The amyloid precursor protein: beyond amyloid

Mutations in PSEN1 and PSEN2 genes account for the majority of cases of early-onset familial Alzheimer disease. Since the first prediction of a genetic link between PSEN1 and PSEN2 with Alzheimer's disease, many research groups from both academia and pharmaceutical industry have sought to unravel how pathogenic mutations in PSEN cause presenile dementia. PSEN genes encode polytopic membrane proteins termed presenilins (PS1 and PS2), which function as the catalytic subunit of γ-secretase, an intramembrane protease that has a wide spectrum of type I membrane protein substrates. Sequential cleavage of amyloid precursor protein by BACE and γ-secretase releases highly fibrillogenic β-amyloid peptides, which accumulate in the brains of aged individuals and patients with Alzheimer's disease. Familial Alzheimer's disease-associated presenilin variants are thought to exert their pathogenic function by selectively elevating the levels of highly amyloidogenic Aβ42 peptides. In addition to Alzheimer's disease, several recent studies have linked PSEN1 to familiar frontotemporal dementia. Here, we review the biology of PS1, its role in γ-secretase activity, and discuss recent developments in the cell biology of PS1 with respect to Alzheimer's disease pathogenesis.     

Involvement of presenilin holoprotein upregulation in calcium dyshomeostasis of Alzheimer's disease

Mutations in presenilins (PS1 and PS2) account for the vast majority of early onset familial Alzheimer's disease cases. Beside the well investigated role of presenilins as the catalytic unit in γ‐secretase complex, their involvement in regulation of intracellular calcium homeostasis has recently come into more focus of Alzheimer's disease research. Here we report that the overexpression of PS1 full‐length holoprotein forms, in particular familial Alzheimer's disease‐causing forms of PS1, result in significantly attenuated calcium release from thapsigargin‐ and bradykinin‐sensitive stores. Interestingly, treatment of HEK293 cells with γ‐secretase inhibitors also leads to decreased amount of calcium release from endoplasmic reticulum (ER) accompanying elevated PS1 holoprotein levels. Similarly, the knockdown of PEN‐2 which is associated with deficient PS1 endoproteolysis and accumulation of its holoprotein form also leads to decreased ER calcium release. Notably, we detected enhanced PS1 holoprotein levels also in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Taken together, the conditions in which the amount of full length PS1 holoprotein is increased result in reduction of calcium release from ER. Based on these results, we propose that the disturbed ER calcium homeostasis mediated by the elevation of PS1 holoprotein levels may be a contributing factor to the pathogenesis of Alzheimer's disease.     

Mutagenesis mapping of the presenilin 1 calcium leak conductance pore

Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) proteins are a major cause of familial Alzheimer disease. Presenilins are proteins with nine transmembrane (TM) domains that function as catalytic subunits of the γ-secretase complex responsible for the cleavage of the amyloid precursor protein and other type I transmembrane proteins. The water-filled cavity within presenilin is necessary to mediate the intramembrane proteolysis reaction. Consistent with this idea, cysteine-scanning mutagenesis and NMR studies revealed a number of water-accessible residues within TM7 and TM9 of mouse PS1. In addition to γ-secretase function, presenilins also demonstrate a low conductance endoplasmic reticulum Ca(2+) leak function, and many familial Alzheimer disease presenilin mutations impair this function. To map the potential Ca(2+) conductance pore in PS1, we systematically evaluated endoplasmic reticulum Ca(2+) leak activity supported by a series of cysteine point mutants in TM6, TM7, and TM9 of mouse PS1. The results indicate that TM7 and TM9, but not TM6, could play an important role in forming the conductance pore of PS1. These results are consistent with previous cysteine-scanning mutagenesis and NMR analyses of PS1 and provide further support for our hypothesis that the hydrophilic catalytic cavity of presenilins may also constitute a Ca(2+) conductance pore.     

