2/6 vectors: wild-type human α1 and α2 forms; T172Dα1 mutant mimicking constitutive phosphorylation of threonine 172; K45Rα2 kinase dead mutant and 1-310α2 truncated form lacking auto-inhibitory loop and ability to integrate in the AMPK complex. b Primary cortical neurons transduced with α-syn-encoding vector (or non-coding vector as control) were co-transduced with AMPKα expressing vectors. Protein analysis by western blotting shows total AMPK (tAMPKα), T172-phosphorylated AMPKα (pAMPKα), α-syn, S79-phosphorylated ACC (pACC) and actin. Analysis is performed at day 7 after infection. c Relative quantification of tAMPK levels normalized to actin. Transduction with AAV-AMPKα increases tAMPK levels. Note that α-syn overexpression leads to a significant decrease in tAMPK protein levels. d Relative quantification of pAMPK level normalized to actin. Note that the overexpression of α-syn does not induce any significant change in T172 phosphorylation and that overexpressed α1 is more efficiently phosphorylated than other AMPKα variants. Statistical analysis: repeated measures two-way ANOVA with Fisher‘s LSD post hoc test; n=3 per condition; *P<0.05; **P<0.01; ***P<0.001. In panels c, d: ‘1-310α2 endo’ refers to the level of endogenous tAMPK and pAMPK (upper bands) in neurons overexpressing the 1-310α2 variant