A Novel Dual Fluorochrome Near-Infrared Imaging Probe for Potential Alzheimer's Enzyme Biomarkers-BACE1 and Cathepsin D.

A molecular imaging probe to fluorescently image the ß-site of the amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) and cathepsin D (CatD) enzymes associated with Alzheimer's disease (AD) was designed and synthesized. This imaging probe was built upon iron oxide nanoparticles (cross-linked dextran iron oxide nanoparticles, or CLIO). Peptide substrates containing a terminal near-infrared fluorochrome (fluorophore emitting at 775 nm for CatD or fluorophore emitting at 669 nm for BACE1) were conjugated to the CLIO nanoparticles. The CatD substrate contained a phenylalanine-phenylalanine cleavage site more specific to CatD than BACE1. The BACE1 substrate contained the sequence surrounding the leucine-asparagine cleavage site of the BACE1 found in the Swedish mutation of APP, which is more specific to BACE1 than CatD. These fluorescently-labeled peptide substrates were then conjugated to the nanoparticle. The nanoparticle probes were purified by gel filtration, and their fluorescence intensities were determined using a fluorescence plate reader. The CatD peptide substrate demonstrated a 15.5-fold increase in fluorescence when incubated with purified CatD enzyme, and the BACE1 substrate exhibited a 31.5-fold increase in fluorescence when incubated with purified BACE1 enzyme. Probe specificity was also demonstrated in the human H4 neuroglioma cells and the H4 cells stably transfected with BACE1 in which the probe monitored enzymatic cleavage. In the H4 and H4-BACE1 cells, BACE1 and active CatD activity increased, an occurrence that was reflected in enzyme expression levels as determined by immunoblotting. These results demonstrate the applicability of this probe for detecting potential Alzheimer's enzyme biomarkers.

Isolation of intramembrane proteases in membrane-like environments.

Intramembrane proteases (IMPs) are proteolytic enzymes embedded in the lipid bilayer, where they cleave transmembrane substrates. The importance of IMPs relies on their role in a wide variety of cellular processes and diseases. In order to study the activity and function of IMPs, their purified form is often desired. The production of pure and active IMPs has proven to be a challenging task. This process unavoidably requires the use of solubilizing agents that will, to some extent, alter the native environment of these proteases. In this review we present the current solubilization and reconstitution techniques that have been applied to IMPs. In addition, we describe how these techniques had an influence on the activity and structural studies of IMPs, focusing on rhomboid proteases and γ-secretase.

Development of an Efficient Enzyme Production and Structure-Based Discovery Platform for BACE1 Inhibitors.

BACE1 (Beta-site Amyloid Precursor Protein (APP) Cleaving Enzyme 1) is a promising therapeutic target for Alzheimer's Disease (AD). However, efficient expression, purification, and crystallization systems are not well described or detailed in the literature nor are approaches for treatment of enzyme kinetic data for potent inhibitors well described. We therefore developed a platform for expression and purification of BACE1, including protein refolding from E.coli inclusion bodies, in addition to optimizing a reproducible crystallization procedure of BACE1 bound with inhibitors. We also report a detailed approach to the proper analysis of enzyme kinetic data for compounds that exhibit either rapid-equilibrium or tight-binding mechanisms. Our methods allow for the purification of ∼15 mg of BACE1 enzyme from 1 L of culture which is higher than reported yields in the current literature. To evaluate the data analysis approach developed here, a well-known potent inhibitor and two of its derivatives were tested, analyzed, and compared. The inhibitory constants (Ki) obtained from the kinetic studies are in agreement with dissociation constants (Kd) that were also determined using isothermal titration calorimetry (ITC) experiments. The X-ray structures of these three compounds in complex with BACE1 were readily obtained and provide important insight into the structure and thermodynamics of the BACE1-inhibitor interactions.

Efficient production of a mature and functional gamma secretase protease.

