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.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

Bip/GRP78-induced production of cytokines and uptake of amyloid-beta(1-42) peptide in microglia

In the brains of Alzheimer's disease (AD) patients, fibrillar amyloid-beta peptides (Abeta) are markedly accumulated and the microglia associate with the amyloid plaques. However, the regulation of Abeta clearance is still unclear. In the present study, we examined the effect of a chaperone protein BiP/GRP78 on the microglial function. Exogenous addition of recombinant BiP/GRP78 induced the production of cytokines such as interleukin-6 and tumor necrosis factor-alpha, but heat treatment of this protein abolished the activity. Although Abeta(1-42) did not induce cytokine production, it was taken up by the microglia. In addition, the amount of Abeta(1-42) uptake and the number of microglia that phagocytosed Abeta(1-42) were markedly increased by BiP/GRP78. Exogenous BiP/GRP78 also translocated to the endoplasmic reticulum (ER). These results suggest that BiP/GRP78 stimulates Abeta clearance in the microglia, and that dysfunction in the ER may cause the accumulation of extracellular Abeta(1-42).     

Aluminium exposure induces Alzheimer's disease-like histopathological alterations in mouse brain

Aluminium (Al) is a neurotoxic metal and Al exposure may be a factor in the aetiology of various neurodegenerative diseases such as Alzheimer's disease (AD). The major pathohistological findings in the AD brain are the presence of neuritic plaques containing beta-amyloid (Abeta) which may interfere with neuronal communication. Moreover, it has been observed that GRP78, a stress-response protein induced by conditions that adversely affect endoplasmic reticulum (ER) function, is reduced in the brain of AD patients. In this study, we investigated the correlation between the expression of Abeta and GRP78 in the brain cortex of mice chronically treated with aluminium sulphate. Chronic exposure over 12 months to aluminium sulphate in drinking water resulted in deposition of Abeta similar to that seen in congophilic amyloid angiopathy (CAA) in humans and a reduction in neuronal expression of GRP78 similar to what has previously been observed in Alzheimer's disease. So, we hypothesise that chronic Al administration is responsible for oxidative cell damage that interferes with ER functions inducing Abeta accumulation and neurodegenerative damage.     

High levels of the molecular chaperone Mdg1/ERdj4 reflect the activation state of endothelial cells

Mdg1/ERdj4, a mammalian chaperone that belongs to the HSP40 protein family, has been reported to be located in the endoplasmic reticulum (ER), is induced by ER stress, and protects ER stressed cells from apoptosis. Here we show that under normal physiological conditions, Mdg1/ERdj4 is expressed at various levels in the vasculature due to different activation states of the endothelium. To elucidate the stimuli that induce ER stress and thus upregulate Mdg1/ERdj4, we investigated the effect of several endothelium specific stressors on its expression. Mdg1/ERdj4 mRNA is induced by activated macrophages, by nitric oxide (NO) and heat shock, and during terminal cell differentiation, whereas shear stress does not affect Mdg1/ERdj4 expression levels. While the mRNA stability of BiP/GRP78 is unaffected in ER stressed cells, the stability of Mdg1/ERdj4 mRNA is prolonged during ER stress resulting in rapid increases and high levels of Mdg1/ERdj4 mRNA. Mdg1/ERdj4 protein is localized in the ER under control conditions. While heat shock induces a rapid translocation of Mdg1/ERdj4 to the nucleoli, no translocation could be observed during ER stress. This indicates that Mdg1/ERdj4 protein has diverse mechanisms to protect stressed cells from apoptosis.     