Presenilins: Role in calcium homeostasis

Mutations in presenilins are responsible for the vast majority of early-onset familial Alzheimer's disease cases. Full-length presenilin structure is composed of nine transmembrane domains which are localized on the endoplasmic reticulum membrane. Upon endoproteolytic cleavage, presenilins assemble into the γ-secretase multiprotein complex and subsequently get transported to the cell surface. There is a wealth of knowledge around the role of presenilins as the catalytic component of γ-secretase, their involvement in amyloid precursor protein processing and generation of neurotoxic β-amyloid species. However recent findings have revealed a wide range of γ-secretase-independent presenilin functions, including involvement in calcium homeostasis. Particularly, familial Alzheimer's disease presenilin mutations have been shown to interfere with the function of several molecular elements involved in endoplasmic reticulum calcium homeostasis. Presenilins modulate the activity of IP(3) and Ryanodine receptor channels, regulate SERCA pump function, affect capacitative calcium entry and function per se as endoplasmic reticulum calcium leak conductance pores.     

Protease digestion indicates that endogenous presenilin 1 is present in at least two physical forms

The membrane-bound protein complex γ-secretase is an intramembranous protease whose substrates are a number of type I transmembrane proteins including the β-amyloid precursor protein (APP). A presenilin molecule is thought to be the catalytic unit of γ-secretase and either of two presenilin homologues, PS1 or PS2, can play this role. Mutations in the presenilins, apparently leading to aberrant processing of APP, have been genetically linked to early-onset familial Alzheimer’s disease. To look for possible molecular heterogeneity in presenilin/γ-secretase we examined the ability of proteinase K (PK) to digest endogenously expressed presenilins in intact endoplasmic reticulum vesicles. We demonstrate the existence of two physically different forms of γ-secretase-associated PS1, one that is relatively PK-sensitive and one that is significantly more PK-resistant. A similarly PK-resistant form of PS2 was not observed. We speculate that the structural heterogeneity we observe may underlie, at least in part, previous observations indicating the physical and functional heterogeneity of γ-secretase. In particular, our results suggest that there are significant differences between γ-secretase complexes incorporating PS1 and PS2. This difference may underlie the more dominant role of PS1 in the generation of β-amyloid peptides and in familial Alzheimer’s disease.     

Role of presenilin 1 in structural plasticity of cortical dendritic spines in vivo

Mutations in presenilins are the major cause of familial Alzheimer's disease (FAD), leading to impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Presenilins are the catalytic subunits of γ-secretase, which itself is critically involved in the processing of amyloid precursor protein to release neurotoxic amyloid β (Aβ). Besides Aβ generation, there is growing evidence that presenilins play an essential role in the formation and maintenance of synapses. To further elucidate the effect of presenilin1 (PS1) on synapses, we performed longitudinal in vivo two-photon imaging of dendritic spines in the somatosensory cortex of transgenic mice over-expressing either human wild-type PS1 or the FAD-mutated variant A246E (FAD-PS1). Interestingly, the consequences of transgene expression were different in two subtypes of cortical dendrites. On apical layer 5 dendrites, we found an enhanced spine density in both mice over-expressing human wild-type presenilin1 and FAD-PS1, whereas on basal layer 3 dendrites only over-expression of FAD-PS1 increased the spine density. Time-lapse imaging revealed no differences in kinetically distinct classes of dendritic spines nor was the shape of spines affected. Although γ-secretase-dependent processing of synapse-relevant proteins seemed to be unaltered, higher expression levels of ryanodine receptors suggest a modified Ca(2+) homeostasis in PS1 over-expressing mice. However, the conditional depletion of PS1 in single cortical neurons had no observable impact on dendritic spines. In consequence, our results favor the view that PS1 influences dendritic spine plasticity in a gain-of-function but γ-secretase-independent manner.     

Presenilins: how much more than γ-secretase?!

