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Fig. 2 | Molecular Neurodegeneration

Fig. 2

From: AMPK hyperactivation promotes dendrite retraction, synaptic loss, and neuronal dysfunction in glaucoma

Fig. 2

Glaucoma-induced energy stress triggers AMPK activation. A, B Western blot and densitometry analysis of retinal homogenates demonstrate a substantial increase in active AMPK (pAMPKThr172), a readout of metabolic stress, as early as 1 week after induction of OHT (Student’s t-test, * = p<0.05, N = 5 mice/group). The lower panel is the same blot probed with an antibody against total AMPK and β-actin for normalization. C, D Western blot and densitometry analysis of retinal homogenates show increased LKB1 activity (pLKB1) in glaucomatous retinas (Student’s t-test, ** = p<0.01, N = 7–9 mice/group). The lower panel is the same blot probed with an antibody against total LKB1 and β-actin for normalization. E, F Immunohistochemical analysis of mouse retinal cross sections with an antibody against pAMPKThr172, reveals robust AMPK activity in retinal cells subjected to OHT. G, H Co-labeling with antibodies against pAMPKThr172 and the RGC-specific marker Brn3a demonstrates AMPK hyperactivity in RGC. I, J Quantification of the number of pAMPKThr172-positive RGC as well as epifluorescence intensity per neuron confirms a significant increase in AMPK activity (Student’s t-test, ** = p<0.01, N = 5 mice/group, n = 50 RGC/group). K, L pAMPKThr172 retinal immunostaining of primary open angle glaucoma patients and age-matched controls (Table 2) reveals increased AMPK function. M, N Co-labeling with anti-pAMPKThr172 and RBPMS, a selective marker for RGC, confirms AMPK overactivation in glaucomatous RGC. O Quantification of epifluorescence intensity in pAMPKThr172-positive RGC demonstrates a two-fold increase in AMPK activity in human glaucomatous retinas relative to age-matched controls (Student’s t-test, ** = p<0.01, glaucoma: N = 27 retinas/group, n = 100 RGC/group; controls: N = 15 retinas/group, n = 100 RGC/group). P Administration of compound C (CC), an inhibitor of AMPK, results a significant decrease in AMPK activity compared to vehicle-treated eyes. (Student’s t-test, *** = p<0.001, N = 5 mice/group). Q, R Representative examples of dendritic arbors from OHT retinas treated with vehicle or CC, an inhibitor of AMPK, visualized at 2 weeks after microbeads injection. S-V Quantitative analysis of dendritic parameters reveals that CC-treated neurons had longer dendrites and markedly larger and more complex arbors than vehicle-treated controls (CC: green, vehicle: grey, sham controls: white) (ANOVA with Tukey’s multiple comparison post-hoc test, * = p<0.05, *** = p<0.001, N = 4–6 mice/group, n = 30–40 RGC/group, Table 3). W, X Brn3a-labeled flat-mounted retinas display greater RGC soma density at 3 weeks of OHT following CC administration compared to vehicle (ANOVA with Tukey’s multiple comparison post-hoc test, ** = p<0.01, N = 5–8 mice/group). Values are expressed as the mean ± S.E.M. ONL: Outer Nuclear Layer, OPL: Outer Plexiform Layer, INL: Inner Nuclear Layer, IPL: Inner Plexiform Layer, GCL: Ganglion Cell Layer

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