Fig. 3
AMPK hyperactivation promotes dendritic retraction and synapse loss in glaucoma. A-C Western blot and densitometry analysis confirm that intravitreal delivery of siAMPK reduced retinal AMPK protein and its function, visualized with an antibody against pAMPKThr172 (active form) (Student’s t-test, * = p < 0.05, N = 4 mice/group). The lower panel is the same blot as in the upper panels but probed with an antibody that recognizes β-actin to ensure equal protein loading. D siAMPK or its control siRNA (siCTL) are administered by intravitreal injection once a week, starting at 3 days after glaucoma induction. RGC dendritic and synaptic analyses are carried out at 2 weeks after injury. E, F siAMPK administration results in marked reduction of AMPK activity in RGC, visualized by co-labeling with pAMPKThr172 and Brn3a on retinal cross sections. G Quantification of pAMPKThr172- and Brn3a-positive cells confirms a significant decrease of AMPK function in glaucomatous eyes treated with siAMPK (Student’s t-test, **p < 0.01, N = 5 mice/group, n = 50 RGC/group). H, I siAMPK-treated RGC have longer dendrites and more elaborate arbors than control neurons treated with siCTL. J-M Quantitative analysis of dendritic parameters and Sholl analysis confirm that siAMPK-mediated AMPK knockdown significantly increased the area, length, number of branches and complexity of RGC dendrites (ANOVA with Tukey’s multiple comparison post-hoc test, *** = p<0.001, ** = p < 0.01, * = p<0.05, N = 5 mice/group, n = 30–50 RGC/group, Table 1). (N-Q) siAMPK also rescues VGLUT1 and PSD95 expression in RGC dendrites, and quantitative analysis of pre- and postsynaptic voxels confirms robust synaptic protection compared to controls (ANOVA with Tukey’s multiple comparison post-hoc test, *** = p<0.001, N = 5 mice/group). Values are expressed as the mean ± S.E.M. INL: Inner Nuclear Layer, IPL: Inner Plexiform Layer, GCL: Ganglion Cell Layer