1. Long-term in vivo inhibition of CNS neurodegeneration by Bcl-XL gene transfer 
J.M.I. Malik, Z. Shevtsova, M. Bähr, S. Kügler 
Molecular Therapy 
Volume 11, Issue 3, Pages (March 2005)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
AAV-2 vectors used in this study. (A) Schematic depiction of the AAV-2 vector genomes. ITR, AAV-2 inverted terminal repeats; hSyn1, human synapsin 1 gene promoter; Bcl-XL, rat Bcl-XL cDNA N-terminally tagged with the FLAG epitope; Int, intron; SV40, SV40 polyadenylation site; TB, synthetic transcription blocker; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; bGH, bovine growth hormone polyadenylation site. (B) Transgene expression analysis by Western blot. Cell lysates from AAV-Bcl-XL- and AAV-EGFP-transduced primary hippocampal neurons were probed with antibodies specific to Bcl-X, FLAG, or EGFP. Size standards in kDa are shown on the right. (C) Experimental schedule given in weeks.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Retinal transduction by intravitreal application of 1 × 107 transducing AAV-2 units. (A) The retinal area typically transduced by injection of 107 transducing AAV-2 units is shown as an overlay of EGFP fluorescence and phase contrast of a flat-mounted retina. (B) A schematic depiction of the transduction areas obtained throughout the experiment and of the sites in which surviving RGCs were counted. Cell counts were performed in individual areas of 92,000 μm2 as shown in (C) for EGFP fluorescence of transduced RGCs and in (D) for FluoroGold (FG) fluorescence of surviving RGCs. Black arrows in (C) and (D) point to the rare large-class RGCs, which show very high levels of EGFP expression. Black circles mark transduced amacrine cells (which do not show FG labeling), which also express high levels of EGFP. Higher magnifications of (C) and (D) are shown in (E) and (F). Black circles again mark amacrine cells, while white arrows point to RGCs without detectable EGFP expression. Note that the majority of RGCs display only moderate EGFP expression compared to amacrine cells or large-class RGCs. Scale bar, 500 μm in (A) and 55 μm in (C) and (D).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Retinal ganglion cell-specific transgene expression from bicistronic AAV-2 vector. (A) RGC specifically labeled by the retrograde fluorescent tracer FG are shown in a retinal cryosection. (B) Expression of the EGFP reporter gene after intravitreal vector injection. (C) Only a few retinal neurons show EGFP expression but are not FG labeled (arrows). (E) Expression of the Bcl-XL transgene is detected by anti Bcl-X antibody staining and colocalizes with (D) EGFP expression from the same vector genome as shown by the overlap in (F). (G) Almost no Bcl-XL expression was detected in retinal sections from either untransduced or AAV-EGFP-transduced animals. (H) While EGFP fluorescence proceeded uniformly in the cytoplasm of transduced RGCs, (K) Bcl-XL expression was detected exclusively in granule-like cytoplasmic structures. (L) In the axons projecting from the transduced retinal area to the optic disc, EGFP fluorescence was readily detected in varicosities, but (M) these structures were always negative for Bcl-XL staining. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer; PE, pigment epithelium.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Representative areas of whole-mounted retinal specimens at 4 weeks after axotomy. EGFP fluorescence of cell bodies or neuritic structures is shown in the left column, FG fluorescence is shown in the right column. The transduced area after AAV-EGFP application is shown in (A), while (B) shows an area outside of AAV-EGFP transduction. The inset in (A) depicts the predominant morphology of FG-labeled cells found at 4 weeks after AAV-EGFP application (microglia that have taken up the FG label during RGC phagocytosis). The transduced area after AAV-Bcl-XL transduction is shown in (C), while (D) shows an area outside of AAV-Bcl-XL transduction from the same retinal specimen. The inset in (C) shows that the majority of FG-labeled cells in the AAV-Bcl-XL-transduced area are RGCs (perinuclear punctate FG label).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Quantification of surviving RGCs after axotomy. The mean number of RGCs/mm2 is shown by the hatched bar. In the central region of retinal transduction, 90 ± 5% of the RGCs are transduced by the Bcl-XL/EGFP-expressing vectors (black bar). The number of surviving RGCs was determined after control vector transduction (AAV-EGFP) and after AAV-Bcl-XL transduction in either the transduced area (gray shaded bars) or the untransduced area (white bars). Bars represent means ± standard deviation. Asterisks denote statistical significant differences (P < 0.001).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Preservation of proximal axons after axotomy of Bcl-XL-expressing RGCs. Flat-mounted retinae transduced with AAV-Bcl-XL (A) without axotomy or (B) at 6 weeks after axotomy are shown as overlays of EGFP reporter gene fluorescence with the phase-contrast image of retinal tissue. Axon bundles emanating from the transduced area are clearly visualized due to their EGFP fluorescence. Higher magnification pictures of axon bundles at (C) 9 weeks after transduction (without axotomy), serving as controls, (D) 2 weeks after axotomy, and (E) 6 weeks after axotomy show that gross axonal morphology is preserved at 2 weeks after axotomy but appears distorted at 6 weeks after axotomy. Arrows point to varicosities clearly visible in controls and at 2 weeks after axotomy but only very rarely at 6 weeks after axotomy. Scale bar, 500 μm in (A) and (B) and 55 μm in (C–E).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions