Philippe Cupers

A presenilin-1-dependent |[gamma]|-secretase-like protease mediates release of Notch intracellular domain

Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of γ-secretase-mediated cleavage of β-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimers disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by γ-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several γ-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of γ-secretase for the treatment of Alzheimers disease may risk toxicity caused by reduced Notch signalling.

The amyloid precursor protein (APP)-cytoplasmic fragment generated by gamma-secretase is rapidly degraded but distributes partially in a nuclear fraction of neurones in culture.

The γ‐secretase cleavage is the last step in the generation of the β‐amyloid peptide (Aβ) from the amyloid precursor protein (APP). The Aβ precipitates in the amyloid plaques in the brain of Alzheimers disease patients. The fate of the intracellular APP carboxy‐terminal stub generated together with Aβ has been, in contrast, only poorly documented. The analogies between the processing of APP and other transmembrane proteins like SREBP and Notch suggests that this intracellular fragment could have important signalling functions. We demonstrate here that APP‐C59 is rapidly degraded (half‐life ∼5 min) when overexpressed in baby hamster kidney cells or primary cultures of neurones by a mechanism that is not inhibited by endosomal/lysosomal or proteasome inhibitors. Furthermore, APP‐C59 binds to the DNA binding protein Fe65, although this does not increase the half‐life of APP‐C59. Finally, we demonstrate that a fraction of APP‐C59 becomes redistributed to the nuclear detergent‐insoluble pellet, in which the transcription factor SP1 is also present. Overall our results reinforce the analogy between Notch and APP processing, and suggest that the APP intracellular domain, like the Notch intracellular domain, could have a role in signalling events from the plasma membrane to the nucleus.

Interaction with telencephalin and the amyloid precursor protein predicts a ring structure for presenilins

The carboxyl terminus of presenilin 1 and 2 (PS1 and PS2) binds to the neuron-specific cell adhesion molecule telencephalin (TLN) in the brain. PS1 deficiency results in the abnormal accumulation of TLN in a yet unidentified intracellular compartment. The first transmembrane domain and carboxyl terminus of PS1 form a binding pocket with the transmembrane domain of TLN. Remarkably, APP binds to the same regions via part of its transmembrane domain encompassing the critical residues mutated in familial Alzheimers disease. Our data surprisingly indicate a spatial dissociation between the binding site and the proposed catalytic site near the critical aspartates in PSs. They provide important experimental evidence to support a ring structure model for PS.

The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein.

We investigated the relationship between PS1 and γ-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent γ-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no γ-secretase processing was observed when holo-APP or APP-C99, a direct substrate for γ-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent γ-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that γ-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the γ-cleavage of APP-C99. In agreement, we found that intracellular γ-secretase processing of APP-C99-KK both at the γ40 and the γ42 site could be restored partially after brefeldin A treatment. Our data confirm the “spatial paradox” and raise several questions regarding the PS1 is γ-secretase hypothesis.

Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells.

Urea-based β-amyloid (Aβ) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of Aβ peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid. A comparable and highly conserved pattern of Aβ peptides, namely, 1–40/42 and carboxyl-terminal-truncated 1–37, 1–38, and 1–39, was found. Besides Aβ1–42, we also observed a consistent elevation of amino-terminal-truncated Aβ2–42 in a detergent-soluble pool in brains of subjects with Alzheimers disease. Aβ2–42 was also specifically elevated in cerebrospinal fluid samples of Alzheimers disease patients. To decipher the contribution of potential different γ-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminal-truncated Aβ peptides, we overexpressed β-amyloid precursor protein (APP)-trafficking mutants in PS1+/+ and PS1−/− neurons. As compared with APP-WT (primary neurons from control or PS1-deficient mice infected with Semliki Forest virus), PS1−/− neurons and PS1+/+ neurons overexpressing APP-Δct (a slow-internalizing mutant) show a decrease of all secreted Aβ peptide species, as expected, because this mutant is processed mainly by α-secretase. This drop is even more pronounced for the APP-KK construct (APP mutant carrying an endoplasmic reticulum retention motif). Surprisingly, Aβ2–42 is significantly less affected in PS1−/− neurons and in neurons transfected with the endocytosis-deficient APP-Δct construct. Our data confirm that PS1 is closely involved in the production of Aβ1–40/42 and the carboxyl-terminal-truncated Aβ1–37, Aβ1–38, and Aβ1–39, but the amino-terminal-truncated and carboxyl-terminal-elongated Aβ2–42 seems to be less affected by PS1 deficiency. Moreover, our results indicate that the latter Aβ peptide species could be generated by a βAsp/Ala-secretase activity.

Presenilin function in APP processing.

