Figure 6. PINK1 activates downstream cytosolic-localized PKA to modulate BDNF signaling.

A. Representative Western blotting for the indicated proteins in lysates extracted at differential time-points from DMSO, BDNF, and forskolin treated WT PCNs. B. Densitometric analysis of the endogenous expression level of MAP2B, TRKβ, phospho-TRKβ, PSD95, Synaptophysin, and BDNF expression levels. Values were normalized to β-Tubulin. Ordinary one-way ANOVA and post-hoc analysis using Dunnett’s multiple comparison test were used for statistical analysis. Bars denote the average ± SEM and are representative of three independent experiments. C. Representative Western blotting of Phospho-PKA (cat ^Thr197 ) and PKA-C expression in cell lysates extracted from DMSO, 6h kinetin, 12h kinetin, and 24h kinetin treated WT PCNs. D. Densitometric analysis on the endogenous expression level of Phospho-PKA-C (Thr¹⁹⁷) levels in DMSO, 6h kinetin, 12h kinetin, and 24h kinetin treated WT PCNs. Values were normalized to β-Tubulin. Ordinary one-way ANOVA and post-hoc analysis using Dunnett’s multiple comparison test were used for statistical analysis. Bars denote the average ± SEM and are representative of three independent experiments. E. Representative Western blotting for LC3 expression in lysates extracted from Control, FCCP treated, kinetin treated, and FCCP+kinetin treated WT PCNs. F. Densitometric quantification of the ratio of LCII to LCI ratio in Control, FCCP treated, kinetin treated, and FCCP+kinetin treated WT PCNs. Values were normalized to β-Tubulin. Ordinary one-way ANOVA and post-hoc analysis using Dunnett’s multiple comparison test were used for statistical analysis. Bars denote the average ± SEM and are representative of three independent experiments. G. Representative Western blotting of BDNF expression in cell lysates extracted from SH-SY5Y cells transfected with plasmids encoding for control (empty vector), PINK1–3XFlag, GFP alone, an inhibitor of Protein Kinase A (PKI) or co-transfected with PINK1–3XFlag and PKI. H. Representative bright-field images of SH-SY5Y cells transfected with plasmids coding for control (empty vector) and PINK1–3XFlag and treated with DMSO (control) and ANA12 (TRKβ antagonist). I. Quantification of mean neurite length in SH-SY5Y cells transfected with plasmids coding for control (empty vector) and PINK1–4XFlag and treated with DMSO (control) and ANA12. Ordinary one-way ANOVA and post-hoc analysis using Dunnett’s multiple comparison test were used for statistical analysis. Bars denote the average ± SEM and are representative of three independent experiments (N=30; 10 neurons per genotype per experiment). *P < 0.05, **P < 0.01, ***P < 0.001.