Summary of main dendritic morphological changes of long-term gene therapy affected retinal ganglion cells (RGCs) compared to appropriate GFP transduced or non-transduced control groups.

<p>Summary of main dendritic morphological changes of long-term gene therapy affected retinal ganglion cells (RGCs) compared to appropriate GFP transduced or non-transduced control groups.</p

Number of RGCs analyzed in each control and experimental group for all RGCs, and for those identified as Type RI cells.

<p>Number of RGCs analyzed in each control and experimental group for all RGCs, and for those identified as Type RI cells.</p

Evidence for morphological differences in RGCs from retinae injected with rAAV2 encoding different transgenes.

<p>A: Plot showing canonical scores 1 (Y axis) and 2 (X axis) from a multivariate discriminant analysis of dendritic morphology of all retinal ganglion cells (RGCs). Plots show the first two canonical scores that together represent more than 80% of the variance. Axes represent units of standard deviation. Circles represent the 95% confidence region to contain the true mean of the treatment groups. Black lines show the coordinate direction (i.e. morphological parameters measured in Neurolucida) in canonical space. Note that the length of the lines is not representative of effect size due to the multidimensional nature of the analysis. B–D: Box plots showing median and quartiles for selected morphological parameters that were significantly different between treatment groups. B: values for the third canonical score which accounts for the significant difference between Saline and rAAV2-GFP injected groups. Means for soma area (C) and dendritic field area (D) are shown for all treatment groups. * p<0.05; **p<0.001; *** P<0.0001. E: Sholl analysis of all RGCs by treatment group. Error bars = standard error of the mean. Asterisk (*) indicates significant (p<0.05) difference between rAAV2-BDNF-GFP and GFP; # indicates significant (p<0.05) difference between rAAV2-CNTF-GFP and GFP.</p

Evidence for morphological differences in type RI-like RGCs from retinae injected with rAAV2 encoding different transgenes.

<p>A: Plots showing canonical scores 1 (X axis) and 2 (Y axis) from multivariate discriminant analysis of dendritic morphology for RI-like retinal ganglion cells (RGCs). Plots show the first two canonical scores that together represent more than 80% of the variance. Axes represent units of standard deviation. Circles represent the 95% confidence region to contain the true mean of the group. Black lines show the coordinate direction (i.e. morphological parameters measured in Neurolucida) in canonical space. Note that the length of the lines is not representative of effect size due to the multidimensional nature of the analysis. B: Box plots showing median and quartiles for selected morphological parameters that were significantly different between treatment groups. Transduced and non-transduced (nt) FG⁺ RGCs in the 4 rAAV2 groups are labelled as GFP/ntGFP, CNTF/ntCNTF, BDNF/ntBDNF or GAP43/ntGAP43 respectively. Asterisk (*) indicates groups that are significantly different from ntGFP RGCs (p<0.05) and # indicates groups that are significantly different from GFP RGCs (p<0.05).</p

Classification of RI-like RGCs.

<p>A: Neurolucida traces of representative RI-like retinal ganglion cells (RGCs) from control and experimental rAAV2 groups. Transduced and non-transduced (nt) FG⁺ RGCs in the 4 rAAV2 groups are labeled as GFP/ntGFP, CNTF/ntCNTF, BDNF/ntBDNF or GAP43/ntGAP43 respectively. B: Clustering of transduced and ntRI-like RGCs based on the number of primary dendrites (X-axis) and soma area (Y axis) in control and experimental groups. RI-like RGCs are denoted by grey circles and remaining cells by white diamonds. C: Plot showing canonical scores 1 (X axis) and 2 (Y axis) from a multivariate discriminant analysis of dendritic morphology of RI-like RGCs and “other” RGCs. Plots show the first two canonical scores that together represent more than 80% of the variance. Axes represent units of standard deviation. Solid black circles represent the 95% confidence region to contain the true mean of the group. Black lines show the coordinate direction (i.e. morphological parameters measured in Neurolucida) in canonical space. Note that the length of the lines is not representative of effect size due to the multidimensional nature of the analysis.</p

Abnormal stratification in RGCs from retinae injected with rAAV2 encoding different transgenes.

