1. ¹Sir James McCusker Alzheimer's Disease Research Unit, University of Western Australia, School of Psychiatry and Clinical Neurosciences, Hollywood Private Hospital, Nedlands, WA, Australia
2. ²Dementia Research Program, Nathan Kline Institute, Orangeburg, NY, USA
3. ³Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada
4. ⁴Departments of Medical Biophysics and Medicine (Neurology), University of Toronto, Toronto, Ontario, Canada
5. ⁵Tranzyme, Inc., Birmingham, AL, USA
6. ⁶Garvan Institute for Medical Research, Department of Biotechnology, Darlinghurst, NSW, Australia
7. ⁷University of New South Wales, Sydney, NSW, Australia
8. ⁸Farber Institute for Neurosciences at Thomas Jefferson University, Philadelphia, PA, USA

Correspondence: R Martins, Sir James McCusker Alzheimer's Disease Research Unit, Hollywood Private Hospital, The University of Western Australia Nedlands, Perth, WA 6009, Australia. E-mail: rmartins@cyllene.uwa.edu.au

Received 17 July 2003; Revised 13 October 2003; Accepted 17 October 2003; Published online 2 March 2004.

Abstract

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid- protein (A) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases A following a reaction catalyzed by an enzyme termed '-secretase'. The molecular mechanism of PS1-mediated, -secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the -secretase protease or whether instead PS1 mediates -secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1E9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an A-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1E9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1E9 molecules expressed in Sf9 cells retain the ability to modulate A levels. Baculoviral-expressed PS1E9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting -secretase activity from its individual purified component parts.