Presenilins as therapeutic targets for the treatment of Alzheimer's disease

Studies demonstrating that accumulation and aggregation of the amyloid beta protein (Abeta) within the brain is likely to cause Alzheimer's disease (AD) have provided the rationale for therapeutic strategies aimed at influencing Abeta production, aggregation and clearance. gamma-secretase catalyzes the final cleavage that releases the Abeta from its precursor; therefore, it is a potential therapeutic target for the treatment of AD. Recent data show that the polytopic membrane proteins presenilin 1 and presenilin 2 are either catalytic components or essential co-factors of a membrane-bound proteolytic complex that possesses gamma-secretase activity. Although recent findings demonstrating that gamma-secretase inhibitors bind directly to presenilins (PSs) further support a catalytic role for PSs in gamma-secretase cleavage, additional studies are still needed to clarify the role of PSs in gamma-secretase cleavage and the use of targeting PSs to reduce Abeta production.     

APH1, PEN2, and Nicastrin increase Abeta levels and gamma-secretase activity

A major component of the amyloid plaque core in Alzheimer's disease (AD) is the 40-42-residue amyloid beta peptide (Abeta). Mutations linked to AD such as those in presenilins 1 (PS1) and 2 (PS2) invariably increase the longer Abeta42 species that forms neurotoxic oligomers. It is believed that PS1/2 constitute the catalytic subunit of the gamma-secretase responsible for the final step in Abeta biogenesis. Recent genetic studies have identified a number of additional genes encoding APH1a, APH1b, PEN2, and Nicastrin proteins, which are part of the gamma-secretase complex with PS1. Further, knockout studies using RNAi showed that these components are essential for gamma-secretase activity. However, the nature of gamma-secretase and how the aforementioned proteins regulate its activity are still incompletely understood. Here we present evidence that unlike PS1, overexpression of these proteins can increase the levels of Abeta, suggesting that these proteins are limiting for gamma-secretase activity. In addition, our studies also suggest that the presenilin partners regulate the relative levels of Abeta40 and Abeta42.     

Bcl-2 family: life-or-death switch

Proteolytic cleavage of the amyloid protein from the amyloid protein precursor (APP) by APP secretases is a key event in Alzheimer's disease (AD) pathogenesis. alpha-Secretases cleave APP within the amyloid sequences, whereas beta- and gamma-secretases cleave on the N- and C-terminal ends respectively. The transmembrane aspartyl protease BACE has been identified as beta-secretase and several proteases (ADAM-10, TACE, PC7) may be alpha-secretases. A number of studies have suggested that presenilins could be gamma-secretases, although this remains to be demonstrated conclusively. Inhibition of beta- and gamma-secretase, or stimulation of alpha-secretase, is a rational strategy for therapeutic intervention in AD.     

L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gamma-secretase activity

Progressive cerebral amyloid beta-protein (A beta) deposition is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). Elevated levels of A beta(42) peptide formation have been linked to early-onset familial AD-causing gene mutations in the amyloid beta-protein precursor (A beta PP) and the presenilins. Sequential cleavage of A beta PP by the beta- and gamma-secretases generates the N- and C-termini of the A beta peptide, making both the beta- and gamma-secretase enzymes potential therapeutic targets for AD. The identity of the A beta PP gamma-secretase and the mechanism by which the C-termini of A beta are formed remain uncertain, although it has been suggested that the presenilins themselves are novel intramembrane-cleaving gamma-secretases of the aspartyl protease class [Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517]. In this study we report the identification of L-685,458 as a structurally novel inhibitor of A beta PP gamma-secretase activity, with a similar potency for inhibition of A beta(42) and A beta(40) peptides. This compound contains an hydroxyethylene dipeptide isostere which suggests that it could function as a transition state analogue mimic of an aspartyl protease. The preferred stereochemistry of the hydroxyethylene dipeptide isostere was found to be the opposite to that required for inhibition of the HIV-1 aspartyl protease, a factor which may contribute to the observed specificity of this compound. Specific and potent inhibitors of A beta PP gamma-secretase activity such as L-685,458 will enable important advances toward the identification and elucidation of the mechanism of action of this enigmatic protease.     