Abstract: Familial Alzheimers disease (FAD) is now linked to at least three genes encoding the amyloid precursor protein (APP) on chromosome 21, and presenilin 1 and 2 on chromosome 14 and 1, respectively. FAD cases in whom presenilin mutations occur are more frequent than those with APP mutations. However, altogether they only account for approximately 0.1% of all the people suffering from Alzheimers disease (AD), 1 and the causes of the remaining 99.9% of the sporadic form of AD or senile dementia remain unknown. Since FAD presents with the same neuropathological features as sporadic AD, i.e., cognitive impairments and the amyloid plaques and tangles in the brain, our working hypothesis is that similar molecular pathogenic mechanisms underly both sporadic and familial AD. It follows that APP and the presenilins must be key players in the disease. Detailed knowledge about the cell biology of these proteins will be a rich source of insight into the pathology of AD, but will also shed light on the fundamental neurobiology of these proteins.

The presenilins as potential drug targets in Alzheimer’s disease

Alzheimer’s disease (AD) is a major neurodegenerative disorder affecting a large proportion of the elderly. Given the increasing life expectancy in developed countries, the generation of new drugs to treat AD represents one of the major challenges for the pharmaceutical industry in the coming decades. Central to the disease is the abnormal processing of amyloid precursor protein (APP) resulting in the release of the β-amyloid peptide (Aβ), the major constituent of plaques in the brains of AD patients. Mis-sense mutations causing AD have been found in the APP gene, and more recently in the presenilin genes. Remarkably, all mutations investigated cause an increased production of a particular form of the Aβ that is more prone to precipitation. Presenilins, which are multi-membrane spanning proteins, appear to specifically regulate the production of Aβ by controlling the intramembranous proteolytic cleavage of APP. Inhibition of presenilin is therefore an obvious drug target, although side-effects caused by i...

Missorting of the Dendritic Cell Adhesion Molecule Telencephalin in Presenilin-Deficient Neurons

Presenilin deficiency abrogates γ-secretase cleavage, thereby preventing the release of the amyloid peptide from the β-clipped APP fragment or the notch intracellular domain. The presenilins are therefore implicated in such diverse processes as the neurodegeneration in Alzheimer’s disease and the regulation of Notch signaling. The question remains whether other type I transmembrane proteins or pathways are subject to presenilin-mediated proteolysis. While the two aspartate residues in presenilin likely are essential for a functional γ-secretase complex, the highly conserved carboxyterminal tail also appears to be important for presenilin function. Therefore we screened a double hybrid library with a short presenilin 1 carboxyterminal fragment as a bait. We identified a type I transmembrane protein that belongs to the family of intercellular cell adhesion molecules, telencephalin. Telencephalin is exclusively expressed in neurons of the telencephalic region, where it is abundantly expressed at the somatodendritic plasmamembrane. Functionally, telencephalin is, like notch, involved in neurite outgrowth. Interestingly, telencephalin is also involved in long-term potentiation. We confirmed the presenilin 1/telencephalin interaction using two other independent approaches. Finally, functional evidence for the physiological relevance of this interaction comes from the observation that telencephalin becomes mislocalized in differentiated hippocampal neurons. Further research into the telencephalin and presenilin 1 interaction may shed some light on the way presenilin 1 influences the processing of other type I transmembrane proteins, such as APP and Notch.

The Putative Role of Presenilins in the Transmembrane Domain Cleavage of Amyloid Precursor Protein and Other Integral Membrane Proteins

Missense mutations in the presenilin (PS)-1 and -2 genes are major causes of familial Alzheimer’s disease (AD), acting apparently in a dominant fashion (Levy-Lahad et al. 1995; Rogaev et al. 1995; Sherrington et al. 1995). Although the exact pathogenic mechanism underlying the disease process remains to be further elucidated, it is fairly established that almost all PS missense mutations affect the processing of the amyloid precursor protein (APP). The result is an increased secretion of the longer form of the amyloid peptide (Borchelt et al. 1996; Duff et al. 1996; Lemere et al. 1996; Tomita et al. 1997; Xia et al. 1997). This peptide constitutes the major component of the amyloid plaques in patients. Interestingly several of the PS mutations appear also to enhance the sensitivity of cells, and in particular neurons, to apoptotic stimuli (Deng et al. 1996; Guo et al. 1997; Janicki and Monteiro 1997; Vito et al. 1997; Wolozin et al. 1996). In principle both mechanisms could contribute to the pathogenesis of AD (Zhang et al. 1998). Insight into the biological functions of the PS in the cell is probably not only important for understanding the pathogenesis of the familial form of AD, but also of the sporadic form of AD. Interestingly, recent findings suggest that PS is involved in the proteolysis of the transmembrane domains of APP, Notch, APLP-1, and possibly Ire-1, and could therefore function as a molecular switch linking proteolysis to intracullular signaling (Annaert and De Strooper 1999).