Baculoviral protein expression in insect cells has been previously used to generate large quantities of a protein of interest for subsequent use in biochemical and structural analyses. The MultiBac baculovirus protein expression system has enabled, the use of a single baculovirus to reconstitute a protein complex of interest, resulting in a larger protein yield. Using this system, we aimed to reconstruct the gamma (γ)-secretase complex, a multiprotein enzyme complex essential for the production of amyloid-ß (Aß) protein. A MultiBac vector containing all components of the γ-secretase complex was generated and expression was observed for all components. The complex was active in processing APP and Notch derived γ-secretase substrates and proteolysis could be inhibited with γ-secretase inhibitors, confirming specificity of the recombinant γ-secretase enzyme. Finally, affinity purification was used to purify an active recombinant γ-secretase complex. In this study we demonstrated that the MultiBac protein expression system can be used to generate an active γ-secretase complex and provides a new tool to study γ-secretase enzyme and its variants.

Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations.

The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analyzed for their function with single-molecule microscopy. Expansion of this approach toward a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilization and reconstitution often cause protein conformational changes and loss of membrane-specific cofactors, which may impair protein function. To overcome this limitation, we here demonstrate how antibody-modified nanoparticles can be used to achieve affinity purification and enrichment of selected integral membrane proteins directly from cell membrane preparations. Nanoparticles were first bound to the ectodomain of ß-secretase 1 (BACE1) contained in cell-derived membrane vesicles. In a subsequent step, these were merged into a continuous supported membrane in a microfluidic channel. Through the extended nanoparticle tag, a weak (∼fN) hydrodynamic force could be applied, inducing directed in-membrane movement of targeted BACE1 exclusively. This enabled selective thousand-fold enrichment of the targeted membrane protein while preserving a natural lipid environment. In addition, nanoparticle-targeting also enabled simultaneous tracking analysis of each individual manipulated protein, revealing how their mobility changed when moved from one lipid environment to another. We therefore believe this approach will be particularly useful for separation in-line with single-molecule analysis, eventually opening up for membrane-protein sorting devices analogous to fluorescence-activated cell sorting.

Expression, Purification, and Enzymatic Characterization of Intramembrane Proteases.

Intramembrane proteases catalyze peptide bond hydrolysis in the lipid bilayer and play a key role in numerous cellular processes. These integral membrane enzymes consist of four classes: site-2 protease (S2P), rhomboid serine protease, Rce1-type glutamyl protease, and aspartyl protease exemplified by presenilin and signal peptide peptidase (SPP). Structural elucidation of these enzymes is important for mechanistic understanding of their functions, particularly their roles in cell signaling and debilitating diseases such as Parkinson's disease and Alzheimer's disease. In the past decade, rigorous effort has led to determination of the crystal structures of S2P from archaebacterium, rhomboid serine protease from E. coli (GlpG), and presenilin/SPP from archaebacterium (PSH). A novel method has been developed to express well-behaved human γ-secretase, which facilitated its structure determination by cryoelectron microscopy (cryo-EM). In this chapter, we will discuss the expression and purification of intramembrane proteases including human γ-secretase and describe the enzymatic activity assays for these intramembrane proteases.

An Inducible Reconstitution System for the Real-Time Kinetic Analysis of Protease Activity and Inhibition Inside the Membrane.

Intramembrane proteases are an ancient and diverse group of multispanning membrane proteins that cleave transmembrane substrates inside the membrane to effect a wide range of biological processes. As proteases, a clear understanding of their function requires kinetic dissection of their catalytic mechanism, but this is difficult to achieve for membrane proteins. Kinetic measurements in detergent systems are complicated by micelle fusion/exchange, which introduces an additional kinetic step and imposes system-specific behaviors (e.g., cooperativity). Conversely, kinetic analysis in proteoliposomes is hindered by premature substrate cleavage during coreconstitution, and lack of methods to quantify proteolysis in membranes in real time. In this chapter, we describe a method for the real-time kinetic analysis of intramembrane proteolysis in model liposomes. Our assay is inducible, because the enzyme is held inactive by low pH during reconstitution, and fluorogenic, since fluorescence emission from the substrate is quenched near lipids but restored upon proteolytic release from the membrane. The precise measurement of initial reaction velocities continuously in real time facilitates accurate steady-state kinetic analysis of intramembrane proteolysis and its inhibition inside the membrane environment. Using real data we describe a step-by-step strategy to implement this assay for essentially any intramembrane protease.

