Studies on the γ-Secretase Complex and Processing of the Alzheimer ’ s Disease-associated Amyloid Precursor Protein

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that causes the most common form of dementia. Pathological lesions, such as plaques consisting of the amyloid βpeptide (Aβ), are found in the brains of AD patients. Aβ is produced by sequential cleavages of the amyloid precursor protein (APP) by βand γ-secretase. Concomitant with γ-cleavage, another cleavage, termed ε-cleavage, occurs seven to nine residues C-terminal of the γ-site, releasing the APP intracellular domain (AICD). The γand ε-cleavages critically require the γ-secretase complex consisting of four well-conserved proteins, namely presenilin (PS), nicastrin, Aph-1 and Pen-2. Interestingly, this processing occurs within the anhydrous environment of the lipid membrane bilayer where PS is proposed to provide the catalytic core of the complex, acting as an aspartyl protease. The work presented in this thesis describes the intramembrane processing of APP and how disease-causing familial AD mutations in the APP protein affect ε-cleavage, and thereby generation of AICD. Also, the intricate biogenesis and assembly of the γ-secretase complex was investigated by detailed studies of the Pen-2 and PS1 proteins. In addition to cleavage of APP, PS is required for the processing of a number of type I membrane proteins, such as the Notch receptors. γ-Secretase provides a potential therapeutic target for AD. However, inhibition of γ-secretase can possibly lead to unwanted side effects due to impaired signaling of other PS substrates. In paper I, a novel γ-secretase reporter assay was developed. The assay specifically and quantitatively records total γ-cleavage occurring in intact cells, enabling detailed studies of the intramembrane processing of APP. In addition, the reporter assay can be used for screening compound libraries for drugs that differentially affect γ-secretase processing of APP and other PS substrates such as Notch. Generation of AICD was characterized in paper II by using the reporter assay developed in paper I. Formation of AICD was found to occur in a compartment downstream of the endoplasmic reticulum (ER) in the secretory pathway, thus overlapping with the reported site of production of Aβ. Furthermore, familial AD mutations showed unchanged levels of AICD generation. Thus, the disease-causing consequences of these APP mutations are unlikely to be mediated by the amount of AICD fragment produced. Understanding of the γ-secretase complex is essential for unraveling the pathogenic mechanism(s) leading to AD. Pen-2, the smallest protein in the γ-secretase complex, was studied in depth in paper III. Presence or absence of PS had a great impact on cellular levels and distribution of Pen-2. In PS null cells, Pen-2 was destabilized and restricted to the ER, compared with cells expressing PS1, where Pen-2 levels were stable and the Pen-2 protein was trafficked further in the secretory pathway. Destabilization of Pen-2 in PS null cells was mediated by ubiquitylation and proteasomal degradation. In the absence of PS, the Pen-2 protein appeared to be retrotranslocated out of the ER into the cytosol prior to ubiquitylation and degradation. These observations suggest an ER-associated proteasomal degradation pathway mediating regulation of protein levels and trafficking of Pen-2, and possibly other components, not incorporated into the γ-secretase complex. Analysis of the γ-secretase complex was continued in paper IV and focused on the C-terminal domain of PS1. Truncations of seven to seventeen residues in the PS1 C terminus resulted in PS1 molecules deficient in supporting γ-secretase activity, with accompanying impairment in γ-secretase complex formation and endoproteolysis of the PS1 molecule. However, intramolecular PS1 heterodimer formation was shown to occur for C-terminally truncated molecules that were unable to associate with nicastrin and Aph-1. On a PS null background the C-terminal fragment of PS1 was by itself able to interact with Aph-1 and nicastrin, thus suggesting an additional function for Aph-1 and nicastrin apart from stabilizing the full-length PS1 molecule. Although the main components of the γ-secretase complex are known, the molecular mechanisms underlying the inter-regulation, assembly and actual stochiometry of the complex are less well understood. The studies presented here provide insights into intramembrane processing of APP and detailed information about the γ-secretase complex components Pen-2 and PS1, thereby providing a framework for future studies of the intricate regulation of the γsecretase complex and its proteolytic function in terms of APP processing. LIST OF ORIGINAL PUBLICATIONS The thesis is based on the following publications, which are referred to in the text by their