Specificity of presenilin‐1‐ and presenilin‐2‐dependent γ‐secretases towards substrate processing

The two presenilin‐1 (PS1) and presenilin‐2 (PS2) homologs are the catalytic core of the γ‐secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1‐ and PS2‐dependent γ‐secretases to the production of β‐amyloid peptide (Aβ) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aβ release without affecting the processing of other γ‐secretase substrates. To that end, we studied PS1‐ and PS2‐dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1‐PS2 double‐KO noted PSdKO) or stably re‐expressing human PS1 or PS2 in an endogenous PS‐null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L‐685,458). We found that murine PS1 γ‐secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ‐secretase. The inhibitors blocked more efficiently murine PS2‐ than murine PS1‐dependent processing. Human PSs, especially human PS1, expression in a PS‐null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1‐ than human PS2‐dependent γ‐secretase activity.     

Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificity and substrate selectivity of γ‐secretase

γ‐Secretase plays a central role in the generation of the Alzheimer disease‐causing amyloid β‐peptide (Aβ) from the β‐amyloid precursor protein (APP) and is thus a major Alzheimer′s disease drug target. As several other γ‐secretase substrates including Notch1 and CD44 have crucial signaling functions, an understanding of the mechanism of substrate recognition and cleavage is key for the development of APP selective γ‐secretase‐targeting drugs. The γ‐secretase active site domain in its catalytic subunit presenilin (PS) 1 has been implicated in substrate recognition/docking and cleavage. Highly critical in this process is its GxGD active site motif, whose invariant glycine residues cannot be replaced without causing severe functional losses in substrate selection and/or cleavage efficiency. Here, we have investigated the contribution of the less well characterized residue x of the motif (L383 in PS1) to this function. Extensive mutational analysis showed that processing of APP was overall well‐tolerated over a wide range of hydrophobic and hydrophilic mutations. Interestingly, however, most L383 mutants gave rise to reduced levels of Aβ_37–39 species, and several increased the pathogenic Aβ_42/43 species. Several of the Aβ_42/43‐increasing mutants severely impaired the cleavages of Notch1 and CD44 substrates, which were not affected by any other L383 mutation. Our data thus establish an important, but compared with the glycine residues of the motif, overall less critical functional role for L383. We suggest that L383 and the flanking glycine residues form a spatial arrangement in PS1 that is critical for docking and/or cleavage of different γ‐secretase substrates.     

γ‐Secretase Inhibition Induces Muscle Hypertrophy in a Notch‐Independent Mechanism

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ‐secretase. Notch‐1 is one of the substrates of γ‐secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch‐1, a number of proteins have been identified to undergo proteolysis by γ‐secretase. To date, the specific role of γ‐secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ‐secretase inhibitor DAPT (N‐N[‐(3,5‐Difluorophenacetyl‐l ‐alanyl)]‐S‐328 phenylglycine‐t‐butyl‐ester) induces muscle hypertrophy. Knockdown of Notch‐1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ‐secretase‐dependent effects on muscle hypertrophy in chick myogenic cells are Notch‐1‐independent. We also investigate the effects of γ‐secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label‐free proteomic approach. Data overview of interaction network obtained from STRING show that after γ‐secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ‐secretase in skeletal muscle hypertrophy.     

Identification of new Presenilin‐1 phosphosites: implication for γ‐secretase activity and Aβ production

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.

     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

Masking of Transmembrane‐Based Retention Signals Controls ER Export of γ‐Secretase

γ‐Secretase is critically involved in the Notch pathway and in Alzheimer's disease. The four subunits of γ‐secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ER‐retention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of γ‐secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER‐retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER‐retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent γ‐secretase complex could mask TMD‐based ER‐retention/retrieval signals to allow plasma membrane transport of fully assembled γ‐secretase.     

Mitofusin‐2 knockdown increases ER–mitochondria contact and decreases amyloid β‐peptide production

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria‐associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM‐associated proteins and enhanced ER to mitochondria Ca²⁺ transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β‐peptide (Aβ)‐related neuronal models. Here, we report that siRNA knockdown of mitofusin‐2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca²⁺ transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra‐ and extracellular Aβ₄₀ and Aβ₄₂. Analysis of γ‐secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ‐secretase complex function. Amyloid‐β precursor protein (APP), β‐site APP‐cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER–mitochondria contact affects γ‐secretase activity and Aβ generation. Increased ER–mitochondria contact results in lower γ‐secretase activity suggesting a new mechanism by which Aβ generation can be controlled.     

γ‐Secretase‐Dependent Cleavage Initiates Notch Signaling from the Plasma Membrane

Notch signaling is critical to animal development, and its dysregulation leads to human maladies ranging from birth defects to cancer. Although endocytosis is currently thought to promote signal activation by delivering activated Notch to endosome‐localized γ‐secretase, the data are controversial and the mechanisms that control Notch endocytosis remain poorly defined. Here, we investigated the relationship between Notch internalization and signaling. siRNA‐mediated depletion studies reveal that Notch endocytosis is clathrin‐dependent and requires epsin1, the adaptor protein complex (AP2) and Nedd4. Moreover, we show that epsin1 interaction with Notch is ubiquitin‐dependent. Contrary to the current model, we show that internalization defects lead to elevated γ‐secretase‐mediated Notch processing and downstream signaling. These results indicate that signal activation occurs independently of Notch endocytosis and that γ‐secretase cleaves Notch at the plasma membrane. These observations support a model where endocytosis serves to downregulate Notch in signal‐receiving cells.     

Conditional inactivation of nicastrin restricts amyloid deposition in an Alzheimer's disease mouse model

Production of Aβ by γ‐secretase is a key event in Alzheimer's disease (AD). The γ‐secretase complex consists of presenilin (PS) 1 or 2, nicastrin (ncstn), Pen‐2, and Aph‐1 and cleaves type I transmembrane proteins, including the amyloid precursor protein (APP). Although ncstn is widely accepted as an essential component of the complex required for γ‐secretase activity, recent in vitro studies have suggested that ncstn is dispensable for APP processing and Aβ production. The focus of this study was to answer this controversy and evaluate the role of ncstn in Aβ generation and the development of the amyloid‐related phenotype in the mouse brain. To eliminate ncstn expression in the mouse brain, we used a ncstn conditional knockout mouse that we mated with an established AD transgenic mouse model (5XFAD) and a neuronal Cre‐expressing transgenic mouse (CamKIIα‐iCre), to generate AD mice (5XFAD/CamKIIα‐iCre/ncstn^f/f mice) where ncstn was conditionally inactivated in the brain. 5XFAD/CamKIIα‐iCre/ncstn^f/f mice at 10 week of age developed a neurodegenerative phenotype with a significant reduction in Aβ production and formation of Aβ aggregates and the absence of amyloid plaques. Inactivation of nctsn resulted in substantial accumulation of APP‐CTFs and altered PS1 expression. These results reveal a key role for ncstn in modulating Aβ production and amyloid plaque formation in vivo and suggest ncstn as a target in AD therapeutics.     

Substrate recruitment of γ‐secretase and mechanism of clinical presenilin mutations revealed by photoaffinity mapping

Intramembrane proteases execute fundamental biological processes ranging from crucial signaling events to general membrane proteostasis. Despite the availability of structural information on these proteases, it remains unclear how these enzymes bind and recruit substrates, particularly for the Alzheimer's disease‐associated γ‐secretase. Systematically scanning amyloid precursor protein substrates containing a genetically inserted photocrosslinkable amino acid for binding to γ‐secretase allowed us to identify residues contacting the protease. These were primarily found in the transmembrane cleavage domain of the substrate and were also present in the extramembranous domains. The N‐terminal fragment of the catalytic subunit presenilin was determined as principal substrate‐binding site. Clinical presenilin mutations altered substrate binding in the active site region, implying a pathogenic mechanism for familial Alzheimer's disease. Remarkably, PEN‐2 was identified besides nicastrin as additional substrate‐binding subunit. Probing proteolysis of crosslinked substrates revealed a mechanistic model of how these subunits interact to mediate a stepwise transfer of bound substrate to the catalytic site. We propose that sequential binding steps might be common for intramembrane proteases to sample and select cognate substrates for catalysis.     

Identifying genes that interact with Drosophila presenilin and amyloid precursor protein

The γ‐secretase complex is involved in cleaving transmembrane proteins such as Notch and one of the genes targeted in Alzheimer's disease known as amyloid precursor protein (APP). Presenilins function within the catalytic core of γ‐secretase, and mutated forms of presenilins were identified as causative factors in familial Alzheimer's disease. Recent studies show that in addition to Notch and APP, numerous signal transduction pathways are modulated by presenilins, including intracellular calcium signaling. Thus, presenilins appear to have diverse roles. To further understand presenilin function, we searched for Presenilin‐interacting genes in Drosophila by performing a genetic modifier screen for enhancers and suppressors of Presenilin‐dependent Notch‐related phenotypes. We identified 177 modifiers, including known members of the Notch pathway and genes involved in intracellular calcium homeostasis. We further demonstrate that 53 of these modifiers genetically interacted with APP. Characterization of these genes may provide valuable insights into Presenilin function in development and disease. genesis 47:246–260, 2009. © 2009 Wiley‐Liss, Inc.     

