A novel sorting nexin modulates endocytic trafficking and alpha-secretase cleavage of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases alpha- and beta-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid beta peptide (Abeta). beta-Secretase catalyzes the first step in Abeta generation, whereas alpha-secretase cleaves within the Abeta domain, prevents Abeta generation, and generates a secreted form of APP with neuroprotective properties. At present, little is known about the cellular mechanisms that control APP alpha-secretase cleavage and Abeta generation. To explore the contributory pathways, we carried out an expression cloning screen. We identified a novel member of the sorting nexin (SNX) family of endosomal trafficking proteins, called SNX33, as a new activator of APP alpha-secretase cleavage. SNX33 is a homolog of SNX9 and was found to be a ubiquitously expressed phosphoprotein. Exogenous expression of SNX33 in cultured cells increased APP alpha-secretase cleavage 4-fold but surprisingly had little effect on beta-secretase cleavage. This effect was similar to the expression of the dominant negative dynamin-1 mutant K44A. SNX33 bound the endocytic GTPase dynamin and reduced the rate of APP endocytosis in a dynamin-dependent manner. This led to an increase of APP at the plasma membrane, where alpha-secretase cleavage mostly occurs. In summary, our study identifies SNX33 as a new endocytic protein, which modulates APP endocytosis and APP alpha-secretase cleavage, and demonstrates that the rate of APP endocytosis is a major control factor for APP alpha-secretase cleavage.     

APP processing is regulated by cytoplasmic phosphorylation

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.     

Cleavage of Alzheimer's amyloid precursor protein by alpha-secretase occurs at the surface of neuronal cells.

The amyloid precursor protein (APP) is proteolytically processed predominantly by alpha-secretase to release the ectodomain (sAPPalpha). In this study, we have addressed the cellular location of the constitutive alpha-secretase cleavage of endogenous APP in a neuronal cell line. Incubation of the neuroblastoma cell line IMR32 at 20 degrees C prevented the secretion into the medium of soluble wild-type APP cleaved by alpha-secretase as revealed by both immunoelectrophoretic blot analysis with a site-specific antibody and immunoprecipitation following metabolic labeling of the cells. No sAPPalpha was detected in the cell lysates following incubation of the cells at 20 degrees C, indicating that alpha-secretase does not cleave APP in the secretory pathway prior to or within the trans-Golgi network. Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible to inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S., et al. (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPPalpha by >92%, indicating that alpha-secretase is cleaving APP almost exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in the secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleaved primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPPalpha.     

PAR-4 is involved in regulation of beta-secretase cleavage of the Alzheimer amyloid precursor protein

Mounting evidence indicates that aberrant production and aggregation of amyloid beta-peptide (Abeta)-(1-42) play a central role in the pathogenesis of Alzheimer disease (AD). Abeta is produced when amyloid precursor protein (APP) is cleaved by beta- and gamma-secretases at the N and C termini of the Abeta domain, respectively. The beta-secretase is membrane-bound aspartyl protease, most commonly known as BACE1. Because BACE1 cleaves APP at the N terminus of the Abeta domain, it catalyzes the first step in Abeta generation. PAR-4 (prostate apoptosis response-4) is a leucine zipper protein that was initially identified to be associated with neuronal degeneration and aberrant Abeta production in models of AD. We now report that the C-terminal domain of PAR-4 is necessary for forming a complex with the cytosolic tail of BACE1 in co-immunoprecipitation assays and in vitro pull-down experiments. Overexpression of PAR-4 significantly increased, whereas silencing of PAR-4 expression by RNA interference significantly decreased, beta-secretase cleavage of APP. These results suggest that PAR-4 may be directly involved in regulating the APP cleavage activity of BACE1. Because the increased BACE1 activity observed in AD patients does not seem to arise from genetic mutations or polymorphisms in BACE1, the identification of PAR-4 as an endogenous regulator of BACE1 activity may have significant implications for developing novel therapeutic strategies for AD.     

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.     

ELISA analysis of beta-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways

Limiting beta amyloid (Abeta) production could become an important therapeutic target in Alzheimer's disease (AD). Abeta is derived by the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. A double missense mutation in APP found in a Swedish pedigree (APPsw) elevates Abeta40 and Abeta42 production. Abeta production and, therefore, beta-secretase cleavage of APPsw reportedly occur in the endoplasmic reticulum (ER), Golgi and endocytic compartments. However, the relative contribution of beta-secretase cleavage occurring in each compartment has not been determined. Experiments described here use a novel ELISA to measure the beta-cleaved product, APPswbeta. Using this ELISA, we provide new information regarding the relative amount of beta-secretase cleavage of APPsw that occurs in secretory and endocytic pathways. Using a dilysine retrieval motif to retain APPsw in the ER, we discovered that less than 15% of the beta-secretase cleavage was still detected. Experiments utilizing endocytosis-impaired mutants of APPsw revealed that little or no beta-secretase cleavage of APPsw appears to take place through endocytosis. Surprisingly, deletion of the entire cytoplasmic tail increased both APPswbeta and Abeta secretion, suggesting that protein interactions with this region normally impede beta-secretase cleavage. These results suggest that APPsw is cleaved by beta-secretase late in the secretory pathway.     

