Katleen Craessaerts

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.

Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.

Point mutations in the presenilin-1 gene (PS1) are a major cause of familial Alzheimers disease. They result in a selective increase in the production of the amyloidogenic peptide amyloid-β(1–42) by proteolytic processing of the amyloid precursor protein (APP). Here we investigate whether PS1 is also involved in normal APP processing in neuronal cultures derived from PS1-deficient mouse embryos. Cleavage by α- and β-secretase of the extracellular domain of APP was not affected by the absence of PS1, whereas cleavage by γ-secretase of the transmembrane domain of APP was prevented, causing carboxyl-terminal fragments of APP to accumulate and a fivefold drop in the production of amyloid peptide. Pulse-chase experiments indicated that PS1 deficiency specifically decreased the turnover of the membrane-associated fragments of APP. As in the regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, PS1 appears to facilitate a proteolytic activity that cleaves the integral membrane domain of APP. Our results indicate that mutations in PS1 that manifest clinically cause a gain of function and that inhibition of PS1 activity is a potential target for anti-amyloidogenic therapy in Alzheimers disease.

Mitochondrial Rhomboid PARL Regulates Cytochrome c Release during Apoptosis via OPA1-Dependent Cristae Remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.

Neuronal membrane cholesterol loss enhances amyloid peptide generation

Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimers disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its β-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1–APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

Phosphorylation, Subcellular Localization, and Membrane Orientation of the Alzheimer's Disease-associated Presenilins

Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Golgi apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37°C instead of 95°C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 → Glu or Cys-410 → Tyr that cause Alzheimers disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.

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.

Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

γ‐Secretase‐dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase‐3β, Neprilysin and APP, we found no consistent effects of γ‐secretase inhibitors or of genetic deficiencies in the γ‐secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD‐binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Aβ generation in Alzheimers disease will not interfere significantly with the function of these genes.

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.

γ-secretase heterogeneity in the Aph1 subunit: relevance for Alzheimer’s Disease

Tactical Target Intramembrane proteolysis by the γ-secretase complex is important during development and in the pathology of Alzheimers disease. γ-Secretase has usually been considered as a homogeneous activity. Serneels et al. (p. 639, published online 19 March; see the Perspective by Golde and Kukar) now show that the Aph1B component of the γ-secretase complex is responsible for the generation of long β-amyloid species involved in Alzheimers disease. In a mouse model of Alzheimers disease, full knockout of Aph1B improved disease phenotypes, without the sort of toxicity previously observed when targeting γ-secretase more generally. Targeted knockout of only part of the γ-secretase complex lessens toxicity and still improves disease phenotypes. The γ-secretase complex plays a role in Alzheimer’s disease and cancer progression. The development of clinically useful inhibitors, however, is complicated by the role of the γ-secretase complex in regulated intramembrane proteolysis of Notch and other essential proteins. Different γ-secretase complexes containing different Presenilin or Aph1 protein subunits are present in various tissues. Here we show that these complexes have heterogeneous biochemical and physiological properties. Specific inactivation of the Aph1B γ-secretase in a mouse Alzheimer’s disease model led to improvements of Alzheimer’s disease–relevant phenotypic features without any Notch-related side effects. The Aph1B complex contributes to total γ-secretase activity in the human brain, and thus specific targeting of Aph1B-containing γ-secretase complexes may help generate less toxic therapies for Alzheimer’s disease.

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.