Additional file 2 of AMPK hyperactivation promotes dendrite retraction, synaptic loss, and neuronal dysfunction in glaucoma
Creators: name: Belforte, Nicolas
name: Agostinone, Jessica
name: Alarcon-Martinez, Luis
name: Villafranca-Baughman, Deborah
name: Dotigny, Florence
name: Cueva Vargas, Jorge L.
name: Di Polo, Adriana
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Physiology

description: Additional file 2: Supplementary Figure 1. (A) The number of YFP-positive RGC does not change at 2 weeks of ocular hypertension (OHT) relative to non-injured controls (Student’s t-test, n.s.: not significant, N = 5 mice/group). (B) Real-time qPCR analysis confirms that retinal YFP gene expression does not change within 2 weeks of OHT damage (Student’s t-test, n.s.: not significant, N = 5 mice/group). (C-E) RGC co-expressing YFP and SMI-32 are selected for dendritic arbor imaging and 3D reconstruction. Supplementary Figure 2. Analysis of whole-mounted retinas immunolabeled with an antibody recognizing pAMPKThr172 revealed a substantial increase of AMPK activity in RGC axons, visualized with SMI-32, at two weeks after glaucoma induction. Scale bars = 25 μm. Supplementary Figure 3. (A, B) Retinal imaging showed no fluorescein extravasation in eyes injected with compound C or vehicle. (C) In contrast, marked fluorescein extravasation was found in ischemic retinas subjected to central retinal artery ligature. (D), a procedure known to compromise the BRB [31] and used as positive control (Fig. S3C). (D, E) T cell infiltration, visualized with markers the MHCII, MPO and CD45, was not detected in retinas treated with compound C or vehicle. (F) Ischemic retinas, used as positive controls, display cells positive for MHCII, MPO and CD45. (Q) Quantification of cells expressing MHCII, MPO and CD45 confirm lack of cellular infiltrates in compound c-treated retinas. (ANOVA with Tukey’s multiple comparison post-hoc test, * = p&lt;0.05, N = 3 mice/group). Scale bars = 10 μm. Values are expressed as the mean ± S.E.M. Supplementary Figure 4. (A) Intravitreal delivery of non-targeting (scrambled) Cy3-tagged control siRNA (siCTL-Cy3) results in rapid uptake by neurons in the ganglion cell layer (GCL), (B-D) identified as RBPMS-positive RGC (B, inset in A) as early as 3 h after injection. Scale bars (A) = 20 μm, (B-D) = 20 μm. OPL: Outer Plexiform Layer, INL: Inner Nuclear Layer, IPL: Inner Plexiform Layer. Supplementary Figure 5. (A) In the inner nuclear layer (INL), pS6Ser240/244 co-localizes with the calcium-binding protein calbindin, a marker of horizontal cells. (B) pS6Ser240/244 expression in horizontal cells remains unchanged after OHT induction (Student’s t-test, n.s.: not significant, N = 5 mice/group). (C, D) Immunohistochemical analysis of PSD95 and VGLUT1 shows no appreciable change in excitatory synapses in sham retinas treated with siAMPK and vehicle or rapamycin (ANOVA with Tukey’s multiple comparison post-hoc test, n.s. = not significant, N = 3 mice/group). (E) The total area of sham-operated versus glaucomatous retinas is similar, ruling out changes caused by ocular hypertension (Student’s t-test, n.s = not significant, N = 5–6 mice/group). Scale bars (A) = 20 μm, (C) = 10 μm. Values are expressed as the mean ± S.E.M. IPL: Inner Plexiform Layer, GCL: Ganglion Cell Layer. Supplementary Figure 6. (A) Immunohistochemistry of optic nerve head (ONH) cross-sections shows that although Iba1-positive cells and GFAP increase with high intraocular pressure, siAMPK treatment did not alter this response relative to control siRNA (siCTL). (B) Quantification of Iba1-positive cells and GFAP fluorescence confirmed that siAMPK does not have a significant effect on the number of Iba1-positive cells or GFAP reactivity (ANOVA with Tukey’s multiple comparison post-hoc test, * = p&lt;0.05, ** = p&lt;0.01, N = 3 mice/group). Scale bars = 50 μm. Values are expressed as the mean ± S.E.M.