Protein kinase C-dependent alpha-secretase competes with beta-secretase for cleavage of amyloid-beta precursor protein in the trans-golgi network

The release of amyloidogenic amyloid-beta peptide (Abeta) from amyloid-beta precursor protein (APP) requires cleavage by beta- and gamma-secretases. In contrast, alpha-secretase cleaves APP within the Abeta sequence and precludes amyloidogenesis. Regulated and unregulated alpha-secretase activities have been reported, and the fraction of cellular alpha-secretase activity regulated by protein kinase C (PKC) has been attributed to the ADAM (a disintegrin and metalloprotease) family members TACE and ADAM-10. Although unregulated alpha-secretase cleavage of APP has been shown to occur at the cell surface, we sought to identify the intracellular site of PKC-regulated alpha-secretase APP cleavage. To accomplish this, we measured levels of secreted ectodomains and C-terminal fragments of APP generated by alpha-secretase (sAPPalpha) (C83) versus beta-secretase (sAPPbeta) (C99) and secreted Abeta in cultured cells treated with PKC and inhibitors of TACE/ADAM-10. We found that PKC stimulation increased sAPPalpha but decreased sAPPbeta levels by altering the competition between alpha- versus beta-secretase for APP within the same organelle rather than by perturbing APP trafficking. Moreover, data implicating the trans-Golgi network (TGN) as a major site for beta-secretase activity prompted us to hypothesize that PKC-regulated alpha-secretase(s) also reside in this organelle. To test this hypothesis, we performed studies demonstrating proteolytically mature TACE intracellularly, and we also showed that regulated alpha-secretase APP cleavage occurs in the TGN using an APP mutant construct targeted specifically to the TGN. By detecting regulated alpha-secretase APP cleavage in the TGN by TACE/ADAM-10, we reveal ADAM activity in a novel location. Finally, the competition between TACE/ADAM-10 and beta-secretase for intracellular APP cleavage may represent a novel target for the discovery of new therapeutic agents to treat Alzheimer's disease.     

A novel gamma -secretase assay based on detection of the putative C-terminal fragment-gamma of amyloid beta protein precursor

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.     

alpha- and beta-secretase: profound changes in Alzheimer's disease

The amyloid plaque, a neuropathological hallmark of Alzheimer's disease, is produced by the deposition of beta-amyloid (Abeta) peptide, which is cleaved from Amyloid Precursor Protein (APP) by the enzyme beta-secretase. Only small amounts of Abeta form in normal brain; more typically this is precluded by the processing of APP by alpha-secretase. Here, we describe a decrease in alpha-secretase (81% of normal) and a large increase in beta-secretase activity (185%) in sporadic Alzheimer's disease temporal cortex. Since alpha-secretase is present principally in neurons known to be vulnerable in Alzheimer's disease, and there is known competition between alpha- and beta-secretase for the substrate APP, it is significant that the majority of Alzheimer samples tested here were low in alpha-secretase. Eighty percent of Alzheimer brains examined had an increase in beta-secretase, a decrease in alpha-secretase, or both; which may account for the means by which the majority of people develop Alzheimer's disease.     

Bcl-2 family: life-or-death switch

Proteolytic cleavage of the amyloid protein from the amyloid protein precursor (APP) by APP secretases is a key event in Alzheimer's disease (AD) pathogenesis. alpha-Secretases cleave APP within the amyloid sequences, whereas beta- and gamma-secretases cleave on the N- and C-terminal ends respectively. The transmembrane aspartyl protease BACE has been identified as beta-secretase and several proteases (ADAM-10, TACE, PC7) may be alpha-secretases. A number of studies have suggested that presenilins could be gamma-secretases, although this remains to be demonstrated conclusively. Inhibition of beta- and gamma-secretase, or stimulation of alpha-secretase, is a rational strategy for therapeutic intervention in AD.     

Protein kinase C regulation of intracellular and cell surface amyloid precursor protein (APP) cleavage in CHO695 cells

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP alpha-secretase cleavage has been shown to involve tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP(695) (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE.     

Effects of peptides derived from BACE1 catalytic domain on APP processing

One of the hallmarks of Alzheimer's disease (AD) is the deposition of beta-amyloid (Abeta) peptides in neuritic plaques. Abeta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. beta-APP cleaving enzyme-1 (BACE1) has been shown to be the major beta-secretase and is a primary therapeutic target for AD. We report here novel BACE1 inhibitory peptidomimetics, which are derived from catalytic domains of BACE1 themselves, instead of APP cleavage sites and are structurally modified by myristoylation in N-terminus for efficient cell permeability. The peptides not only inhibited the formation of APPbeta (a soluble N-terminal fragment of APP cleaved by beta-secretase), but also significantly reduced Abeta40 production. Our results suggest a new approach for identifying inhibitory agents for the treatment of AD.     

