A new functional screening system for identification of regulators for the generation of amyloid beta-protein

Presenilin (PS) is essential for gamma-cleavage, which is required for the generation of amyloid beta-protein (Abeta) from the beta-amyloid precursor protein. However, it remains to be clarified how gamma-cleavage is regulated. To elucidate the regulation of PS-mediated gamma-cleavage, we developed a new functional screening method for identifying cDNA that enhances gamma-cleavage. This screening system utilizes our own developed cell line, where the expression of cDNA that enhances gamma-cleavage confers puromycin resistance. The cDNA library is retrovirally delivered to the above-mentioned cell line, allowing the identification of our target cDNAs by a combination of puromycin resistance selection and Abeta assay screening. With this screening method, we isolated several cDNAs enhancing gamma-cleavage, including the previously reported Herp. Here we also demonstrate that Rab1A, identified with this screening, can be a regulator of Abeta generation. Thus, our established screening method is a powerful tool for identifying multiple regulators involved in gamma-cleavage in the Abeta generation pathway, including modulators of gamma-secretase activity or the intracellular trafficking of factors necessary for gamma-cleavage.     

The ubiquitin-like domain of Herp is involved in Herp degradation, but not necessary for its enhancement of amyloid beta-protein generation

Herp is an endoplasmic reticulum (ER)-stress-inducible membrane protein, which has a ubiquitin-like domain (ULD). However, its biological function is as yet unknown. Previously, we reported that a high expression level of Herp in cells increases the generation of amyloid beta-protein (Abeta) and that Herp interacts with presenilin (PS). Here, we addressed the role of the ULD of Herp in Abeta generation and intracellular Herp stability. We found that the ULD is not essential for the enhancement of Abeta generation by Herp expression and the interaction of Herp with PS, but is involved in the rapid degradation of Herp, most likely via the ubiquitin/proteasome pathway. Thus, the ULD of Herp most likely plays a role in the regulation of the intracellular level of Herp under ER stress.     

Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress

Hyperhomocysteinemia, a risk factor for vascular disease, injures endothelial cells through undefined mechanisms. We previously identified several homocysteine-responsive genes in cultured human vascular endothelial cells, including the endoplasmic reticulum (ER)-resident molecular chaperone GRP78/BiP. Here, we demonstrate that homocysteine induces the ER stress response and leads to the expression of a novel protein, Herp, containing a ubiquitin-like domain at the N terminus. mRNA expression of Herp was strongly up-regulated by inducers of ER stress, including mercaptoethanol, tunicamycin, A23187, and thapsigargin. The ER stress-dependent induction of Herp was also observed at the protein level. Immunochemical analyses using Herp-specific antibodies indicated that Herp is a 54-kDa, membrane-associated ER protein. Herp is the first integral membrane protein regulated by the ER stress response pathway. Both the N and C termini face the cytoplasmic side of the ER; this membrane topology makes it unlikely that Herp acts as a molecular chaperone for proteins in the ER, in contrast to GRP78 and other ER stress-responsive proteins. Herp may, therefore, play an unknown role in the cellular survival response to stress.     

PEN-2 enhances gamma-cleavage after presenilin heterodimer formation

The presenilin (PS) complex, including PS, nicastrin, APH-1 and PEN-2, is essential for gamma-secretase activity, which is required for amyloid beta-protein (Abeta) generation. However, the precise individual roles of the three cofactors in the PS complex in Abeta generation remain to be clarified. Here, to distinguish the roles of PS cofactors in gamma-secretase activity from those in PS endoproteolysis, we investigated their roles in the gamma-secretase activity reconstituted by the coexpression of PS N- and C-terminal fragments (NTF and CTF) in PS-null cells. We demonstrate that the coexpression of PS1 NTF and CTF forms the heterodimer and restores Abeta generation in PS-null cells. The generation of Abeta was saturable at a certain expression level of PS1 NTF/CTF, while the overexpression of PEN-2 alone resulted in a further increase in Abeta generation. Although PEN-2 did not enhance PS1 NTF/CTF heterodimer formation, PEN-2 expression reduced the IC50 of a specific gamma-secretase inhibitor, a transition state analogue, for Abeta generation, suggesting that PEN-2 expression enhances the affinity or the accessibility of the substrate to the catalytic site. Thus, our results strongly suggest that PEN-2 is not only an essential component of the gamma-secretase complex but also an enhancer of gamma-cleavage after PS heterodimer formation.     

Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase

We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.     

DAPT-induced intracellular accumulations of longer amyloid beta-proteins: further implications for the mechanism of intramembrane cleavage by gamma-secretase

Gamma-secretase cleaves the transmembrane domain of beta-amyloid precursor protein at multiple sites. These are referred to as gamma-, zeta-, and epsilon-cleavages. We showed previously that DAPT, a potent dipeptide gamma-secretase inhibitor, caused differential accumulations of longer amyloid beta-proteins (Abetas) (Abeta43 and Abeta46) in CHO cells that are induced to express the beta C-terminal fragment (CTF). To learn more about the cleavage mechanism by gamma-secretase, CHO cell lines coexpressing betaCTF and wild-type or mutant presenilin (PS) 1/2 were generated and treated with DAPT. In all cell lines treated with DAPT, as the levels of Abeta40 decreased, Abeta46 accumulated to varying extents. In wild-type PS1 or M146L mutant PS1 cells, substantial amounts of Abeta43 and Abeta46 accumulated. In contrast, this was not the case with wild-type PS2 cells. In M233T mutant PS1 cells, significant amounts of Abeta46 and Abeta48 accumulated differentially, whereas in N141I mutant PS2 cells, large amounts of Abeta45 accumulated concomitantly with a large decrease in Abeta42 levels. Most interestingly, in G384A mutant PS1 cells, there were no significant accumulations of longer Abetas except for Abeta46. Abeta40 was very susceptible to DAPT, but other Abetas were variably resistant. Complicated suppression and accumulation patterns by DAPT may be explained by stepwise processing of betaCTF from a zeta- or epsilon-cleavage site to a gamma-cleavage site and its preferential suppression of gamma-cleavage over zeta- or epsilon-cleavage.     

Novel cadherin-related membrane proteins,Alcadeins, enhance the X11-like protein-mediated stabilization of amyloid beta-protein precursor metabolism

Previously we found that X11-like protein (X11L) associates with amyloid beta-protein precursor (APP). X11L stabilizes APP metabolism and suppresses the secretion of the amyloid beta-protein (Abeta) that are the pathogenic agents of Alzheimer's disease (AD). Here we found that Alcadein (Alc), a novel membrane protein family that contains cadherin motifs and originally reported as calsyntenins, also interacted with X11L. Alc was abundant in the brain and occurred in the same areas of the brain as X11L. X11L could simultaneously associate with APP and Alc, resulting in the formation of a tripartite complex in brain. The tripartite complex stabilized intracellular APP metabolism and enhanced the X11L-mediated suppression of Abeta secretion that is due to the retardation of intracellular APP maturation. X11L and Alc also formed another complex with C99, a carboxyl-terminal fragment of APP cleaved at the beta-site (CTFbeta). The formation of the Alc.X11L.C99 complex inhibited the interaction of C99 with presenilin, which strongly suppressed the gamma-cleavage of C99. In AD patient brains, Alc and APP were particularly colocalized in dystrophic neurites in senile plaques. Deficiencies in the X11L-mediated interaction between Alc and APP and/or CTFbeta enhanced the production of Abeta, which may be related to the development or progression of AD.     