Insulin induces chaperone and CHOP gene expressions in adipocytes

Adipocyte secretes bioactive proteins called adipocytokines, and biosynthesis of secretory proteins requires molecular chaperones and folding enzymes in endoplasmic reticulum (ER). ER chaperones are known to be induced by unfolded protein response (UPR) and growth factors, however, it has not been determined how ER chaperones expression is regulated in adipocytes. Here we show that insulin treatment induced GRP78 and ERO1L mRNA levels in 3T3-L1 adipocytes. Insulin also upregulated CHOP mRNA levels, but did not induce phosphorylation of eIF2α. Pretreatment with insulin protected 3T3-L1 adipocytes against thapsigargin-mediated phosphorylation of eIF2α but did not against DTT-mediated one. In vivo mice study showed that GRP78 and CHOP expressions were regulated by feeding conditions. These results suggest that insulin signaling is important to induce mRNA expressions of GRP78 and CHOP, and may have a protective role against UPR.     

Abeta42 is essential for parenchymal and vascular amyloid deposition in mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.     

Inhibition of PTEN Attenuates Endoplasmic Reticulum Stress and Apoptosis via Activation of PI3K/AKT Pathway in Alzheimer’s Disease

Amyloid plaques and neurofibrillary tangles are pathologic hallmarks of Alzheimer’s disease (AD). Endoplasmic reticulum (ER) stress has been implicated in the loss of neurons in AD. The phosphatase and tensin homolog deleted on chromosome ten (PTEN) plays an important role in regulating neuronal survival processes. However, the direct effects of the PTEN on ER stress and apoptosis in AD have not been elucidated. In this study, we demonstrate that the expression of PTEN and ER stress related proteins, GRP78 and CHOP, increased in APP/PS1 transgenic AD mice compared with WT mice. A PTEN inhibitor, dipotassium bisperoxo-(5-hydroxypyridine-2-carboxyl)-oxovanadate (bpv) could decrease apoptosis, induce AKT phosphorylation and inhibit the ER stress response proteins in hippocampus in APP/PS1 transgenic AD model mice. Furthermore, treatment with the specific PI3K inhibitor, LY294002, significantly blocked the anti-apoptotic effects of bpv in AD mice. The expression in GRP78, CHOP and apoptosis levels by bpv was reversed after PI3K inhibitor treatment. Taken together, our results indicate that the neuroprotective role of bpv involves the suppression of ER stress via the activation of the PI3K/AKT signalling pathways in APP/PS1 transgenic AD model mice.     

Altered APP processing in PDAPP (Val717 --> Phe) transgenic mice yields extended-length Abeta peptides

Central to the pathology of Alzheimer's disease (AD) is the profuse accumulation of amyloid-beta (Abeta) peptides in the brain of affected individuals, and several amyloid precursor protein (APP) transgenic (Tg) mice models have been created to mimic Abeta deposition. Among these, the PDAPP Tg mice carrying the familial AD APP 717 Val --> Phe mutation have been widely used to test potential AD therapeutic interventions including active and passive anti-Abeta immunizations. The structure and biochemistry of the PDAPP Tg mice Abeta-related peptides were investigated using acid and detergent lysis of brain tissue, ultracentrifugation, FPLC, HPLC, enzymatic and chemical cleavage of peptides, Western blot, immunoprecipitation, and MALDI-TOF and SELDI-TOF mass spectrometry. Our experiments reveal that PDAPP mice produce a variety of C-terminally elongated Abeta peptides in addition to Abeta n-40 and Abeta n-42, as well as N-terminally truncated peptides, suggesting anomalous proteolysis of both APP and Abeta. Important alterations in the overall APP degradation also occur in this model, resulting in a striking comparative lack of CT83 and CT99 fragments, which may be inherent to the strain of mice, a generalized gamma-secretase failure, or the ultimate manifestation of the overwhelming amount of expressed human transgene; these alterations are not observed in other strains of APP Tg mice or in sporadic AD. Understanding at the molecular level the nature of these important animal models will permit a better understanding of therapeutic interventions directed to prevent, delay, or reverse the ravages of sporadic AD.     