AD (Alzheimer's disease) is a neurodegenerative disease characterized by a gradual loss of neurons and the accumulation of neurotoxic Aβ (amyloid β-peptide) and hyperphosphorylated tau. The discovery of mutations in three genes, PSEN1 (presenilin 1), PSEN2 (presenilin 2) and APP (amyloid precursor protein), in patients with FAD (familial AD) has made an important contribution towards an understanding of the disease aetiology; however, a complete molecular mechanism is still lacking. Both presenilins belong to the γ-secretase complex, and serve as the catalytic entity needed for the final cleavage of APP into Aβ. PSEN only functions within the γ-secretase complex through intra- and inter-molecular interactions with three other membrane components, including nicastrin, Aph-1 (anterior pharynx defective-1) and Pen-2 (PSEN enhancer-2). However, although the list of γ-secretase substrates is still expanding, other non-catalytic activities of presenilins are also increasing the complexity behind its molecular contribution towards AD. These γ-secretase-independent roles are so far mainly attributed to PSEN1, including the transport of membrane proteins, cell adhesion, ER (endoplasmic reticulum) Ca(2+) regulation and cell signalling. In the present minireview, we discuss the current understanding of the γ-secretase-independent roles of PSENs and their possible implications in respect of AD.     

γ-Secretase in biology and medicine

γ-Secretase is a membrane-embedded proteolytic complex composed of presenilin and three other subunits. The γ-secretase complex generates the amyloid β-peptide of Alzheimer's disease but also plays important roles in normal physiology, especially in signaling from the Notch receptor. How this hydrolytic enzyme works in a hydrophobic environment is largely unanswered, but mutagenesis and chemical probes have offered insight. γ-Secretase is an important therapeutic target, although mechanism-based toxicity presents a serious obstacle. Agents that lower amyloid β-peptide production while leaving important normal functions of γ-secretase intact are promising therapeutic leads. Inhibition of Notch signaling by γ-secretase inhibitors, which is undesirable for the prevention or treatment of Alzheimer's disease, may be beneficial for the treatment of a variety of cancers.     

Synthesis of a potent photoreactive acidic γ-secretase modulator for target identification in cells

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimer´s disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Compounds named γ-secretase modulators (GSMs) can shift the substrate cleavage specificity of γ-secretase toward the production of non-amyloidogenic, shorter Aβ fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of γ-secretase), supporting a mode of action involving binding to γ-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in γ-secretase proteolytic specificity should pave the way for the development of improved drugs against AD.     

Rac1 changes the substrate specificity of gamma-secretase between amyloid precursor protein and Notch1

Beta amyloid peptide is generated from amyloid precursor protein (APP) by proteolytic cleavage of β- and γ-secretases, and plays a critical role in the pathogenesis of Alzheimer’s disease. Since γ-secretase cleaves several proteins including APP and Notch in a number of cell types, it is important to understand the conditions determining γ-secretase substrate specificity. In the present study, inhibition of Rac1 attenuated γ-secretase activity for APP, resulting in decreased production of the APP intracellular domain but accumulated C-terminal fragments (APP-CTF). In contrast, Rac1 inhibitor, NSC23766 increased production of the Notch1 intracellular domain but slightly decreased the ectodomain-shed form of Notch1 (NotchΔE). To elucidate the mechanism underlying these observations, we performed co-immunoprecipitation experiments to analyze the interaction between Rac1 and presenilin1 (PS1), a component of the γ-secretase complex. Inhibition of Rac1 enhanced its interaction with PS1. Under the same condition, PS1 interacted more strongly with NotchΔE than with APP-CTF. Our results suggested that PS1 determines the preferred substrate for γ-secretase between APP and Notch1, depending on the activation status of Rac1.     

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production

In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.     

治疗阿尔茨海默病的早老素/γ-分泌酶抑制剂和调节剂

阿尔茨海默病（Alzheimer’s disease,AD）又称老年痴呆症,是一种中枢神经系统（central nervous system,CNS）退行性疾病。β-淀粉样蛋白（β-amyloid,Aβ42）被认为在阿尔茨海默病（AD）的发生、发展过程中起核心作用。Aβ42由APP经β-和γ-分泌酶相继切割产生。γ-分泌酶是一个蛋白酶复合体,早老素（presenilin,PS）是γ-分泌酶的催化组分。因此,抑制PS/γ分泌酶的活性是治疗AD的关键,因而PS/γ分泌酶也是治疗AD的主要靶点。根据这些理论,人们提出了一些治疗AD的新方法,其中PS/γ-分泌酶抑制剂和调节剂成为近年来人们关注的焦点。     

