1. Activation of the Extracellular Signal-Regulated Kinase 1/2 Pathway by AAV Gene Transfer Protects Retinal Ganglion Cells in Glaucoma 
Yu Zhou, Vincent Pernet, William W. Hauswirth, Adriana Di Polo 
Molecular Therapy 
Volume 12, Issue 3, Pages (September 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Outline of the experimental protocol used to test the effect of AAV.MEK-CA on RGC survival in experimental glaucoma. Following intraocular injection of viral vectors, RGCs were back-labeled with the fluorescent tracer DiI. Episcleral vein injection was performed 1 week after DiI application to ensure that all RGCs were labeled prior to intraocular pressure increase. Retinas were examined histologically at 5 and 7 weeks following ocular hypertension surgery to determine the density of surviving RGCs.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
AAV mediates MEK-CA gene product expression in adult RGCs. (A–C) AAV-mediated MEK-CA and (D–F) MEK-WT were visualized using an antibody against the HA tag present only in MEK1 transgenes. Robust HA staining was observed in a large number of cell bodies in the ganglion cell layer (GCL) and dendrites in the inner plexiform layer (IPL) (A and D). RGCs were visualized using the retrograde tracer FluoroGold (FG) applied to the superior colliculus, the main target for these neurons in the rat brain (B and E). Superimposition of the HA and FG staining demonstrated that the vast majority of RGCs expressed MEK-CA (C) or MEK-WT (F) gene product. HA immunoreactivity was not detected in retinal sections after intraocular injection of AAV.GFP (data not shown). High-power magnification demonstrated HA labeling on RGC soma and dendritic processes after infection with (G) AAV.MEK-CA or (J) AAV.MEK-WT. (H and K) Retrograde labeling with FG confirmed that (I and L) RGCs expressed HA-tagged MEK proteins. (M) In vivo activation of Erk1/2 kinases was detected in retinal homogenates at 4 weeks after injection of AAV.MEK-CA compared to control eyes. Western blots of total retinal extracts were probed with an antibody that selectively recognizes both Erk1 and Erk2 phosphorylated on Thr202/Tyr204 residues. The bottom blots show the same blot reprobed with an antibody to visualize total Erk1/2 protein or the HA tag, present only in the MEK1 transgenes. Scale bars: A–F, 100 μm; G–L, 25 μm. PS, photoreceptors segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
AAV.MEK-CA protects RGCs from hypertension-induced death. Increased expression of phospho-Erk1/2 in RGCs was observed in (A) the superior (dorsal) hemisphere and compared to (B) the inferior (ventral) hemisphere of AAV.MEK-CA-injected eyes. Scale bars: 50 μm. Quantitative analysis of RGC survival following injection of AAV.MEK-CA, AAV.MEK-WT, or AAV.GFP is shown for (C) whole retina or (D) superior hemisphere only, where AAV vectors were injected (n = 8–13 rats per group). The densities of RGCs in intact, untreated retinas (Intact) or glaucomatous, untreated retinas (No treatment) are shown as references. MEK-CA gene transfer markedly increased the number of RGCs that survived at 5 or 7 weeks after ocular hypertension surgery (ANOVA, **P < 0.001; ***P < 0.0001). AAV.MEK-CA injection into normal, intact eyes did not increase the number of RGCs, strongly suggesting that AAV.MEK-CA promotes survival, but not proliferation, of adult RGCs. Data are expressed as the means ± SEM. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
AAV.MEK-CA protects RGC soma from hypertension damage. Fluorescence photomicrographs of flat-mounted retinas showing DiI-labeled RGCs in (A) intact and glaucomatous retinas treated with (B) AAV.MEK-CA or (C) AAV.GFP or (D) left untreated at 5 weeks after ocular hypertension surgery. Images were taken from the superior, central retina. AAV.MEK-CA treatment led to higher neuronal densities and better preservation of cellular integrity. Scale bar: 100 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Erk1/2 activation protects intraretinal RGC axons in glaucoma. Confocal microscopy images of intraretinal RGC axons visualized on flat-mounted retinas stained with RT-97, an antibody that recognizes the phosphorylated 200-kDa neurofilament H subunit. (A) Immunoreactive axons coursed in organized bundles toward the optic nerve head in normal retinas. (B) Treatment with AAV.MEK-CA remarkably preserved the overall structure of RGC axon bundles, while (C) retinas treated with the control vector AAV.GFP suffered significant fiber loss at 5 weeks after hypertension surgery. Many remaining fibers had a beaded appearance, confirming the progressive axonal degeneration after glaucomatous injury. Scale bars: 20 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
AAV.MEK-CA treatment reduces optic nerve damage in glaucoma. Cross sections of optic nerve segments from (A) intact and glaucomatous eyes treated with (B) AAV.MEK-CA or (C) AAV.MEK-WT at 5 weeks after ocular hypertension surgery. AAV.MEK-CA-treated eyes displayed a larger number of axonal fibers with normal morphology compared to AAV.MEK-WT-treated control eyes, which showed extensive axon degeneration, including disarray of fascicular organization and degradation of myelin sheaths. (D) Quantitative analysis of RGC axons in the optic nerve following injection of AAV.MEK-CA, AAV.MEK-WT, or AAV.GFP (n = 4–7 rats per group). The number of axons in the intact, uninjured optic nerve is shown as reference. MEK-CA gene transfer markedly protected RGC axons at 5 weeks post-ocular hypertension surgery (ANOVA, *P < 0.05). Data are expressed as the means ± SEM.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

8. Supplementary data associated with this article can be found, in the online version, at doi: /j.ymthe
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

9. Supplementary data associated with this article can be found, in the online version, at doi: /j.ymthe
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions