Characterization of the reconstituted gamma-secretase complex from Sf9 cells co-expressing presenilin 1, nicastrin [correction of nacastrin], aph-1a, and pen-2

Gamma-secretase catalyzes the proteolytic processing of a number of integral membrane proteins, including amyloid precursor protein (APP) and Notch. The native gamma-secretase is a heterogeneous population of large membrane protein complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b, nicastrin, and pen-2. Here we report the reconstitution of a gamma-secretase complex in Sf9 cells by co-infection with baculoviruses carrying the PS1, nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and the Notch-like substrate N160 and displays the characteristic features of gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor, upregulation of Abeta42 production by a familial Alzheimer's disease (FAD) mutation in the APP gene, and downregulation of Notch processing by PS1 FAD mutations. However, the ratio of Abeta42:Abeta40 production by the reconstituted gamma-secretase is significantly higher than that of the native enzyme from 293 cells. Unlike in mammalian cells where PS1 FAD mutations cause an increase in Abeta42 production, PS1 FAD missense mutations in the reconstitution system alter the cleavage sites in the C99 substrate without changing the Abeta42:Abeta40 ratio. In addition, PS1DeltaE9 is a loss-of-function mutation in both C99 and N160 processing. Reconstitution of gamma-secretase provides a homogeneous system for studying the individual gamma-secretase complexes and their roles in Abeta production, Notch processing and AD pathogenesis. These studies may provide important insight into the development of a new generation of selective gamma-secretase inhibitors with an improved side effect profile.     

Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease

Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.     

Reconstitution of gamma-secretase activity

gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.     

Presenilin-1-mediated retention of APP derivatives in early biosynthetic compartments

Processing of the amyloid precursor protein (APP) leads to the production of amyloid-beta (Abeta), the major component of extracellular plaques in the brains of Alzheimer's disease (AD) patients. Presenilin-1 (PS-1) plays a key role in the final step of Abeta formation, the gamma-secretase cleavage. Previously, we showed that PS-1 is retained in pre-Golgi compartments by incorporation into COPI-coated membranes of the vesicular tubular clusters (VTCs) between endoplasmic reticulum (ER) and Golgi complex. Here, we show that PS-1 also mediates the retention of the beta-cleavage-derived APP-C-terminal fragment (CTFbeta) and/or Abeta in pre-Golgi membranes. Overexpression of PS-1 increased the percentage of CTFbeta and/or Abeta in VTCs as well as their distribution to COPI-coated VTC membranes. By contrast, overexpression of the dominant-negative aspartate mutant PS-1(D257A) or PS-knockout decreased incorporation of these APP derivatives into COPI-coated membranes. Sorting of APP derivatives to COPI-coated VTC membranes was not depending on the APP cytosolic tail. In post-Golgi compartments, PS-1 expression enhanced the association of full-length APP/APPs with endosomal compartments at the expense of plasma membrane-bound APP. We conclude that PS-1, in addition to its role in gamma-secretase cleavage, is also required for the subcellular routing of APP and its derivatives. Malfunctioning of PS-1 in this role may have important consequences for the progress of AD.     

Retention of the Alzheimer's amyloid precursor fragment C99 in the endoplasmic reticulum prevents formation of amyloid beta-peptide

gamma-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's beta-amyloid precursor protein (APP), resulting in the release of amyloid beta-peptide (Abeta). The molecular identity of gamma-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that gamma-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of Abeta. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the Abeta(42)/Abeta(40) ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of gamma-secretase cleavage. Biogenesis of both Abeta(40) and Abeta(42) was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for gamma-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as gamma-secretase. The results suggest that presenilins may acquire the characteristics of gamma-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.     

Ubiquitin-proteasome pathway mediates degradation of APH-1

Gamma-secretase catalyzes intramembraneous proteolysis of several type I transmembrane proteins, including beta-amyloid precursor protein (APP), to generate amyloid beta protein (Abeta), a key player in the pathogenesis of Alzheimer's disease (AD). The critical components of the gamma-secretase complex include presenilin (PS), nicastrin (NCT), presenilin enhancer-2 (PEN-2) and anterior pharynx defective-1 (APH-1). Abnormalities of the ubiquitin-proteasome pathway have been implicated in the pathogenesis of AD; while PS and PEN-2 turnover is regulated by this pathway, it is unknown whether the ubiquitin-proteasome pathway is also involved in the degradation of APH-1 protein. In this study, we found that the expression of endogenous and exogenous APH-1 significantly increased in cells treated with proteasome-specific inhibitors. The effect of the proteasome inhibitors on APH-1 was dose- and time-dependent. APH-1 protein was ubiquitinated. Pulse-chase metabolic labeling experiments showed that the degradation of newly synthesized radiolabeled APH-1 proteins was inhibited by lactacystin. Disruption of the PS1 and PS2 genes did not affect the degradation of APH-1 by the ubiquitin-proteasome pathway. Furthermore, over-expression of APH-1 and inhibition of proteasomal APH-1 degradation facilitated gamma-secretase cleavage of APP to generate Abeta. These results demonstrate that the degradation of APH-1 protein is mediated by the ubiquitin-proteasome pathway.     

{gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE

Mutation of the amyloid precursor protein (APP), presenilin-1, or presenilin-2 results in the development of early onset autosomal dominant forms of Alzheimer disease (AD). These mutations lead to an increased Abeta42/Abeta40 ratio that correlates with the onset of disease. However, it remains unknown how these mutations affect gamma-secretase, a protease that generates the termini of Abeta40 and Abeta42. Here we have determined the reaction mechanism of gamma-secretase with wild type and three mutated APP substrates. Our findings indicate that despite the overall outcome of an increased Abeta42/Abeta40 ratio, these mutations each display rather distinct reactivity to gamma-secretase. Intriguingly, we found that the ratio of Abeta42/Abeta40 is variable with substrate concentration; increased substrate concentrations result in higher ratios of Abeta42/Abeta40. Moreover, we demonstrated that reduction of gamma-secretase substrate concentration by BACE1 inhibition in cells decreased the Abeta42/Abeta40 ratio. This study indicates that biological factors affecting targets such as BACE1 and APP, which ultimately cause an increased concentration of gamma-secretase substrate, can augment the Abeta42/Abeta40 ratio and may play a causative role in sporadic AD. Therefore, strategies lowering the Abeta42/Abeta40 ratio through partial reduction of gamma-secretase substrate production may introduce a practical therapeutic modality for treatment of AD.     

BACE1 interacts with nicastrin

Beta-amyloid peptide (Abeta) is generated through the proteolytic cleavage of beta-amyloid precursor protein (APP) by beta- and gamma-secretases. The beta-secretase, BACE1, initiates Abeta formation followed by gamma-cleavage within the APP transmembrane domain. Although BACE1 localizes in the transGolgi network (TGN), its physiological substrates and modulators are not known. In addition, the relationship to other secretase(s) also remains unidentified. Here, we demonstrate that BACE1 binds to nicastrin, a component of gamma-secretase complexes, in vitro, and that nicastrin activates beta-secretase activity in COS-7 cells.     

Cellular localization of Nicastrin affects amyloid beta species production

The gamma-secretase complex, composed by presenilin, nicastrin, APH-1 and PEN-2, is involved in intramembranous proteolysis of membrane proteins, such as amyloid precursor protein or Notch. Cleavage occurs in multiple cellular compartments. Here, nicastrin mutants containing targeting signals to the endoplasmic reticulum, trans-Golgi network, lysosomes, or plasma membrane have been shown to yield active gamma-secretase complexes with different activities and specificities: wild-type and plasma membrane nicastrin complexes yielded the highest amounts of secreted amyloid-beta peptide (Abeta), predominantly Abeta40, whereas intracellular targeted mutants produced intracellular Abeta, with a comparatively higher amount of Abeta42. These results suggest that compartmental microenvironments play a role in gamma-secretase activity and specificity.     

Partial purification and characterization of gamma-secretase from post-mortem human brain

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.     

gamma-Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site

gamma-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified gamma-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Abeta, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified gamma-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified gamma-secretase preparations; the labeling was blocked by ATP itself and APP-selective gamma-secretase inhibitors. We concluded that a nucleotide-binding site exists within gamma-secretase, and certain compounds that bind to this site can specifically modulate the generation of Abeta while sparing Notch. Drugs targeting the gamma-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.     

Purification and characterization of the human gamma-secretase complex

Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.     

Mammalian APH-1 interacts with presenilin and nicastrin and is required for intramembrane proteolysis of amyloid-beta precursor protein and Notch

Presenilin and nicastrin are essential components of the gamma-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-beta precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, we now show that mammalian APH-1 (mAPH-1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin amino- and carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of gamma-secretase substrates (APP carboxyl-terminal fragments), and reduction of gamma-secretase products (amyloid-beta peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the gamma-secretase complex required for the intramembrane proteolysis of APP and Notch.     

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.     

Presenilin 1 is required for maturation and cell surface accumulation of nicastrin

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.     

aph-1 and pen-2 are required for Notch pathway signaling,gamma-secretase cleavage of betaAPP,and presenilin protein accumulation

Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.     

Nicastrin functions as a gamma-secretase-substrate receptor

gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.     

gamma-Secretase, evidence for multiple proteolytic activities and influence of membrane positioning of substrate on generation of amyloid beta peptides of varying length

gamma-Secretase activity is the final cleavage event that releases the amyloid beta peptide (Abeta) from the beta-secretase cleaved carboxyl-terminal fragment of the amyloid beta protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of gamma-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing Abeta production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. Abeta production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in gamma-secretase cleavage in the presence or absence of pepstatin, an inhibitor of gamma-secretase activity. These studies demonstrate that gamma-secretase's cleavage specificity is primarily determined by location of the gamma-secretase cleavage site of APP with respect to the membrane, and that gamma-secretase activity is due to the action of multiple proteases exhibiting both a pepstatin- sensitive activity and a pepstatin-insensitive activity. Given that gamma-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of Abeta production that will direct efforts to isolate the gamma-secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant gamma-secretase activities.