Figure 4.

BRAF overexpression protects against DYNC1I1 deficiency induced dendritic atrophy by activating MEK-dependent autophagy. (A–D) BRAF overexpression enhances protective autophagy, which is MEK dependent. Primary hippocampal neurons are co-transfected GFP-LC3 with blank vector, shRNA1-6, shRNA1-6 and BRAF treated without or with 100 nmol/L of MEK inhibitor trametinib at DIV6, cultured additional 5 days and imaged at DIV11. Autophagosomes (green puncta) are labeled with GFP-LC3. All photos of GFP-LC3 are imaged with confocal microscope and further deconvolved for clarity. The scale bars represent 2 μm. (E) Scatterplots with boxplots show the number distribution of GFP-LC3 puncta in the soma of neurons transfected with control vector treated without (n = 41) or with 10 nmol/L of trametinib (n = 22), shRNA1-6 (n = 48), shRNA1-6 co-transfected with BRAF treated without (n = 39) or with 100 nmol/L of trametinib (n = 44). (F–I) Lysosomal protease inhibitors (E64D and pepstatin A) can inhibit the protective role of BRAF in dendritic atrophy caused by DYNC1I1 knockdown with shRNA1-6. Neurons are transfected and treated without or with 1 μmol/L of E64D and pepstatin A at DIV6, cultured additional 5 days and imaged at DIV11.The scale bars represent 20 μm. (J) Scatterplots with boxplots show the total dendritic length distribution of neurons transfected with control vector treated without (n = 29, gray) or with 1 μmol/L of E64D and pepstatin A (n = 30, green), shRNA1-6 (n = 30, red), shRNA1-6 co-transfected with BRAF treated without (n = 30, purple) or with 1 μmol/L of E64D and pepstatin A (n = 30, blue). *, P < 0.001