Homocysteine-induced endoplasmic reticulum protein (Herp) is up-regulated in sporadic inclusion-body myositis and in endoplasmic reticulum stress-induced cultured human muscle fibers

Herp is a stress-response protein localized in the endoplasmic reticulum (ER) membrane. Herp was proposed to improve ER-folding, decrease ER protein load, and participate in ER-associated degradation (ERAD). Intra-muscle-fiber ubiquitinated multiprotein-aggregates containing, among other proteins, either amyloid-beta (Abeta) or phosphorylated tau are characteristic of sporadic inclusion-body myositis (s-IBM). ER stress and proteasome inhibition appear to play a role in s-IBM pathogenesis. We have now studied Herp in s-IBM muscle fibers and in ER-stress-induced or proteasome-inhibited cultured human muscle fibers. In s-IBM muscle fibers: (i) Herp was strongly immunoreactive in the form of aggregates, which co-localized with Abeta, GRP78, and beta2 proteasome subunit; (ii) Herp mRNA and protein were increased. In ER-stress-induced cultured human muscle fibers: (i) Herp immunoreactivity was diffusely increased; (ii) Herp mRNA and protein were increased. In proteasome-inhibited cultured human muscle fibers: (i) Herp immunoreactivity was in the form of aggregates; (ii) Herp protein was increased, but its mRNA was not. Accordingly, in s-IBM muscle fibers: (i) increase of Herp might be due to both ER-stress and proteasome inhibition; (ii) co-localization of Herp with Abeta, proteasome, and ER-chaperone GRP78 could reflect its possible role in processing and degradation of cytotoxic proteins in ER.     

Hydrogen peroxide promotes Abeta production through JNK-dependent activation of gamma-secretase

Accumulation of senile plaques composed of amyloid beta-peptide (Abeta) is a pathological hallmark of Alzheimer disease (AD), and Abeta is generated through the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase. Although oxidative stress has been implicated in the AD pathogenesis by inducing Abeta production, the underlying mechanism remains elusive. Here we show that the pro-oxidant H(2)O(2) promotes Abeta production through c-Jun N-terminal kinase (JNK)-dependent activation of gamma-secretase. Treatment with H(2)O(2) induced significant increase in the levels of intracellular and secreted Abeta in human neuroblastoma SH-SY5Y cells. Although gamma-secretase-mediated cleavage of APP or C99 was enhanced upon H(2)O(2) treatment, expression of APP or its alpha/beta-secretase-mediated cleavage was not affected. Silencing of the stress-activated JNK by small interfering RNA or the specific JNK inhibitor SP600125 reduced H(2)O(2)-induced gamma-secretase-mediated cleavage of APP. JNK activity was augmented in human brain tissues from AD patients and active JNK located surrounding the senile plaques in the brain of AD model mouse. Our data suggest that oxidative stress-activated JNK may contribute to senile plaque expansion through the promotion of gamma-secretase-mediated APP cleavage and Abeta production.     

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.     

Specific interaction of VEGF165 with beta-amyloid, and its protective effect on beta-amyloid-induced neurotoxicity

beta-amyloid (Abeta) is a major component of senile plaques that is commonly found in the brain of Alzheimer's disease (AD) patient. In the previous report, we showed that an important angiogenic factor, vascular endothelial growth factor (VEGF) interacts with Abeta and is accumulated in the senile plaques of AD patients' brains. Here we show that Abeta interacts with VEGF(165) isoform, but not with VEGF(121). Abeta binds to the heparin-binding domain (HBD) of VEGF(165) with similar affinity as that of intact VEGF(165). Abeta binds mostly to the C-terminal subdomain of HBD, but with greatly reduced affinity than HBD. Therefore, the full length of HBD appears to be required for maximal binding of Abeta. Although Abeta binds to heparin-binding sequence of VEGF, it does not bind to other heparin-binding growth factors except midkine. Thus it seems that Abeta recognizes unique structural features of VEGF HBD. VEGF(165) prevents aggregation of Abeta through its HBD. We localized the core VEGF binding site of Abeta at around 26-35 region of the peptide. VEGF(165) and HBD protect PC12 cells from the Abeta-induced cytotoxicity. The mechanism of protection appears to be inhibition of both Abeta-induced formation of reactive oxygen species and Abeta aggregation.     

