Date of Award

5-2005

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Xuemin Xu

Committee Members

Mei-Zhen Cui, Robert Donnell, Karla Mattesson, Hildegard M. Schuller

Abstract

Accumulation of Aβ is widely believed to play an early and crucial role in the pathogenesis of Alzheimer’s disease (AD). The central problem in AD research, the mechanism of Aβ generation mediated by γ-secretase, remains unclear. The present study has identified a novel cleavage site, ζ-cleavage, within the transmembrane domain of amyloid precursor protein (APP) and examined the role of ζ-cleavage in Aβ generation. A novel of Aβ species is produced without γ-secretase inhibitor treatment, but is increased with the treatments in several cell lines. Both MALDI-Mass spectrum and western blot demonstrate that the novel Aβ is Aβ46. Both dominant negative form of PS1 (D385A) and double deficiencies of PS1/PS2 abolish Aβ46 generation, demonstrating the dependence of ζ-cleavage on PS. The Indiana mutation of APP enhances Aβ46 generation. Interestingly, transition state analogues L685,458 and 31C block Aβ46 generation, whereas applications of non-transition state γ-secretase inhibitors lead to an elevated level of Aβ46.

Aβ40/42 can be generated from pre-accumulated Aβ46 in the presence of L685,458. Aβ46 is co-immunoprecipicated with PS1. Moreover, Aβ species which shows the same migration rate in SDS-PAGE as synthetic Aβ49 was observed, suggesting that it is the long-sought Aβ49. Artificially-made Aβ49 is processed into Aβ46. Inhibition of Aβ46 generation from Aβ49 by L685,458 diminishes Aβ40/42 production. These findings suggest the sequences of ε-, ζ-, and γ-cleavages within the TM domain of APP.

Aβ46 is present in 20,000 x g pellet. The majority of Aβ46 is resistant to cell surface trypsinization. Treatment with BFA completely blocks Aβ46 production. A high dose of monesin causes partial inhibition of Aβ46 generation, whereas treatments with NH4Cl, lactcysitn, or tunicamycin do not affect Aβ46 level. Furthermore, retention of APP in the ER with an ER sorting signal abolishes Aβ46 generation, whereas addition of a TGN sorting motif does not affect Aβ46 level. These observations demonstrate that the major subcellular sites for Aβ46 generation are the Golgi and TGN.

Collectively, the present study provides new insights into the underlying mechanism of Aβ generation.

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