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.     

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase

Cleavage of amyloid precursor protein (APP) by the Alzheimer's beta-secretase (BACE1) is a key step in generating amyloid beta-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with beta-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by alpha-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.     

Inhibition of receptor-mediated endocytosis demonstrates generation of amyloid beta-protein at the cell surface

Sequential cleavages of the amyloid beta-protein precursor (APP) by the beta- and gamma-secretases generate the amyloid beta-protein (A beta), which plays a central role in Alzheimer's disease. Previous work provided evidence for involvement of both the secretory and endocytic pathways in A beta generation. Here, we used HeLa cells stably expressing a tetracycline-regulated dominant-negative dynamin I (dyn K44A), which selectively inhibits receptor-mediated endocytosis, and analyzed the effects on the processing of endogenous APP. Upon induction of dyn K44A, levels of mature APP rose at the cell surface, consistent with retention of APP on the plasma membrane. The alpha-secretase cleavage products of APP were increased by dyn K44A, in that alpha-APPs in medium and the C83 C-terminal stub in the membrane both rose. The beta-secretase cleavage of APP, C99, also increased modestly. The use of specific gamma-secretase inhibitors to study the accumulation of alpha- and beta-cleavage products independent of their processing by gamma-secretase confirmed that retention of APP on the plasma membrane results in increased processing by both alpha- and beta-secretases. Unexpectedly, endogenous A beta secretion was significantly increased by dyn K44A, as detected by three distinct methods: metabolic labeling, immunoprecipitation/Western blotting, and enzyme-linked immunosorbent assay. Levels of p3 (generated by sequential alpha- and gamma-cleavage) also rose. We conclude that endogenous A beta can be produced directly at the plasma membrane and that alterations in the degree of APP endocytosis may help regulate its production. Our findings are consistent with a role for the gamma-secretase complex in the processing of numerous single-transmembrane receptors at the cell surface.     

Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.     

Inhibition of cathepsin B reduces beta-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate beta-secretase of Alzheimer's disease

The regulated secretory pathway of neurons is the major source of extracellular A beta that accumulates in Alzheimer's disease (AD). Extracellular A beta secreted from that pathway is generated by beta-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major beta-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with A beta in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to A beta in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular A beta released by the regulated secretory pathway, but CA074Me had no effect on A beta released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal beta-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate beta-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as beta-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce A beta in AD.     

RNA interference-mediated silencing of X11alpha and X11beta attenuates amyloid beta-protein levels via differential effects on beta-amyloid precursor protein processing

Processing of the beta-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by beta- and alpha-secretase to produce APP-C99 and APP-C83, which are further cleaved by gamma-secretase to produce amyloid beta-protein (Abeta) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11alpha (MINT1, encoded by gene APBA1) and X11beta (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11alpha and X11beta alters APP processing and Abeta production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11alpha and X11beta expression on APP processing and Abeta production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Abeta levels in both cell lines by inhibiting gamma-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Abeta levels, but apparently not via attenuation of gamma-secretase cleavage of APP. The notion of attenuating gamma-secretase cleavage of APP via the APP adaptor protein X11alpha is particularly attractive with regard to therapeutic potential given that side effects of gamma-secretase inhibition due to impaired proteolysis of other gamma-secretase substrates, e.g. Notch, might be avoided.     

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.     

RNA interference silencing of the adaptor molecules ShcC and Fe65 differentially affect amyloid precursor protein processing and Abeta generation

The amyloid precursor protein (APP) and its pathogenic by-product amyloid-beta protein (Abeta) play central roles in Alzheimer disease (AD) neuropathogenesis. APP can be cleaved by beta-secretase (BACE) and alpha-secretase to produce APP-C99 and APP-C83. These C-terminal fragments can then be cleaved by gamma-secretase to produce Abeta and p3, respectively. p3 has been reported to promote apoptosis, and Abeta is the key component of senile plaques in AD brain. APP adaptor proteins with phosphotyrosine-binding domains, including ShcA (SHC1), ShcC (SHC3), and Fe65 (APBB1), can bind to and interact with the conserved YENPTY motif in the APP-C terminus. Here we have described for the first time the effects of RNA interference (RNAi) silencing of ShcA, ShcC, and Fe65 expression on APP processing and Abeta production. RNAi silencing of ShcC led to reductions in the levels of APP-C-terminal fragments (APP-CTFs) and Abeta in H4 human neuroglioma cells stably overexpressing full-length APP (H4-FL-APP cells) but not in those expressing APP-C99 (H4-APP-C99 cells). RNAi silencing of ShcC also led to reductions in BACE levels in H4-FL-APP cells. In contrast, RNAi silencing of the homologue ShcA had no effect on APP processing or Abeta levels. RNAi silencing of Fe65 increased APP-CTF levels, although also decreasing Abeta levels in H4-FL-APP cells. These findings suggest that pharmacologically blocking interaction of APP with ShcC and Fe65 may provide novel therapeutic strategies against AD.