Table 1.
Animal experimental groups used for different analysis techniques.
Table 2.
Primers and PCR conditions.
Figure 1.
C100 and Aβ protein production in vitro after plasmid transfection.
Analysis of C100 and Aβ protein production in vitro after transfection with plasmids expressing Aβ40-GFP, Aβ42-GFP, C100-GFP or C100V717F-GFP in cell homogenates (A) and media (B). C100 protein was only detected in cell homogenates after transfection with C100-GFP and C100V717F-GFP plasmids. No Aβ was detected in cell homogenates after transfection with any plasmid. APPSWE 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 C100V717F-GFP plasmids. Control refers to non-transfected cells.
Figure 2.
Transduction in the hippocampus and cerebellum as indicated by GFP expression.
Low magnification images show widespread GFP protein throughout the hippocampus (A) and cerebellar cortex (C). GFP protein was also observed in the contralateral hippocampus (B). Double immunostaining for GFP (green) and NeuN (red) revealed that neurons were specifically transduced (D–O). Merged images (F, I, L, O) also show Hoechst counterstaining. Neurons in many hippocampal sub-regions were transduced including Amon’s horn (D–F) and dentate gyrus (G–I). GFP was observed in Purkinje cells and other neurons in the cerebellum (M–O). Scale bar = 500 µm (A–C) and 50 µm (D–O).
Figure 3.
Aβ and C100 mRNA and protein expression in the hippocampus and cerebellum.
mRNA for Aβ and C100 transgenes was detected using PCR (A). Immunostaining for C100 using CT20 antibody revealed C100 protein in GFP positive neurons (B–G). C100 protein was also detected using western blot (H). 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-C100V717F-GFP, lanes 2, 5, 8, 11; representative brain injected with AAV2-Aβ42-GFP, lanes 3, 6, 9, 12; representative brain injected with AAV2-GFP.
Figure 4.
Immunostaining of microglia using IBA-1 antibody.
Representative figures show microglial staining at low (A–B, E–F, I–J) and high magnification (C–D, G–H, K–L) in injected (INJ) and contralateral (CONTRA) hippocampi at 3 months post-injection with AAV2-GFP (A–D), AAV2-C100V717F-GFP (E–H) and AAV2-Aβ42-GFP (I–L). IBA-1 staining density was quantified in the hippocampus (M) and cerebellum (N) at 3 and 6 months post-injection. Data shown as density of injected region as a percentage of the corresponding contralateral region and is presented as mean ± standard deviation. ***p<0.001, **p<0.01, *p<0.05. Scale bar = 300 µm (A–B, E–F, I–J) and 20 µm (C–D, G–H, K–L).
Figure 5.
Increased mouse IgG staining in the hippocampus and cerebellum.
Example images show normal IgG staining following immunohistochemistry for NeuN after AAV2-GFP injection (A–D) and intense IgG staining after injection of AAV2-Aβ42-GFP (E, G). Increased IgG staining was only observed surrounding the injection site and not in the contralateral hemisphere (F, H). INJ; injected region, CONTRA; contralateral region. Scale bar = 500 µm.
Figure 6.
IgG positive cells in the hippocampus and cerebellum.
Mouse IgG positive cells (red) were frequently observed in the hippocampus (B) and cerebellum (C) after injection with AAV2-Aβ40-GFP, AAV2-Aβ42-GFP, AAV2-C100-GFP and AAV2-C100V717F-GFP in comparison to after AAV2-GFP injection (A). 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 D–G (D: IgG staining, E: IBA-1 staining, F: Hoechst, G: merged image). The total number of IgG positive cells was counted in 3 sections per brain in both the hippocampus (H) and cerebellum (I) in the injected hemisphere at 3 and 6 months post-injection. Scale bar = 200 µm (A–C) and 10 µm (D–G). INJ: injected hemisphere, CONTRA: contralateral hemisphere, ***p<0.001, *p<0.05.