Are presenilins intramembrane-cleaving proteases? Implications for the molecular mechanism of Alzheimer's disease.

The amyloid-beta protein (Abeta) is strongly implicated in the pathogenesis of Alzheimer's disease. The final step in the production of Abeta from the amyloid precursor protein (APP) is proteolysis by the unidentified gamma-secretases. This cleavage event is unusual in that it apparently occurs within the transmembrane region of the substrate. Studies with substrate-based inhibitors together with molecular modeling and mutagenesis of the gamma-secretase cleavage site of APP suggest that gamma-secretases are aspartyl proteases that catalyze a novel intramembranous proteolysis. This proteolysis requires the presenilins, proteins with eight transmembrane domains that are mutated in most cases of autosomal dominant familial Alzheimer's disease. Two conserved transmembrane aspartates in presenilins are essential for gamma-secretase activity, suggesting that presenilins themselves are gamma-secretases. Moreover, presenilins also mediate the apparently intramembranous cleavage of the Notch receptor, an event critical for Notch signaling and embryonic development. Thus, if presenilins are gamma-secretases, then they are also likely the proteases that cleave Notch within its transmembrane domain. Another protease, S2P, involved in the processing of the sterol regulatory element binding protein, is also a multipass integral membrane protein which cleaves within or very close to the transmembrane region of its substrate. Thus, presenilins and S2P appear to be members of a new type of polytopic protease with an intramembranous active site.     

Presenilin-directed inhibitors of gamma-secretase trigger caspase 3 activation in presenilin-expressing and presenilin-deficient cells

The amyloid beta peptide (Abeta) is generated by subsequent cleavages by beta- and gamma-secretases. Therefore, these two enzymes are putative therapeutic targets to prevent Abeta production, and hopefully to slow down or even stop the Alzheimer's disease (AD) neurodegenerative process. Several studies have revealed that gamma-secretase hydrolyses other important substrates besides beta-amyloid precursor protein (betaAPP) thus adding another level of complexity to designing fully AD-specific interfering drugs. Here we demonstrate that three distinct presenilin-directed gamma-secretase inhibitors as well as JLK compounds indirectly potentiate caspase 3 activity, the effector caspase of the apoptotic cascade. Thus, inhibitors were shown to drastically stimulate caspase 3 activity in wild-type mice blastocyst-derived and fibroblast cells. Interestingly, some of these inhibitors known to interact with presenilins also trigger caspase activation in presenilin-deficient cells. However, inhibitors do not affect recombinant caspase 3 activity, indicating that the effect on this enzyme was indirect. Furthermore, we established that caspase 3 activation was not due to an effect of gamma-secretase inhibitors on calpains, a family of proteolytic enzymes able to modulate caspase 3 activity. Altogether, our data demonstrate that presenilin-directed gamma-secretase inhibitors affect caspase 3 activity in a presenilin-independent manner. Therefore, as presenilin-dependent gamma-secretase activity is not specific for betaAPP and because its inhibitors clearly affect other vital cell functions, care should be taken in considering 'gamma-secretase' inhibitors as putative therapeutic tools to interfere with AD pathology.     

