Wim Annaert

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.

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.