<p>A–C: Side views of Neurolucida traces showing examples of abnormal stratification in each rAAV2 treatment group. Arrows indicate axons and “s” is immediately below the soma. The scale bar in A applies to the three traces. D: Neurolucida trace of an rAAV2-GAP43-GFP transduced retinal ganglion cell (RGC). Ai and Aii are side views. E: confocal image of the same cell as in D; pink is Cy3 immunofluorescence for lucifer yellow, wholemount counterstained with Hoechst 33342 (blue). Areas a and b are shown in more detail. Area a is shown in panels ai-aiv. Panel ai shows a higher power magnification of a and panels aii and aiii show the XZ and YZ projections respectively at the crosshairs shown on panel ai. Panel aiv shows the full YZ projection of panel a and shows that dendritic processes descend through the inner plexiform layer and run along the border of the inner nuclear layer (INL), but they do not enter the INL. Area b is shown in panel bi as an XZ projection showing the relationship of this dendritic branch to the INL. GCL, ganglion cell layer. Scale bar in ai: 25 µm.</p

Transduction in the hippocampus and cerebellum as indicated by GFP expression.

<p>Low magnification images show widespread GFP protein throughout the hippocampus (<b>A</b>) and cerebellar cortex (<b>C</b>). GFP protein was also observed in the contralateral hippocampus (<b>B</b>). Double immunostaining for GFP (green) and NeuN (red) revealed that neurons were specifically transduced (<b>D–O</b>). Merged images (<b>F, I, L, O</b>) also show Hoechst counterstaining. Neurons in many hippocampal sub-regions were transduced including Amon’s horn (<b>D–F</b>) and dentate gyrus (<b>G–I</b>). GFP was observed in Purkinje cells and other neurons in the cerebellum (<b>M–O</b>). Scale bar  = 500 µm (<b>A–C</b>) and 50 µm (<b>D–O</b>).</p

IgG positive cells in the hippocampus and cerebellum.

<p>Mouse IgG positive cells (red) were frequently observed in the hippocampus (<b>B</b>) and cerebellum (<b>C</b>) after injection with AAV2-Aβ40-GFP, AAV2-Aβ42-GFP, AAV2-C100-GFP and AAV2-C100^V717F-GFP in comparison to after AAV2-GFP injection (<b>A</b>). Sections were counterstained with Hoechst (blue). Inserts show distinctive morphology of IgG positive cells. High magnification images of mouse IgG positive cells co-labelled with IBA-1 are shown in <b>D–G</b> (<b>D</b>: IgG staining, <b>E</b>: IBA-1 staining, <b>F</b>: Hoechst, <b>G</b>: merged image). The total number of IgG positive cells was counted in 3 sections per brain in both the hippocampus (<b>H</b>) and cerebellum (<b>I</b>) in the injected hemisphere at 3 and 6 months post-injection. Scale bar  = 200 µm (<b>A–C</b>) and 10 µm (<b>D–G</b>). INJ: injected hemisphere, CONTRA: contralateral hemisphere, ***p<0.001, *p<0.05.</p

Increased mouse IgG staining in the hippocampus and cerebellum.

<p>Example images show normal IgG staining following immunohistochemistry for NeuN after AAV2-GFP injection (<b>A–D</b>) and intense IgG staining after injection of AAV2-Aβ42-GFP (<b>E, G</b>). Increased IgG staining was only observed surrounding the injection site and not in the contralateral hemisphere (<b>F, H</b>). INJ; injected region, CONTRA; contralateral region. Scale bar  = 500 µm.</p

C100 and Aβ protein production <i>in vitro</i> after plasmid transfection.

<p>Analysis of C100 and Aβ protein production <i>in vitro</i> after transfection with plasmids expressing Aβ40-GFP, Aβ42-GFP, C100-GFP or C100^V717F-GFP in cell homogenates (<b>A</b>) and media (<b>B</b>). C100 protein was only detected in cell homogenates after transfection with C100-GFP and C100^V717F-GFP plasmids. No Aβ was detected in cell homogenates after transfection with any plasmid. APP^SWE brain homogenate was used as a positive control for Aβ detection. Strong GFP protein expression indicated successful transfection using all plasmids and β-actin was used as a loading control. Immunoprecipitation of Aβ from the cell culture media revealed Aβ in the media from cells transfected with C100-GFP and C100^V717F-GFP plasmids. Control refers to non-transfected cells.</p