Preparation and biological evaluation of conformationally constrained BACE1 inhibitors.

The BACE1 enzyme is a key target for Alzheimer's disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core. Pursuit of S3-binding groups produced low micromolar inhibitor 6, which informed the S3-design for constrained analogs 7 and 8, themselves prepared via independent, multi-step synthetic routes. Biological characterization of BACE inhibitors 6-8 is described.

Generation of monoclonal antibody fragments binding the native γ-secretase complex for use in structural studies.

A detailed understanding of γ-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimer's disease. γ-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.

Effects of membrane lipids on the activity and processivity of purified γ-secretase.

The 19-transmembrane multisubunit γ-secretase complex generates the amyloid ß-peptide (Aß) of Alzheimer's disease (AD) by intramembrane proteolysis of the ß-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on γ-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human γ-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerization, and membrane lipid polar headgroups on γ-secretase function. We analyzed γ-secretase activity and processivity [i.e., sequential cleavages in the APP transmembrane domain that convert longer Aß species (e.g., Aß(46)) into shorter ones (e.g., Aß(40))] by quantifying the APP intracellular domain (AICD) and various Aß peptides, including via a bicine/urea gel system that detects multiple Aß lengths. These assays revealed several trends. (1) Switching from a cis to a trans isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased γ-activity, did not affect Aß(42):Aß(40) ratios, but decreased the ratio of long (≥42) versus short (≤41) Aß peptides. (2) Increasing the FA carbon chain length (14, 16, 18, and 20) increased γ-activity, reduced longer Aß species, and reduced the Aß(42):Aß(40) ratio. (3) Shifting the position of the double bond in 18:1(Δ9-cis) PC to the Δ6 position substantially reduced activity. (4) Gangliosides increased γ-activity but decreased processivity, thus elevating the Aß(42):Aß(40) ratio. (5) Phosphatidylserine decreased γ-activity but increased processivity. (6) Phosphatidylinositol strongly inhibited γ-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence γ-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as presenilin or APP mutations do.

Identification of two novel synaptic γ-secretase associated proteins that affect amyloid ß-peptide levels without altering Notch processing.

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease (AD) and results in loss of cognitive function. One of the causative agents for the synaptic degeneration is the amyloid ß-peptide (Aß), which is formed from its precursor protein by two sequential cleavages mediated by ß- and γ-secretase. We have earlier shown that γ-secretase activity is enriched in synaptic compartments, suggesting that the synaptotoxic Aß is produced locally. Proteins that interact with γ-secretase at the synapse and regulate the production of Aß can therefore be potential therapeutic targets. We used a recently developed affinity purification approach to identify γ-secretase associated proteins (GSAPs) in synaptic membranes and synaptic vesicles prepared from rat brain. Liquid chromatography-tandem mass spectrometry analysis of the affinity purified samples revealed the known γ-secretase components presenilin-1, nicastrin and Aph-1b along with a number of novel potential GSAPs. To investigate the effect of these GSAPs on APP processing, we performed siRNA experiments to knock down the expression of the GSAPs and measured the Aß levels. Silencing of NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 (NDUFS7) resulted in a decrease in Aß levels whereas silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aß levels. Treatment with γ-secretase inhibitors often results in Notch-related side effects and therefore we also studied the effect of the siRNAs on Notch processing. Interestingly, silencing of TPPP or NDUFS7 did not affect cleavage of Notch. We also studied the expression of TPPP and NDUFS7 in control and AD brain and found NDUFS7 to be highly expressed in vulnerable neurons such as pyramidal neurons in the hippocampus, whereas TPPP was found to accumulate in intraneuronal granules and fibrous structures in hippocampus from AD cases. In summary, we here report on two proteins, TPPP and NDUFS7, which interact with γ-secretase and alter the Aß levels without affecting Notch cleavage.