Association between Presenilin-1 and TRAF6 modulates regulated intramembrane proteolysis of the p75NTR neurotrophin receptor

The p75 neurotrophin receptor (p75^NTR) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners including the tumour necrosis factor receptor-associated factor 6 (TRAF6) E3 ubiquitin ligase to produce cellular responses. Recently, p75^NTR was also shown to undergo presenilin-dependent, γ-secretase-mediated regulated intramembrane proteolysis. In this study, we report the characterization of a highly conserved TRAF6-binding site (PxExxAr/Ac) in presenilin-1 (PS1) that mediates nerve growth factor (NGF)-induced association between PS1 and TRAF6. We demonstrate that disruption of this interaction between PS1 and TRAF6 inhibits TRAF6 autoubiquitination and γ-secretase cleavage of p75^NTR. Additionally, we show that PS1-deficiency antagonizes NGF-induced I-κB degradation. Finally, we also show that p75^NTR is a substrate for TRAF6-mediated ubiquitination and that TRAF6 E3 ligase activity is required for regulated intramembrane proteolysis of p75^NTR. In summary, our data suggest that an NGF-induced association between PS1 and TRAF6 influences regulated intramembrane proteolysis of p75^NTR.     

Toward the structure of presenilin/γ-secretase and presenilin homologs

Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer’s disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein–protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases.     

The very many faces of presenilins and the γ-secretase complex

Presenilin is a central, catalytic component of the γ-secretase complex which conducts intramembrane cleavage of various protein substrates. Although identified and mainly studied through its role in the development of amyloid plaques in Alzheimer disease, γ-secretase has many other important functions. The complex seems to be evolutionary conserved throughout the Metazoa, but recent findings in plants and Dictyostelium discoideum as well as in archeons suggest that its evolution and functions might be much more diversified than previously expected. In this review, a selective survey of the multitude of functions of presenilins and the γ-secretase complex is presented. Following a brief overview of γ-secretase structure, assembly and maturation, three functional aspects are analyzed: (1) the role of γ-secretase in autophagy and phagocytosis; (2) involvement of the complex in signaling related to endocytosis; and (3) control of calcium fluxes by presenilins.     

Presenilin 1 modifies lipid raft composition of neuronal membranes

Protein-lipid interactions in the nervous system may provide insight into the causes of neurological disorders. In this study, we elucidated if expression of human presenilin 1 (PS1) in a mouse model changes the physico-chemical properties of brain membranes. PS1 is a multifunctional transmembrane protein and part of the γ-secretase complex. This complex is critical for the production of the Alzheimer related amyloid beta peptide. Brain membranes isolated from mice expressing a human wild-type PS1 transgene are less fluid and contain higher cholesterol and sphingomyelin levels. Moreover, our data reveal significant changes in membrane micro-domains and indicate that PS1 induces the formation of lipid rafts.     

Minor contribution of presenilin 2 for γ-secretase activity in mouse embryonic fibroblasts and adult mouse brain

γ-Secretase plays an important function in the development of Alzheimer disease, since it participates in the production of the toxic amyloid β-peptide (Aβ) from the amyloid precursor protein (APP). Besides APP, γ-secretase cleaves many other substrates resulting in adverse side effects when γ-secretase inhibitors are used in clinical trials. γ-Secretase is a membrane bound protein complex consisting of at least four subunits, presenilin (PS), nicastrin, Aph-1 and Pen-2. PS and Aph-1 exist as different homologs (PS1/PS2 and Aph-1a/Aph-1b, respectively), which generates a variation in complex composition. PS1 and PS2 appears to have distinct roles since PS1 is essential during embryonic development whereas PS2 deficient mice are viable with a mild phenotype. The molecular mechanism behind this diversity is, however, largely unknown. In order to investigate whether PS1 and PS2 show different substrate specificity, we used PS1 or PS2 deficient mouse embryonic fibroblasts to study the processing on the γ-secretase substrates APP, Notch, N-cadherin, and ephrinB. We found that whereas depletion of PS1 severely affected the cleavage of all substrates, the effect of PS2 depletion was minor. In addition, less PS2 was found in active γ-secretase complexes. We also studied the effect of PS2 depletion in adult mouse brain and, in concordance with the results from the mouse embryonic fibroblasts, PS2 deficiency did not alter the cleavage of the two most important substrates, APP and Notch. In summary, this study shows that the contribution of PS2 on γ-secretase activity is of less importance, explaining the mild phenotype of PS2-deficient mice.     

