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

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

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

Aβ and C100 mRNA and protein expression in the hippocampus and cerebellum.

<p>mRNA for Aβ and C100 transgenes was detected using PCR (<b>A</b>). Immunostaining for C100 using CT20 antibody revealed C100 protein in GFP positive neurons (<b>B–G</b>). C100 protein was also detected using western blot (<b>H</b>). HIP (INJ): injected hippocampus, HIP (CONTRA) contralateral hippocampus, CB: cerebellum, ROB: rest of the brain. Lanes 1, 4, 7, 10; representative brain injected with AAV2-C100^V717F-GFP, lanes 2, 5, 8, 11; representative brain injected with AAV2-Aβ42-GFP, lanes 3, 6, 9, 12; representative brain injected with AAV2-GFP.</p

Animal experimental groups used for different analysis techniques.

<p>Animal experimental groups used for different analysis techniques.</p