Identification of novel γ-secretase-associated proteins in detergent-resistant membranes from brain.

In Alzheimer disease, oligomeric amyloid ß-peptide (Aß) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aß from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aß production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aß production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aß production (Aß40 and Aß42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aß production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.

Expression analysis of human ß-secretase in transgenic tomato fruits.

An emerging strategy in biomanufacturing involves using transgenic plants to express recombinant pharmaceutical and industrial proteins in large quantities. ß-Site APP cleaving enzyme 1 (ß-secretase 1, BACE1) is an enzyme involved in the abnormal production of Aß42, the major component of senile plaques in Alzheimer's disease (AD). Thus, BACE1 represents a key target protein in the development of new potential drugs to treat Alzheimer's disease. We aimed to develop a tomato-derived recombinant BACE1 (rBACE1) protein to serve as a vaccine antigen that would promote an immune response. We utilized a plant expression cassette, pE8BACE, to optimize BACE1 expression in tomato fruits. Polyemerase chain reaction and Southern blot analyses verified integration of the BACE1 gene into the plant genome. Northern and Western blot analyses demonstrated successful mRNA and protein expression of rBACE1, respectively; the Sensizyme assay kit estimated the expression level of rBACE1 protein at 136 ± 7 ng mg⁻¹ total soluble protein. The tomato-derived rBACE1 retains its activity for a long storage period at cool or room temperature, and is highly resistant to degradation in conditions such as low acidity. Tomato-derived rBACE1 was severely degraded by heat or boiling. The proteolytic activity of tomato-derived rBACE1, confirmed by fluorescence resonance transfer assay, was similar to that of a commercial sample of Escherichia coli-derived BACE1.

Identification and biology of α-secretase.

'Secretase' is a generic term coined more than 20 years ago to refer to a group of proteases responsible for the cleavage of a vast number of membrane proteins. These endoproteolytic events result in the extracellular or intracellular release of soluble metabolites associated with a broad range of intrinsic physiological functions. α-Secretase refers to the activity targeting the amyloid precursor protein (APP) and generating sAPPα, a soluble extracellular fragment potentially associated with neurotrophic and neuroprotective functions. Several proteases from the a disintegrin and metalloproteinase (ADAM) family, including ADAM10 and ADAM17, have been directly or indirectly associated with the constitutive and regulated α-secretase activities. Recent evidence in primary neuronal cultures indicates that ADAM10 may represent the genuine constitutive α-secretase. Mainly because α-secretase cleaves APP within the sequence of Aß, the core component of the cerebral amyloid plaques in Alzheimer's disease, α-secretase activation is considered to be of therapeutic value. In this article, we will provide a historical perspective on the characterization of α-secretase and review the recent literature on the identification and biology of the current α-secretase candidates.

Cathepsins S, B and L with aminopeptidases display ß-secretase activity associated with the pathogenesis of Alzheimer's disease.

ß-site APP-cleaving enzyme (BACE1) cleaves the wild type (WT) ß-site very slowly (k(cat)/K(m): 46.6 m(-1) s(-1)). Therefore we searched for additional ß-secretases and identified three cathepsins that split the WT ß-site much faster. Human cathepsin S cleaves the WT ß-site (k(cat)/K(m): 54 700 m(-1) s(-1)) 1170-fold faster than BACE1 and cathepsins B and L are 440- and 74-fold faster than BACE1, respectively. These cathepsins split two bonds flanking the WT ß-site (K-MD-A), where the K-M bond (85%) is cleaved more efficiently than the D-A bond (15%). Cleavage at the major K-M bond yields Aß (amyloid ß-peptide) extended by N-terminal Met that should be removed to generate Aß initiated by Asp1. The activity of cytosol and microsomal aminopeptidases on relevant peptides revealed rapid removal of N-terminal Met but not N-terminal Asp. Brain aminopeptidases showed similar specificity. Thus, aminopeptidases would convert Aß extended by Met into regular Aß (Asp1) found in amyloid plaques. Earlier studies indicate that Aß is likely produced in the endosome and lysosome system where cathepsins S, B and L are localized and cysteine cathepsin inhibitors reduce the level of Aß in cells and animals. Taken together, cathepsins S, B and L deserve further evaluation as therapeutic targets to develop disease modifying drugs to treat Alzheimer's disease.

A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane.

A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (ß-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.

beta-Secretase inhibitor potency is decreased by aberrant beta-cleavage location of the "Swedish mutant" amyloid precursor protein.

The amyloid-beta (Abeta) peptide, widely known as the causative molecule of Alzheimer disease (AD), is generated by the sequential cleavage of amyloid precursor protein (APP) by the aspartyl proteases BACE1/beta-secretase and presenilin/gamma-secretase. Inhibition of BACE1, therefore, is a promising strategy for preventing the progression of AD. However, beta-secretase inhibitors (BSIs) exhibit unexpectedly low potency in cells expressing "Swedish mutant" APP (APPswe) and in the transgenic mouse Tg2576, an AD model overexpressing APPswe. The Swedish mutation dramatically accelerates beta-cleavage of APP and hence the generation of Abeta; this acceleration has been assumed to underlie the poor inhibitory activity of BSI against APPswe processing. Here, we studied the mechanism by which the Swedish mutation causes this BSI potency decrease. Surprisingly, decreased BSI potency was not observed in an in vitro assay using purified BACE1 and substrates, indicating that the accelerated beta-cleavage resulting from the Swedish mutation is not its underlying cause. By focusing on differences between the cell-based and in vitro assays, we have demonstrated here that the potency decrease is caused by the aberrant subcellular localization of APPswe processing and not by accelerated beta-cleavage or the accumulation of the C-terminal fragment of beta-cleaved APP. Because most patients with sporadic AD express wild type APP, our findings suggest that the wild type mouse is superior to the Tg2576 mouse as a model for determining the effective dose of BSI for AD patients. This work provides novel insights into the potency decrease of BSI and valuable suggestions for its development as a disease-modifying agent.

Purification, pharmacological modulation, and biochemical characterization of interactors of endogenous human gamma-secretase.

Gamma-secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimer's disease-associated beta-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human gamma-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of gamma-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of approximately 500 kDa. Purified gamma-secretase was capable of producing the major amyloid-beta peptide (Abeta) species, such as Abeta40 and Abeta42, from a recombinant APP substrate in physiological ratios. Abeta generation could be modulated by pharmacological gamma-secretase modulators. Moreover, the Abeta42/Abeta40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimer's disease mutant. Tandem mass spectrometry analysis revealed the consistent coisolation of several proteins with the known gamma-secretase core subunits. Among these were the previously described gamma-secretase interactors CD147 and TMP21 as well as other known interactors of these. Interestingly, the Niemann-Pick type C1 protein, a cholesterol transporter previously implicated in gamma-secretase-mediated processing of APP, was identified as a major copurifying protein. Affinity capture experiments using a biotinylated transition-state analogue inhibitor of gamma-secretase showed that these proteins are absent from active gamma-secretase complexes. Taken together, we provide an effective procedure for isolating endogenous gamma-secretase in considerably high grade, thus aiding further characterization of this pivotal enzyme. In addition, we provide evidence that the copurifying proteins identified are unlikely to be part of the active gamma-secretase enzyme.

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

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