Fig. 4

AMPK knockdown rescues RGC dendrites and synapses through mTORC1 activation. A Immunolabeling of non-injured sham retinas with pS6^Ser240/244 reveals two cell populations endowed with robust constitutively active mTORC1: one located in the ganglion cell layer and another in the inner nuclear layer. B Co-labeling with antibodies against pS6^Ser240/244 and RBPMS show robust mTORC1 activity in these neurons. C-E OHT markedly reduces pS6^Ser240/244 labeling in RGC, indicating loss of mTORC1 function. F-H Administration of siAMPK in glaucomatous retinas restores mTORC1 activity in RGC relative to controls (Student’s t-test, ** = p<0.01, N = 5 mice/group). I, J Co-administration of siAMPK and rapamycin (Rap), an inhibitor of mTORC1, blocks the effect of siAMPK on dendritic rescue. K-N Quantification of dendritic parameters confirms a substantial reduction in area, process length, number of branches, and complexity (Sholl analysis) in rapamycin-treated retinas compared to vehicle-treated controls (ANOVA with Tukey’s multiple comparison post-hoc test, *** = p<0.001, ** = p < 0.01, * = p<0.05, N = 4 mice/group, n = 30–50 RGC/group, Table 1). O, P Rapamycin also blocks siAMPK-mediated rescue of synapses, visualized with the post- and pre-synaptic markers PSD95 and VGLUT1, respectively. Q Quantification of synaptic voxels confirms that siAMPK-induced synaptic protection is abolished by rapamycin, confirming that this process is mTORC1 dependent (Student’s t-test, ***p < 0,001, N = 5 mice/group). Values are expressed as the mean ± S.E.M. IPL: Inner Plexiform Layer, GCL: Ganglion Cell Layer