Involvement of presenilin 1 in the generation of the amyloid beta-peptide : lessons from a cellular model lacking the amyloidogenic pathway

Alzheimer's disease (AD) is the most common form of dementia, affecting 15 million people worldwide, a figure quickly increasing with the ageing of Western populations. As the cause of the disease is still unclear, our possibilities of therapeutic intervention are unfortunately limited to symptomatic handling. Nevertheless, evidence accumulated over the last few years that the accumulation of amyloid beta-peptide (Ab) oligomers and fibrils could be a cornerstone in the development of neurodegeneration. The mechanisms leading to the liberation of the Ab peptide from its precursor by the beta- and gamma-secretase activities have been investigated in this thesis using a cellular model devoid of such activities. More particularly, the function of presenilin 1 (PS1), mutations of which are the main cause of early-onset familial AD, in the ultimate cleavage leading to Ab production was scrutinized. This polytopic protein, the first one to be shown mandatory for gamma-secretase activity, has recently been shown to be dispensable for the production of some Ab species inside as well as outside cells. As gamma-secretase inhibition is a promising way to lower Ab production and treat patients, it was thus important to assess if PS1 is indeed a new kind of protease responsible for a crucial step in Ab generation. In this thesis, we explored the function of PS1 in insect and Chinese hamster cells, as well as in rat and mouse neurons. We show that PS1 binds to the substrates of gamma-secretase, stabilizing them and increasing their cellular steady-states. This results in their increased availability for gamma-secretase cleavage and therefore in an increased production of Ab. Hence, we concluded that the influence of PS1 on the generation of Ab occurs upstream of the gamma-secretase activity and suggest that another protease is responsible for the ultimate cleavage of the amyloid precursor protein. Moreover, the fact that PS1 binds to the gamma-secretase substrates without cleaving them suggests it could have a chaperone role. As the maturation and trafficking of PS1 in the secretory pathway is known to be tightly regulated, PS1 could be involved in the transportation of gamma-secretase substrates towards compartments where gamma-secretase activity takes place, without excluding it could participate in the recruitment/regulation of gamma-secretase activity or in the presentation of the substrate to the enzyme. These advances in understanding the role of PS1 in Ab generation might well reconciliate numerous discordant data published in this field.La maladie d'Alzheimer est la plus fréquente des démences : elle affecte 15 millions de personnes à travers le monde, un chiffre en progression rapide dans nos sociétés occidentales vieillisantes. La cause exacte de la pathologie nous échappant toujours, nos possibilités d'interventions thérapeutiques se limitent malheureusement essentiellement à un traitement symptômatique. Ces dernières années ont néanmoins vu s'accumuler les expériences suggérant que le peptide amyloïde (Ab), et plus particulièrement ses formes oligomérisées, soit un évênement clef dans la pathogenèse. Dans cette thèse, nous avons investigué les mécanismes menant à la libération de l'Ab à partir de son précurseur par les activités beta- et gamma-sécrétases dans un modèle cellulaire dépourvu de telles activités. Nous nous sommes plus particulièrement intéressés à la fonction jouée par la préséniline 1 (PS1), dont les mutations sont la première cause des formes héréditaires de la maladie, dans l'activité gamma-sécrétase libérant finalement l'Ab. Cette proteine à multiples domaines transmembranaires a en effet été la première à être démontrée indispensable au clivage de type gamma-sécrétase, bien qu'il ait récemment été observé qu'elle n'était pas nécessaire à la production de certains types d'Ab, à l'intérieur comme à l'extérieur des cellules. Puisque inhiber l'activité gamma-sécrétase est potentiellement une stratégie de choix pour traiter les patients, il était donc important de déterminer avec certitude si PS1 représentait ou non une protéase d'un nouveau type jouant un rôle crucial dans la production d'Ab. Lors de cette thèse, nous avons donc investigué la fonction de PS1 aussi bien dans des modèles cellulaires d'insectes et de de cobayes que dans des neurones de rats et de souris. Ceci nous a permis de démontrer que PS1 lie les substrats de la gamma-sécrétase, ce qui les stabilise et permet leur accumulation intracellulaire, augmentant leur disponibilité pour l'activité gamma-sécrétase et donc in fine la production d'Ab. Ceci nous amène à penser que l'influence de PS1 sur la génération d'Ab s'exerce en amont de l'activité gamma-sécrétase et qu'une autre protéase est responsable du clivage final du précurseur du peptide amyloïde. De plus, puisque PS1 se lie aux substrats de la gamma-sécrétase sans les cliver, elle pourrait jouer un rôle de chaperonne. Au vu de la stricte régulation de la maturation et du transport de PS1 vers la voie sécrétoire, il est possible que PS1 soit impliquée dans le transport des substrats de la gamma-sécrétase vers les compartiments cellulaires où résident les activités sécrétases, sans néanmoins exclure qu'elle puisse intervenir dans la régulation de telles activités ou dans la présentation des substrats aux enzymes. Cette nouvelle compréhension de la fonction de PS1 dans la production d'Ab pourrait permettre de réconcilier de nombreuses données divergentes dans ce domaine clef de la pathologie.Thèse de doctorat en sciences pharmaceutiques (FARM 3)--UCL, 200

Presenilin 1 stabilizes the C-terminal fragment of the amyloid precursor protein independently of gamma-secretase activity.

The cleavage of the transmembrane amyloid precursor protein (APP) by beta-secretase leaves the C-terminal fragment of APP, C99, anchored in the plasma membrane. C99 is subsequently processed by gamma-secretase, an unusual aspartyl protease activity largely dependent on presenilin (PS), generating the amyloid beta-peptide (Abeta) that accumulates in the brain of patients with Alzheimer's disease. It has been suggested that PS proteins are the catalytic core of this proteolytic activity, but a number of other proteins mandatory for gamma-secretase cleavage have also been discovered. The exact role of PS in the gamma-secretase activity remains a matter of debate, because cells devoid of PS still produce some forms of Abeta. Here, we used insect cells expressing C99 to demonstrate that the expression of presenilin 1 (PS1), which binds C99, not only increases the production of Abeta by these cells but also increases the intracellular levels of C99 to the same extent. Using pulse-chase experiments, we established that this results from an increased half-life of C99 in cells expressing PS1. In Chinese hamster ovary cells producing C99 from full-length human APP, similar results were observed. Finally, we show that a functional inhibitor of gamma-secretase does not alter the ability of PS1 to increase the intracellular levels of C99. This finding suggests that the binding of PS1 to C99 does not necessarily lead to its immediate cleavage by gamma-secretase, which could be a spatio-temporally regulated or an induced event, and provides biochemical evidence for the existence of a substrate-docking site on PS1

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells.

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase