Endoproteolysis Of Presenilin Research Articles

Visualization of PS/γ-Secretase Activity in Living Cells.

SummaryA change in Presenilin (PS)/γ-secretase activity is linked to essential biological events as well as to the progression of many diseases. However, not much is known about how PS/γ-secretase activity is spatiotemporally regulated in cells. One of the limitations is lack of tools to directly monitor dynamic behavior of the PS/γ-secretase in intact/live cells. Here we present successful development and validation of the Förster resonance energy transfer (FRET)-based biosensors that enable quantitative monitoring of endogenous PS/γ-secretase activity in live cells longitudinally on a cell-by-cell basis. Using these FRET biosensors, we uncovered that PS/γ-secretase activity is heterogeneously regulated among live neurons.

Two novel presenilin-1 mutations (I249L and P433S) in early onset Chinese Alzheimer's pedigrees and their functional characterization

Clinical case study and functional characterization of the disease-associated presenilin-1 (PSEN1) mutations may help reveal the roles of PSEN1 in the pathogenesis of Alzheimer's disease (AD). By mutation screening of PSEN1, presenilin-2, and amyloid precursor protein genes in two Chinese Alzheimer's pedigrees, we identified two novel PSEN1 mutations, I249L and P433S. The two probands presented with progressive memory decline and subsequent psychiatric symptoms, with the age of onset at 54 and 34 years old, respectively. The effects of these two mutations on presenilin-1 endoproteolysis and β-amyloid (Aβ) production were examined in SH-SY5Y neuroblastoma cells infected with lentiviruses expressing presenilin-1 wild type (WT), I249L and P433S mutants. Both mutants showed increased Aβ42 levels and Aβ42/Aβ40 ratios. However, the I249L did not affect presenilin-1 endoproteolysis or Aβ43 production, whereas the P433S mutant inhibited presenilin-1 endoproteolysis and enhanced Aβ43 production. Our findings suggest that both I249L and P433S are pathogenic for early onset of AD by increasing Aβ42 production and Aβ42/Aβ40 ratios. Furthermore, P433S may contribute to the very early onset of AD by inhibiting PS1 endoproteolysis and enhancing the production of longer Aβ peptide Aβ43.

HUMAN IPSC-DERIVED ALZHEIMER’S DISEASE ASTROCYTES RECAPITULATE DISEASE-RELATED PHENOTYPES

The importance of astrocytes in neurodegenerative disorders, including Alzheimer's disease (AD), is well recognized. Yet, their exact role in the disease pathogenesis and progression remains unclear. Interestingly, human astrocytes have been found to be far larger and more complex than those of infraprimate mammals, making it necessary to concentrate research on diseased human astrocytes rather than animal cells. In this study, patient-specific induced pluripotent stem cells (iPSCs) were used as a source for human astrocytes. iPSCs were reprogrammed from skin fibroblasts of familial AD patients with deletion of exon 9 in the PSEN1gene (n=3) and from healthy age-matched controls (n=3). The deletion of exon 9 was corrected with CRISPR/Cas9 genome editing, enabling generation of isogenic control lines for the diseased lines. iPSCs were then further differentiated to GFAP and S100b-positive forebrain astrocytes according to our optimized protocol. iPSC-derived astrocytes are functional cells that are able to propagate calcium waves, uptake glucose, produce cytokines and glutathione, and release gliotransmitters, such as GABA and ATP. In addition, AD astrocytes show calcium leakage from ER and reduced presenilin endoproteolysis, both well-characterized defects linked to PSEN1mutations. Interestingly, inflammatory stimulation of AD astrocytes resulted in increased release of proinflammatory cytokines when compared to control cells. AD astrocytes also have alterations in mitochondrial mass, cellular metabolism and glutathione levels. Most importantly, astrocytes contribute to amyloid pathology and are able to both produce and uptake beta-amyloid. AD astrocytes secrete significantly higher amounts of the 1–42 form and show an elevated 1–42/1–40 ratio as compared to controls, but respond well to treatment with gamma-secretase inhibitor DAPT. Our data show that pathological findings commonly seen in AD patients are recapitulated in the iPSC-derived astrocytes. Thus, these cells offer a valuable tool for studying the disease mechanisms and for drug screening and testing novel therapeutic approaches in a cell-type specific manner.

Attenuated presenilin-1 endoproteolysis enhances store-operated calcium currents in neuronal cells.

Presenilins have been reported to regulate calcium homeostasis in the endoplasmic reticulum, and dysregulation of intracellular calcium has been implicated in the pathogenesis of Alzheimer's disease (AD). Reduced endoproteolysis levels of presenilin-1 (PS1) have been detected in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. This study deals with the effect of attenuated endoproteolysis of PS1 on store-operated calcium (SOC) entry in neuronal cells and mouse fibroblasts with double knockouts of PS1 and PS2. Significant enhancement of SOC channel activation has been detected by electrophysiological measurements in cells with reduced PS1 endoproteolysis. The increase in SOC entry was not accompanied by any changes in protein levels of channels subunits or stromal interaction molecule. These data are important for understanding the role of PS1 in AD, apart from its involvement in γ-secretase cleavage of amyloid precursor protein into Aβ. Taking into account that most of familial AD-connected mutations in PS1 are loss-of-function, the observed effects may well be general for familial AD. Reduced endoproteolysis levels of presenilin-1 (PS1) have been detected in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Significant enhancement of SOC channel activation has been detected by electrophysiological measurements in cells with reduced PS1 endoproteolysis. The data obtained shed light on Alzheimer's disease pathogenesis and implicates to the future drugs development.

G206D Mutation of Presenilin-1 Reduces Pen2 Interaction, Increases Aβ42/Aβ40 Ratio and Elevates ER Ca(2+) Accumulation.

Early-onset familial Alzheimer's disease (AD) is most commonly associated with the mutations in presenilin-1 (PS1). PS1 is the catalytic component of the γ-secretase complex, which cleaves amyloid precursor protein to produce amyloid-β (Aβ), the major cause of AD. Presenilin enhancer 2 (Pen2) is critical for activating γ-secretase and exporting PS1 from endoplasmic reticulum (ER). Among all the familial AD-linked PS1 mutations, mutations at the G206 amino acid are the most adjacent position to the Pen2 binding site. Here, we characterized the effect of a familial AD-linked PS1 G206D mutation on the PS1-Pen2 interaction and the accompanied alteration in γ-secretase-dependent and -independent functions. We found that the G206D mutation reduced PS1-Pen2 interaction, but did not abolish γ-secretase formation and PS1 endoproteolysis. For γ-secretase-dependent function, the G206D mutation increased Aβ42 production but not Notch cleavage. For γ-secretase-independent function, this mutation disrupted the ER calcium homeostasis but not lysosomal calcium homeostasis and autophagosome maturation. Impaired ER calcium homeostasis may due to the reduced mutant PS1 level in the ER. Although this mutation did not alter the cell survival under stress, both increased Aβ42 ratio and disturbed ER calcium regulation could be the mechanisms underlying the pathogenesis of the familial AD-linked PS1 G206D mutation.

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.

Disregulation of Calcium Homeostasis Connected with Familial Alzheimer's Disease

Familial Alzheimer's disease (FAD) caused by mutations in presenilin-1 (PS1) gene in approximately 50% of cases and in Amyloid precursor protein (APP) gene in 13 % cases. It was found that FAD genes mutants affect calcium homeostasis in hippocampal neurons by disrupting Ca2+ storage in the lumen of endoplasmic reticulum (ER). In our study we used electrophysiological recordings with a patch-clamp technique in whole-cell mode and calcium imaging with Fura-2AM to study calcium channels activity, ER calcium load and ER calcium leak. We observed different effects of FAD mutated genes expressions on activity of store-operated and L-type voltage-gated calcium channels in mouse hippocampal neurons, mouse neuroblastoma Neuro2a and human neuroblastoma SK-N-SH cell lines. We found that effects of FAD PS1 mutants on calcium channels were driven by misbalance in activity of ER calcium sensors STIM1 and STIM2. The different effects were connected with calcium sensors STIM1 or STIM2 impaired signal transduction from ER to calcium channels in plasmatic membrane (PM) under control of ER calcium levels. The impaired signal transduction was revealed in live confocal imaging experiments. The enzyme activity of PS1 was involved in regulation of store-operated calcium entry in two ways: the activity of channels was depended on PS1 endoproteolysis level and APP cleavage.This work was supported by the program of “Molecular and Cellular Biology” RAS, research grants from the Russian Basic Research Foundation, grant ERA.Net RUS, research grants from the OPTEC LLC and the President of Russia Scholarship.

γ-Secretase Modulators and Presenilin 1 Mutants Act Differently on Presenilin/γ-Secretase Function to Cleave Aβ42 and Aβ43

Deciphering the mechanism by which the relative Aβ42(43) to total Aβ ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aβ42(43) ratio caused by familial mutations and γ-secretase modulators (GSMs) are unclear. Here, we show in vitro and in living cells that presenilin (PS)/γ-secretase cleaves Aβ42 into Aβ38, and Aβ43 into Aβ40 or Aβ38. Approximately 40% of Aβ38 is derived from Aβ43. Aβ42(43) cleavage is involved in the regulation of the Aβ42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aβ42 (increase k(cat)) and slow its dissociation from the enzyme (decrease k(b)), whereas PS1 mutants and inverse GSMs show the opposite effects. Therefore, we suggest a concept to describe the Aβ42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aβ42(43) may initiate AD and might represent a therapeutic target.

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.

Structure of γ-Secretase and Its Trimeric Pre-activation Intermediate by Single-particle Electron Microscopy

The γ-secretase membrane protein complex is responsible for proteolytic maturation of signaling precursors and catalyzes the final step in the production of the amyloid β-peptides implicated in the pathogenesis of Alzheimer disease. The incorporation of PEN-2 (presenilin enhancer 2) into a pre-activation intermediate, composed of the catalytic subunit presenilin and the accessory proteins APH-1 (anterior pharynx-defective 1) and nicastrin, triggers the endoproteolysis of presenilin and results in an active tetrameric γ-secretase. We have determined the three-dimensional reconstruction of a mature and catalytically active γ-secretase using single-particle cryo-electron microscopy. γ-Secretase has a cup-like shape with a lateral belt of ∼40-50 Å in height that encloses a water-accessible internal chamber. Active site labeling with a gold-coupled transition state analog inhibitor suggested that the γ-secretase active site faces this chamber. Comparison with the structure of a trimeric pre-activation intermediate suggested that the incorporation of PEN-2 might contribute to the maturation of the active site architecture.

Genetic Ablation of Apolipoprotein A-IV Accelerates Alzheimer's Disease Pathogenesis in a Mouse Model

The link between lipoprotein metabolism and Alzheimer's disease (AD) has been established. Apolipoprotein A-IV (apoA-IV), a component of lipoprotein particles similar to apolipoprotein E, has been suggested to play an important role in brain metabolism. Although there are clinical debates on the function of its polymorphism in AD, the pathologic role of apoA-IV in AD is still unknown. Here, we report that genetic ablation of apoA-IV is able to accelerate AD pathogenesis in mice. In a mouse model that overexpresses human amyloid precursor protein (APP) and presenilin 1, genetic reduction of apoA-IV augments extracellular amyloid-β peptide (Aβ) burden and aggravates neuron loss in the brain. In addition, genetic ablation of apoA-IV also accelerates spatial learning deficits and increases the mortality of mice. We have found that apoA-IV colocalizes within Aβ plaques in APP/presenilin 1 transgenic mice and binds to Aβ in vitro. Subsequent studies show that apoA-IV in this model facilitates Aβ uptake in the Aβ clearance pathway mediated by astrocytes rather than the amyloidogenic pathway of APP processing. Taken together, we conclude that apoA-IV deficiency increases Aβ deposition and results in cognitive damage in the mouse model. Enhancing levels of apoA-IV may have therapeutic potential in AD treatment.

Mutation Analysis of the Presenilin 1 N-terminal Domain Reveals a Broad Spectrum of γ-Secretase Activity toward Amyloid Precursor Protein and Other Substrates

The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.

A Presenilin-1 Mutation Identified in Familial Alzheimer Disease with Cotton Wool Plaques Causes a Nearly Complete Loss of γ-Secretase Activity

Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit gamma-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by gamma-secretase produces beta-amyloid peptides (Abeta40 and Abeta42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on gamma-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of gamma-secretase activity, including >90% reductions in the generation of Abeta40, Abeta42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of gamma-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Abeta40, Abeta42, and the APP and Notch intracellular domains. These results argue against overproduction of Abeta42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function.

Three-Amino Acid Spacing of Presenilin Endoproteolysis Suggests a General Stepwise Cleavage of -Secretase-Mediated Intramembrane Proteolysis

Presenilin (PS1 or PS2) is the catalytic component of the gamma-secretase complex, which mediates the final proteolytic processing step leading to the Alzheimer's disease (AD)-characterizing amyloid beta-peptide. PS is cleaved during complex assembly into its characteristic N- and C-terminal fragments. Both fragments are integral components of physiologically active gamma-secretase and harbor the two critical aspartyl residues of the active site domain. While the minimal subunit composition of gamma-secretase has been defined and numerous substrates were identified, the cellular mechanism of the endoproteolytic cleavage of PS is still unclear. We addressed this pivotal question by investigating whether familial AD (FAD)-associated PS1 mutations affect the precision of PS endoproteolysis in a manner similar to the way that such mutations shift the intramembrane cleavage of gamma-secretase substrates. We demonstrate that all FAD mutations investigated still allow endoproteolysis to occur. However, the precision of PS1 endoproteolysis is affected by PS1 mutations. Comparing the cleavage products generated by a variety of PS1 mutants revealed that specifically cleavages at positions 293 and 296 of PS1 are selectively affected. Systematic mutagenesis around the cleavage sites revealed a stepwise three amino acid spaced cleavage mechanism of PS endoproteolysis reminiscent to the epsilon-, zeta-, and gamma-cleavages described for typical gamma-secretase substrates, such as the beta-amyloid precursor protein. Our findings therefore suggest that intramembranous cleavage by gamma-secretase and related intramembrane-cleaving proteases may generally occur via stepwise endoproteolysis.

The Large Hydrophilic Loop of Presenilin 1 Is Important for Regulating γ-Secretase Complex Assembly and Dictating the Amyloid β Peptide (Aβ) Profile without Affecting Notch Processing

Gamma-secretase is an enzyme complex that mediates both Notch signaling and beta-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid beta peptide (Abeta), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of gamma-secretase gives rise to Abeta peptides of different lengths, where Abeta42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320-374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of gamma-secretase, for gamma-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, gamma-secretase complex formation, and had a differential effect on Abeta-peptide production. Although the production of Abeta38, Abeta39, and Abeta40 was severely impaired, the effect on Abeta42 was affected to a lesser extent, implying that the production of the AD-related Abeta42 peptide is separate from the production of the Abeta38, Abeta39, and Abeta40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the epsilon/S3 and gamma sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Abeta peptides without affecting Notch processing, two parameters of significance when considering gamma-secretase as a target for pharmaceutical intervention in AD.

