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Fig. 2 | Translational Neurodegeneration

Fig. 2

From: Targeting γ-secretase triggers the selective enrichment of oligomeric APP-CTFs in brain extracellular vesicles from Alzheimer cell and mouse models

Fig. 2

EVs from γ-secretase inhibitor-treated AAV-C99 and 3xTgAD mice are enriched in APP-CTFs including high molecular weight APP-CTFs. a-d Brain EVs from 2 month-old vehicle- or D6-treated AAV-free or AAV-C99 mice were validated by electron microscopy (a) and immunoblot for the detection of the exosomal markers Hsc70 and Flotilin2 (b). a Ultramicrophotographs of vesicles purified from D6-treated AAV-C99 brain showed the presence of mainly vesicles with sizes around 100 nm. Bar scales correspond to 500 nm and 100 nm, respectively. b-d APP-CTFs in EVs and whole brain (tissue) were detected by immunoblot analysis. Proteins were stained by photoactivation using the Bio-Rad prestain method (protein stain) and immunoblots were used for the detection of APP-CTFs and exosomal markers (Hsc70 and Flotilin-2). The α-APPct antibody detected some non-specific immunoreactivites in tissue extracts (asterix). Low and high correspond to low or high exposures of the same immnoblot. The lower blot illustrates the presence of HMW APP-CTFs at higher magnification. Bars in c-d correspond to the levels of APP-CTFs in EVs or whole brain (Tissue) that were normalized to either endogenous Hsc70 (c) or full-length APP (d). Data represent means ± SEM, *p < 0.001 as analyzed by 2-way ANOVA followed by Dunetts posthoc analysis. e Brain sections of 5 month-old 3xTgAD vehicle- or D6-treated mouse were immunostained with 4G8 and microphotographs illustrates the staining at the level of the subiculum. Note the strong extracellular staining in the D6-treated mouse arrow heads. f-g, EVs were purified from hippocampi of vehicle- or D6-treated Wild-type (WT) and 3xTgAD mice and used for immunoblot analysis of APP-CTFs. Blots in (g) allow the comparison of the sizes of HMW APP-CTFs detected in 3xTgAD and AAV-C99 mouse brain vesicles. h Co-immunostaining of APP-CTFs with 4G8 (green) and α-Iba1 (red) on brain sections of D6-treated AAV-C99 animals. Arrows in the merged image point to APP-CTF positive microglia. Bar in e and h corresponds to 100 μm and 10 μm, respectively

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Translational Neurodegeneration

ISSN: 2047-9158

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