Attenuated hippocampus-dependent learning and memory decline in transgenic TgAPPswe Fischer-344 rats

Alzheimer's disease (AD) is characterized by increased beta amyloid (Abeta) levels, extracellular Abeta deposits in senile plaques, neurofibrillary tangles, and neuronal loss. However, the physiological role of normal levels of Abeta and its parent protein, the amyloid precursor protein (APP) are unknown. Here we report that low-level transgenic (Tg) expression of the Swedish APP mutant gene (APPswe) in Fischer-344 rats results in attenuated age-dependent cognitive performance decline in 2 hippocampus-dependent learning and memory tasks compared with age-matched nontransgenic Fischer-344 controls. TgAPPswe rats exhibit mild increases in brain APP mRNA (56.8%), Abeta-42 (21%), and Abeta-40 (6.1%) peptide levels at 12 mo of age, with no extracellular Abeta deposits or senile plaques at 6, 12, and 18 mo of age, whereas 3- to 6-fold increases in Abeta levels are detected in plaque-positive human AD patients and transgenic mouse models. The data support the hypothesis that a threshold paradigm underlies Abeta-related pathology, below which APP expression may play a physiological role in specific hippocampus-dependent tasks, most likely related to its neurotrophic role.     

Isoaspartate formation and neurodegeneration in Alzheimer's disease

We reviewed here that protein isomerization is enhanced in amyloid-beta peptides (Abeta) and paired helical filaments (PHFs) purified from Alzheimer's disease (AD) brains. Biochemical analyses revealed that Abeta purified from senile plaques and vascular amyloid are isomerized at Asp-1 and Asp-7. A specific antibody recognizing isoAsp-23 of Abeta further suggested the isomerization of Abeta at Asp-23 in vascular amyloid as well as in the core of senile plaques. Biochemical analyses of purified PHFs also revealed that heterogeneous molecular weight tau contains L-isoaspartate at Asp-193, Asn-381, and Asp-387, indicating a modification, other than phosphorylation, that differentiates between normal tau and PHF tau. Since protein isomerization as L-isoaspartate causes structural changes and functional inactivation, or enhances the aggregation process, this modification is proposed as one of the progression factors in AD. Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of isomerized proteins containing L-isoaspartate. We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in neurofibrillary tangles (NFTs) in AD. Taken together with the enhanced protein isomerization in AD brains, it is implicated that the upregulated PIMT may associate with increased protein isomerization in AD. We also reviewed studies on PIMT-deficient mice that confirmed that PIMT plays a physiological role in the repair of isomerized proteins containing L-isoaspartate. The knockout study also suggested that the brain of PIMT-deficient mice manifested neurodegenerative changes concomitant with accumulation of L-isoaspartate. We discuss the pathological implications of protein isomerization in the neurodegeneration found in model mice and AD.     

Potential link between amyloid beta-protein 42 and C-terminal fragment gamma 49-99 of beta-amyloid precursor protein

A novel cleavage of beta-amyloid precursor protein (APP), referred to as epsilon-cleavage, occurs downstream of the gamma-cleavage and generates predominantly a C-terminal fragment (CTFgamma) that begins at Val-50, according to amyloid beta-protein (Abeta) numbering. Whether this cleavage occurs independently of, or is coordinated with, gamma-cleavage is unknown. Using a cell-free system, we show here that, although Abeta40 and CTFgamma 50-99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTFgamma 49-99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of Abeta42. Furthermore, a gamma-secretase inhibitor, which suppresses Abeta40 production and paradoxically enhances Abeta42 production at low concentrations, caused the proportion of CTFgamma 50-99 to decrease and that of CTFgamma 49-99 to increase significantly. These results strongly suggest a link between the production of Abeta42 and CTFgamma 49-99 and provide an important insight into the mechanisms of altered gamma-cleavage caused by mtAPP and mtPS1/2.     