Random mutagenesis of presenilin-1 identifies novel mutants exclusively generating long amyloid beta-peptides

Familial Alzheimer disease-causing mutations in the presenilins increase production of longer pathogenic amyloid beta-peptides (A beta(42/43)) by altering gamma-secretase activity. The mechanism underlying this effect remains unknown, although it has been proposed that heteromeric macromolecular complexes containing presenilins mediate gamma-secretase cleavage of the amyloid beta-precursor protein. Using a random mutagenesis screen of presenilin-1 (PS1) for PS1 endoproteolysis-impairing mutations, we identified five unique mutants, including R278I-PS1 and L435H-PS1, that exclusively generated a high level of A beta43, but did not support physiological PS1 endoproteolysis or A beta40 generation. These mutants did not measurably alter the molecular size or subcellular localization of PS1 complexes. Pharmacological studies indicated that the up-regulation of activity for A beta43 generation by these mutations was not further enhanced by the difluoroketone inhibitor DFK167 and was refractory to inhibition by sulindac sulfide. These results suggest that PS1 mutations can lead to a wide spectrum of changes in the activity and specificity of gamma-secretase and that the effects of PS1 mutations and gamma-secretase inhibitors on the specificity are mediated through a common mechanism.     

Localization of presenilin-nicastrin complexes and gamma-secretase activity to the trans-Golgi network

Abundant biochemical and genetic evidence suggests that presenilins are catalytic components of gamma-secretase, the protease responsible for generating the Alzheimer amyloid beta-protein. However, the differential localization of presenilins to early secretory compartments and gamma-secretase substrates to late secretory compartments and the plasma membrane (the "spatial paradox") argues against this view. We investigated this issue by studying the localization of nicastrin, another putative gamma-secretase component, and its association with presenilin-1 into proteolytically active complexes. Glycosidase digests revealed that nicastrin exists in multiple glycoforms and is terminally sialylated, a modification often associated with the trans-Golgi network. Trafficking of nicastrin to the trans-Golgi network was confirmed by density gradient fractionation and immunofluorescence microscopy. In presenilin-deficient cells, however, nicastrin trafficking and maturation were abnormal, as the protein was restricted to early secretory compartments and failed to be sialylated. Mature sialylated nicastrin in trans-Golgi network fractions was complexed quantitatively with N- and C-terminal fragments of presenilin-1, whereas immature nicastrin present in early secretory compartments was not. Additionally, trans-Golgi network fractions contained the gamma-secretase substrate beta-amyloid precursor protein C83 and were enriched in presenilin-dependent gamma-secretase proteolytic activity. The results resolve the apparent spatial paradox by demonstrating that presenilin-nicastrin complexes and presenilin-dependent gamma-secretase activity are co-localized to a late secretory compartment. The findings provide further evidence that presenilin-containing complexes are the gamma-secretase, and indicate that presenilins also regulate gamma-secretase assembly.     

Is gamma-secretase a multienzyme complex for membrane protein degradation? Models and speculations

The beta- and gamma-secretases cleave the amyloid protein precursor (APP) to release the amyloid protein (Abeta). While the beta-secretase has now been identified, the gamma-secretase remains an enigma. A number of mutations in the presenilins (PS) and APP have been shown to alter the cleavage specificity of gamma-secretase. However, the relationship between PS and gamma-secretase remains unclear. This article presents some models of gamma-secretase and suggests that the simplest interpretation of current data is that gamma-secretase is a complex of several proteases located in the lumen of secretory vesicles.     

Characterization of a presenilin-mediated amyloid precursor protein carboxyl-terminal fragment gamma. Evidence for distinct mechanisms involved in gamma -secretase processing of the APP and Notch1 transmembrane domains

A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, gamma-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursor protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxyl-terminal fragment gamma (CTFgamma) derivatives are dependent on PS expression and inhibited by a highly selective and potent gamma-secretase inhibitor. Unexpectedly, the APP-CTFgamma derivative is generated by processing between Leu-645 and Val-646 (of APP(695)), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid beta protein peptides. Although the relationship of APP-CTFgamma to the production of amyloid beta protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent gamma-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP transmembrane domain.     