MUC1 is a substrate for γ‐secretase

Understanding the underlying mechanisms by which a normal cell avoids the oncogenic potential of MUC1 signaling requires further definition of the pathways by which the MUC1 cytoplasmic tail is processed in both normal and tumor‐derived cells. In the present study we describe the processing pathway initiated by TACE/ADAM17 cleavage of MUC1. Utilizing the human uterine epithelial cell line, HES, derived from normal endometrium, we show that endogenous full length MUC1 undergoes regulated intramembranous proteolysis mediated by presenillin‐dependent γ‐secretase. Cytokine‐stimulated HES cells exposed to γ‐secretase inhibitors accumulated a membrane‐associated 15 kDa fragment of the MUC1 C‐terminal subunit (CTF15). Inhibitors of TACE/ADAM17‐mediated shedding inhibited accumulation of MUC1‐CTF15 and MUC1 ectodomain release to a similar extent consistent with MUC1‐CTF15 being a product of TACE/ADAM17 action. Reduction of catalytically active γ‐secretase complex by nicastrin siRNA treatment also resulted in CTF15 accumulation. Furthermore, mature nicastrin, the substrate receptor for γ‐secretase, co‐immunoprecipitated with CTF15 in the presence of γ‐secretase inhibitors indicating the formation of CTF15: nicastrin complexes. MUC1‐CTF15 accumulation in response to γ‐secretase inhibition was demonstrated in both normal and tumor‐derived cells from humans and mice indicating that this processing pathway exists in many cell contexts. We did not detect products of MUC1 cleavage by γ‐secretase in the presence of various proteasomal inhibitors indicating that subsequent degradation is either non‐proteasomal or extremely efficient. We suggest that this efficient pathway attenuates potential signaling mediated by cytoplasmic tail fragments. J. Cell. Biochem. 108: 802–815, 2009. © 2009 Wiley‐Liss, Inc.     

Aph‐1 is required to regulate Presenilin‐mediated γ‐secretase activity and cell survival in Drosophila wing development

Aph‐1 is a multipass transmembrane protein and an essential component of the Presenilin (Psn)‐mediated γ‐secretase complex. During protease assembly, Aph‐1 stabilizes the newly synthesized Psn holoprotein to facilitate generation of the active form of Psn, which is a Psn‐NTF/Psn‐CTF heterodimer produced through a Presenilinase‐initiated endoproteolytic cleavage of the Psn holoprotein. Although it is clear that loss of Aph‐1 activity leads to failure of Psn heterodimer formation, little is understood about whether Aph‐1 plays a role in regulating γ‐secretase activity in addition to assisting Psn maturation. Using various modified Psn forms that do not require endoproteolysis or have a large deletion of the cytosolic loop, we show that in Drosophila Aph‐1 is still required for γ‐secretase activity independent of its role in promoting Psn endoproteolysis. In addition, our results indicate that Aph‐1 is required to promote cell survival in the wing imaginal disc; aph‐1 mutant cells are lost either through cell death or because of a defect in cell proliferation. This function of Aph‐1 is independent of its role in regulating γ‐secretase activity, but possibly involves downregulating the activity of uncleaved Psn holoprotein. genesis 47:169–174, 2009. © 2009 Wiley‐Liss, Inc.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Functional analysis and purification of a Pen‐2 fusion protein for γ‐secretase structural studies

The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.

     

Pen‐2 is dispensable for endoproteolysis of presenilin 1, and nicastrin‐Aph subcomplex is important for both γ‐secretase assembly and substrate recruitment

γ‐secretase is a protease complex with at least four components: presenilin, nicastrin (NCT), anterior pharynx‐defective 1 (Aph‐1), and presenilin enhancer 2 (Pen‐2). In this study, using knockout cell lines and small interfering RNA technology, our data demonstrated that the disappeared presenilin 1 C‐terminal fragment (PS1C) caused by knockdown of pen‐2 or knockout of NCT or Aph‐1 was recovered by the addition of proteasome inhibitors, indicating that Pen‐2, as well as NCT and Aph‐1α, is dispensable for presenilin endoproteolysis. Our data also demonstrate that the formation of the nicastrin‐Aph‐1 subcomplex plays not only an important role in γ‐secretase complex assembly but also in recruiting substrate C‐terminal fragment of amyloid precursor protein generated by β‐cleavage. Ablating any one component resulted in the instability of other components of the γ‐secretase complex, and the presence of all three of the other components is required for full maturation of NCT.     

Probing γ‐secretase–substrate interactions at the single amino acid residue level

Intramembrane proteolysis by γ‐secretases plays major roles in disease pathology and cellular signalling, yet the dynamics of these enzyme complexes and how they recognize substrates remains poorly understood. New work in The EMBO Journal utilizes photo‐affinity cross‐linking to map APP interactions to different γ‐secretase subunits, suggesting a succession of recruitment and engagement steps that lead up to substrate cleavage.     

The mechanism of γ-Secretase dysfunction in familial Alzheimer disease

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid β (Aβ)42 relative to Aβ40 by an unknown, possibly gain‐of‐toxic‐function, mechanism. However, many PSEN mutations paradoxically impair γ‐secretase and ‘loss‐of‐function’ mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aβ generation via three different mechanisms, resulting in qualitative changes in the Aβ profiles, which are not limited to Aβ42. Loss of ε‐cleavage function is not generally observed among FAD mutants. On the other hand, γ‐secretase inhibitors used in the clinic appear to block the initial ε‐cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase‐like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aβ products, and suggest fundamental improvements for current drug development efforts.