Glu-333 of Nicastrin Directly Participates in γ-Secretase Activity

gamma-Secretase is a proteolytic membrane complex that processes a variety of substrates including the amyloid precursor protein and the Notch receptor. Earlier we showed that one of the components of this complex, nicastrin (NCT), functions as a receptor for gamma-secretase substrates. A recent report challenged this, arguing instead that the Glu-333 residue of NCT predicted to participate in substrate recognition only participates in gamma-secretase complex maturation and not in activity per se. Here, we present evidence that Glu-333 directly participates in gamma-secretase activity. By normalizing to the active pool of gamma-secretase with two separate methods, we establish that gamma-secretase complexes containing NCT-E333A are indeed deficient in intrinsic activity. We also demonstrate that the NCT-E333A mutant is deficient in its binding to substrates. Moreover, we find that the cleavage of substrates by gamma-secretase activity requires a free N-terminal amine but no minimal length of the extracellular N-terminal stub. Taken together, these studies provide further evidence supporting the role of NCT in substrate recognition. Finally, because gamma-secretase cleaves itself during its maturation and because NCT-E333A also shows defects in gamma-secretase complex maturation, we present a model whereby Glu-333 can serve a dual role via similar mechanisms in the recruitment of both Type 1 membrane proteins for activity and the presenilin intracellular loop during complex maturation.

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 γ-Secretase Complexes

gamma-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Abeta42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of gamma-secretase, invariably increase the Abeta42:Abeta40 ratio. However, the mechanism by which these mutations affect gamma-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Abeta42:Abeta40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active gamma-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Abeta42:Abeta40 ratios. These data suggest that perturbations to gamma-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Abeta42:Abeta40 ratio.

Identification of γ-Secretase Inhibitor Potency Determinants on Presenilin

Production of amyloid beta peptides (Abeta), followed by their deposition in the brain as amyloid plaques, contributes to the hallmark pathology of Alzheimer disease. The enzymes responsible for production of Abeta, BACE1 and gamma-secretase, are therapeutic targets for treatment of Alzheimer disease. Two presenilin (PS) homologues, referred to as PS1 and PS2, comprise the catalytic core of gamma-secretase. In comparing presenilin selectivity of several classes of gamma-secretase inhibitors, we observed that sulfonamides in general tend to be more selective for inhibition of PS1-comprising gamma-secretase, as exemplified by ELN318463 and BMS299897. We employed a combination of chimeric constructs and point mutants to identify structural determinants for PS1-selective inhibition by ELN318463. Our studies identified amino acid residues Leu(172), Thr(281), and Leu(282) in PS1 as necessary for PS1-selective inhibition by ELN318463. These residues also contributed in part to the PS1-selective inhibition by BMS299897. Alanine scanning mutagenesis of areas flanking Leu(172), Thr(281), and Leu(282) identified additional amino acids that affect inhibitor potency of not only these sulfonamides but also nonsulfonamide inhibitors, without affecting Abeta production and presenilin endoproteolysis. Interestingly, many of these same residues have been identified previously to be important for gamma-secretase function. These findings implicate TM3 and a second region near the carboxyl terminus of PS1 aminoterminal fragment in mediating the activity of gamma-secretase inhibitors. Our observations demonstrate that PS-selective inhibitors of gamma-secretase are feasible, and such inhibitors may allow differential inhibition of Abeta peptide production and Notch signaling.

An AICD-based functional screen to identify APP metabolism regulators

BackgroundA central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered.ResultsTo validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis.ConclusionTaken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.

Putting presenilins centre stage

Genetic analysis of autosomal dominant Alzheimer disease has identified the amyloid precursor protein (APP) and the presenilins as its pathogenic loci (Rogaeva, 2002). APP is normally cleaved by γ‐secretase into the APP intracellular domain (AICD) and the 40‐residue amyloid β peptide (Aβ40) or, less frequently, the 42‐residue Aβ42. Analyses of blood plasma from mutation carriers shows that all of the pathogenic mutations cause an increase in the Aβ42/Aβ40 ratio, suggesting that they alter the position of the γ‐secretase cleavage of APP (Scheuner et al , 1996; Haass & Selkoe, 1993). Gene‐ablation studies and focused mutagenesis experiments by De Strooper, Wolfe and their colleagues (De Strooper et al , 1998; Wolfe et al , 1999) shows that the presenilins—along with other accessory proteins (Edbauer et al , 2003)—form the γ‐secretase complex. This work is a good example of how genetic analysis can give direct mechanistic insight into the pathogenesis of …