Gamma-secretase inhibition

Development of Alzheimer's disease (AD) pathology appears to be causally related to age-dependent changes in the metabolism of the amyloid-beta peptide (A beta), leading to its enhanced aggregation and deposition. gamma-Secretase is a crucial enzyme for the generation of A beta from the amyloid-beta precursor protein and thus represents a valid potential therapeutic target for the treatment or prevention of AD. Enzyme activity has been shown to be dependent on the expression of presenilins and the identification of inhibitors containing transition-state analogue mimics, together with mutagenesis and knockout studies, confirms that presenilins may provide at least a component of the catalytic site for this putative aspartyl protease. Considerable effort has been expended to identify compounds which specifically reduce gamma-secretase activity in the central nervous system, and those with the appropriate properties are being utilized in on-going proof-of-concept studies in animals and humans, to determine the extent and duration of gamma-secretase inhibition required to elicit therapeutic benefits. gamma-Secretase-mediated substrate cleavage appears to fall into the category of 'regulated intramembrane proteolysis'. By virtue of its mechanistic similarities, the effects of gamma-secretase inhibitors on proteolysis and signalling through other substrates, such as Notch, has to be determined carefully, since this is likely to impact on the clinically safe dose of these compounds.     

Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."     

Active gamma-secretase is localized to detergent-resistant membranes in human brain

Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.     

The transmembrane aspartates in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid beta-protein generation

The discovery that a deficiency of presenilin 1 (PS1) decreases the production of amyloid beta-protein (Abeta) identified the presenilins as important mediators of the gamma-secretase cleavage of beta-amyloid precursor protein (APP). Recently, we found that two conserved transmembrane (TM) aspartates in PS1 are critical for Abeta production, providing evidence that PS1 either functions as a required diaspartyl cofactor for gamma-secretase or is itself gamma-secretase. Presenilin 2 (PS2) shares substantial sequence and possibly functional homology with PS1. Here, we show that the two TM aspartates in PS2 are also critical for gamma-secretase activity, providing further evidence that PS2 is functionally homologous to PS1. Cells stably co-expressing TM Asp --> Ala mutations in both PS1 and PS2 show further accumulation of the APP-derived gamma-secretase substrates, C83 and C99. The production of Abeta is reduced to undetectable levels in the conditioned media of these cells. Furthermore, endoproteolysis of the exogenous Asp mutant PS2 is absent, and endogenous PS1 C-terminal fragments are diminished to undetectable levels. Therefore, the co-expression of PS1 and PS2 TM Asp --> Ala mutants suppresses the formation of any detectable PS1 or PS2 heterodimeric fragments and essentially abolishes the production of Abeta. These results explain the residual Abeta production seen in PS1-deficient cells and demonstrate the absolute requirement of functional presenilins for Abeta generation. We conclude that presenilins, and their TM aspartates in particular, are attractive targets for lowering Abeta therapeutically to prevent Alzheimer's disease.     

Peptide-based,irreversible inhibitors of gamma-secretase activity

The characterization of the enzymes responsible for amyloid beta-peptide (Abeta) production is considered to be a primary goal towards the development of future therapeutics for the treatment of Alzheimer's disease. Inhibitors of gamma-secretase activity were critical in demonstrating that the presenilins (PSs) likely comprised at least part of the active site of the gamma-secretase enzyme complex, with two highly conserved membrane aspartates presumably acting as catalytic residues. However, whether or not these aspartates are actually the catalytic residues of the enzyme complex or are merely essential for normal PS function and/or maturation is still unknown. In this paper, we report the development of reactive inhibitors of gamma-secretase activity that are functionally irreversible. Since such inhibitors have been shown to bind catalytic residues in other aspartyl proteases (e.g., HIV protease), they might be used to determine if the transmembrane aspartates of PSs are involved directly in substrate cleavage.     

Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1

The beta-amyloid precursor protein (beta-APP), which is involved in the pathogenesis of Alzheimer's disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown gamma-secretases. Here we show that an affinity reagent designed to interact with the active site of gamma-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer's and are known mediators of gamma-secretase cleavage of both beta-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of gamma-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer's disease.     

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.