Pin1 promotes production of Alzheimer's amyloid beta from beta-cleaved amyloid precursor protein

Here we show that prolyl isomerase Pin1 is involved in the Abeta production central to the pathogenesis of Alzheimer's disease. Enzyme immunoassay of brains of the Pin1-deficient mice revealed that production of Abeta40 and Abeta42 was lower than that of the wild-type mice, indicating that Pin1 promotes Abeta production in the brain. GST-Pin1 pull-down and immunoprecipitation assay revealed that Pin1 binds phosphorylated Thr668-Pro of C99. In the Pin1-/- MEF transfected with C99, Pin1 co-transfection enhanced the levels of Abeta40 and Abeta42 compared to that without Pin1 co-transfection. In COS7 cells transfected with C99, Pin1 co-transfection enhanced the generation of Abeta40 and Abeta42, and reduced the expression level of C99, facilitating the C99 turnover. Thus, Pin1 interacts with C99 and promotes its gamma-cleavage, generating Abeta40 and Abeta42. Further, GSK3 inhibitor lithium blocked Pin1 binding to C99 by decreasing Thr668 phosphorylation and attenuated Abeta generation, explaining the inhibitory effect of lithium on Abeta generation.     

BACE1 cytoplasmic domain interacts with the copper chaperone for superoxide dismutase-1 and binds copper

The amyloidogenic pathway leading to the production and deposition of Abeta peptides, major constituents of Alzheimer disease senile plaques, is linked to neuronal metal homeostasis. The amyloid precursor protein binds copper and zinc in its extracellular domain, and the Abeta peptides also bind copper, zinc, and iron. The first step in the generation of Abeta is cleavage of amyloid precursor protein by the aspartic protease BACE1. Here we show that BACE1 interacts with CCS (the copper chaperone for superoxide dismutase-1 (SOD1)) through domain I and the proteins co-immunoprecipitate from rat brain extracts. We have also been able to visualize the co-transport of membranous BACE1 and soluble CCS through axons. BACE1 expression reduces the activity of SOD1 in cells consistent with direct competition for available CCS as overexpression of CCS restores SOD1 activity. Finally, we demonstrate that the twenty-four residue C-terminal domain of BACE1 binds a single Cu(I) atom with high affinity through cysteine residues.     

Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer's disease

The processing of amyloid precursor protein (APP) generates amyloid-beta (Abeta) peptides 1-40 and 1-42. The latter is neurotoxic and its accumulation results in amyloid fibril formation and the generation of senile plaques, the hallmark of Alzheimer's disease (AD). Whilst there has been considerable progress made in understanding the generation of Abeta by alpha-, beta- and gamma-secretase activity on APP, recently enzymes involved in the degradation of Abeta have been identified including neprilysin and insulin-degrading enzyme (IDE). We review the pathways involved in proteolytic processing of APP and discuss the potential implications of aberrant proteolysis on neurodegeneration. It is conceivable that single nucleotide polymorphisms (SNPs) in the regulatory regions of genes in these proteolytic cascades, which alter their expression, could contribute to some of the age-related changes seen in AD.     

Herp stabilizes neuronal Ca2+ homeostasis and mitochondrial function during endoplasmic reticulum stress

In response to endoplasmic reticulum (ER) stress, cells launch homeostatic and protective responses, but can also activate cell death cascades. A 54 kDa integral ER membrane protein called Herp was identified as a stress-responsive protein in non-neuronal cells. We report that Herp is present in neurons in the developing and adult brain, and that it is regulated in neurons by ER stress; sublethal levels of ER stress increase Herp levels, whereas higher doses decrease Herp levels and induce apoptosis. The decrease in Herp protein levels following a lethal ER stress occurs prior to mitochondrial dysfunction and cell death, and is mediated by caspases which generate a 30-kDa proteolytic Herp fragment. Mutagenesis of the caspase cleavage site in Herp enhances its neuroprotective function during ER stress. While suppression of Herp induction by RNA interference sensitizes neural cells to apoptosis induced by ER stress, overexpression of Herp promotes survival by a mechanism involving stabilization of ER Ca(2+) levels, preservation of mitochondrial function and suppression of caspase 3 activation. ER stress-induced activation of JNK/c-Jun and caspase 12 are reduced by Herp, whereas induction of major ER chaperones is unaffected. Herp prevents ER Ca(2+) overload under conditions of ER stress and agonist-induced ER Ca(2+) release is attenuated by Herp suggesting a role for Herp in regulating neuronal Ca(2+) signaling. By stabilizing ER Ca(2+) homeostasis and mitochondrial functions, Herp serves a neuroprotective function under conditions of ER stress.