Interleukin-1 receptor type 1 is a substrate for gamma-secretase-dependent regulated intramembrane proteolysis

Biochemical and genetic studies have revealed that the presenilins interact with several proteins and are involved in the regulated intramembrane proteolysis of numerous type 1 membrane proteins, thereby linking presenilins to a range of cellular processes. In this study, we report the characterization of a highly conserved tumor necrosis factor receptor-associated factor-6 (TRAF6) consensus-binding site within the hydrophilic loop domain of presenilin-1 (PS-1). In coimmunoprecipitation studies we indicate that presenilin-1 interacts with TRAF6 and interleukin-1 receptor-associated kinase 2. Substitution of presenilin-1 residues Pro-374 and Glu-376 by site-directed mutagenesis greatly reduces the ability of PS1 to associate with TRAF6. By studying these interactions, we also demonstrate that the interleukin-1 receptor type 1 (IL-1R1) undergoes intramembrane proteolytic processing, mediated by presenilin-dependent gamma-secretase activity. A metalloprotease-dependent proteolytic event liberates soluble IL-1R1 ectodomain and produces an approximately 32-kDa C-terminal domain. This IL-1R1 C-terminal domain is a substrate for subsequent gamma-secretase cleavage, which generates an approximately 26-kDa intracellular domain. Specific pharmacological gamma-secretase inhibitors, expression of dominant negative presenilin-1, or presenilin deficiency independently inhibit generation of the IL-1R1 intracellular domain. Attenuation of gamma-secretase activity also impairs responsiveness to IL-1beta-stimulated activation of the MAPKs and cytokine secretion. Thus, TRAF6 and interleukin receptor-associated kinase 2 are novel binding partners for PS1, and IL-1R1 is a new substrate for presenilin-dependent gamma-secretase cleavage. These findings also suggest that regulated intramembrane proteolysis may be a control mechanism for IL-1R1-mediated signaling.     

Identification of presenilin 1-selective γ-secretase inhibitors with reconstituted γ-secretase complexes

Accumulation of the β-amyloid (Aβ) peptides is one of the major pathologic hallmarks in the brains of Alzheimer's disease (AD) patients. Aβ is generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) catalyzed by β- and γ-secretases. Inhibition of Aβ production by γ-secretase inhibitors (GSIs) is thus being pursued as a target for treatment of AD. In addition to processing APP, γ-secretase also catalyzes proteolytic cleavage of other transmembrane substrates, with the best characterized one being the cell surface receptor Notch. GSIs reduce Aβ production in animals and humans but also cause significant side effects because of the inhibition of Notch processing. The development of GSIs that reduce Aβ production and have less Notch-mediated side effect liability is therefore an important goal. γ-Secretase is a large membrane protein complex with four components, two of which have multiple isoforms: presenilin (PS1 or PS2), aph-1 (aph-1a or aph-1b), nicastrin, and pen-2. Here we describe the reconstitution of four γ-secretase complexes in Sf9 cells containing PS1--aph-1a, PS1--aph-1b, PS2--aph-1a, and PS2--aph-1b complexes. While PS1--aph-1a, PS1--aph-1b, and PS2--aph-1a complexes displayed robust γ-secretase activity, the reconstituted PS2--aph-1b complex was devoid of detectable γ-secretase activity. γ-Secretase complexes containing PS1 produced a higher proportion of the toxic species Aβ42 than γ-secretase complexes containing PS2. Using the reconstitution system, we identified MRK-560 and SCH 1500022 as highly selective inhibitors of PS1 γ-secretase activity. These findings may provide important insights into developing a new generation of γ-secretase inhibitors with improved side effect profiles.