Abstract
Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.
Highlights
Amyloid-b-protein (Ab), the key component of senile plaques in Alzheimer’s disease (AD) neuropathology, was first isolated from meningovascular amyloid deposits in AD and Down’s syndrome [1,2], and has been reported to be the subunit of the plaque amyloid [2,3,4]
Immunoblot analysis of amyloid precursor protein (APP)-Carboxylterminal fragment (CTF) revealed increases in levels of APP-C99 and APP-C83 in the cells treated with Dab Small interfering RNA (siRNA) as compared to the cells treated with control siRNA (Figure 1A)
No significant differences in FLAPP levels were observed between Dab siRNA- and control siRNA-treated cells
Summary
Amyloid-b-protein (Ab), the key component of senile plaques in Alzheimer’s disease (AD) neuropathology, was first isolated from meningovascular amyloid deposits in AD and Down’s syndrome [1,2], and has been reported to be the subunit of the plaque amyloid [2,3,4]. APP-C99 is further cleaved to release a ~4kDa peptide, Ab, and the amyloid precursor protein intracellular domain (AICD). The effects of reduced expression of Dab and Numb on APP processing and Ab production, the key components of AD neuropathogenesis, have not been assessed. For this purpose, we established RNAi knockdown of Dab and Numb in H4 human neuroglioma cells overexpressing full-length (FL)-APP (H4-FL-APP cells) and C-99 (H4-APP-C99 cells), and evaluated the effects of RNAi-mediated knock-down of Dab and Numb on APP processing and Ab levels
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