Intracellularly generated amyloid-beta peptide counteracts the antiapoptotic function of its precursor protein and primes proapoptotic pathways for activation by other insults in neuroblastoma cells

Most mutations in amyloid precursor proteins (APPs) linked to early onset familial Alzheimer's disease (FAD) increase the production of amyloid-beta peptides ending at residue 42 (Abeta42), which are released from APP by beta- and gamma-secretase cleavage. Stably transfected cells expressing wild-type human APP (APP(WT)) were more resistant to apoptosis-inducing treatments than cells expressing FAD-mutant human APP (APP(FAD)). Preventing Abeta42 production with an M596I mutation (beta-), which blocks beta-secretase cleavage of APP, or by treatment with a gamma-secretase inhibitor increased the resistance of APP(FAD)-expressing cells to apoptosis. Exposing hAPP(FAD/beta-) cells to exogenous Abeta42 or conditioned medium from Abeta42-producing APP(FAD) cells did not diminish their resistance to apoptosis. Preventing APP from entering the distal secretory pathway, where most Abeta peptides are generated, by retaining APP in the endoplasmic reticulum (ER)/intermediate compartment (IC) increased the resistance of APP(FAD)-expressing cells to apoptosis and did not alter the resistance of APP(WT)-expressing cells. p53-mediated gene transactivation after apoptosis-inducing treatments was much stronger in APP(FAD) cells than in hAPP(WT) or hAPP(FAD/beta-) cells. In contrast, upon induction of ER stress, cells expressing APP(FAD), hAPP(FAD/beta-), or APP(WT) had comparable levels of glucose-regulated protein-78 mRNA, an unfolded protein response indicator. We conclude that Abeta, especially intracellular Abeta, counteracts the antiapoptotic function of its precursor protein and predisposes cells to p53-mediated, and possibly other, proapoptotic pathways.     

The Possible Mechanism of Advanced Glycation End Products (AGEs) for Alzheimer’s Disease

Amyloid precursor protein (APP) has been modified by β and γ-secretase that cause amyloid deposits (plaques) in neuronal cells. Glyceraldhyde-derived AGEs has been identified as a major source of neurotoxicity in Alzheimer’s disease (AD). In a previous study, we demonstrated that glyceraldehyde-derived AGEs increase APP and Aβ via ROS. Furthermore, the combination of AGEs and Aβ has been shown to enhance neurotoxicity. In mice, APP expression is increased by tail vein injection of AGEs. This evidence suggests a correlation between AGEs and the development of AD. However, the role played by AGEs in the pathogenesis of AD remains unclear. In this report, we demonstrate that AGEs up-regulate APP processing protein (BACE and PS1) and Sirt1 expression via ROS, but do not affect the expression of downstream antioxidant genes HO-1 and NQO-1. Moreover, we found that AGEs increase GRP78 expression and enhance the cell death-related pathway p53, bcl-2/bax ratio, caspase 3. These results indicate that AGEs impair the neuroprotective effects of Sirt1 and lead to neuronal cell death via ER stress. Our findings suggest that AGEs increase ROS production, which stimulates downstream pathways related to APP processing, Aβ production, Sirt1, and GRP78, resulting in the up-regulation of cell death related pathway. This in-turn enhances neuronal cell death, which leads to the development of AD.     

Rab5-stimulated up-regulation of the endocytic pathway increases intracellular beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta production

We previously identified abnormalities of the endocytic pathway in neurons as the earliest known pathology in sporadic Alzheimer's disease (AD) and Down's syndrome brain. In this study, we modeled aspects of these AD-related endocytic changes in murine L cells by overexpressing Rab5, a positive regulator of endocytosis. Rab5-transfected cells exhibited abnormally large endosomes immunoreactive for Rab5 and early endosomal antigen 1, resembling the endosome morphology seen in affected neurons from AD brain. The levels of both Abeta40 and Abeta42 in conditioned medium were increased more than 2.5-fold following Rab5 overexpression. In Rab5 overexpressing cells, the levels of beta-cleaved amyloid precursor protein (APP) carboxyl-terminal fragments (betaCTF), the rate-limiting proteolytic intermediate in Abeta generation, were increased up to 2-fold relative to APP holoprotein levels. An increase in beta-cleaved soluble APP relative to alpha-cleaved soluble APP was also observed following Rab5 overexpression. BetaCTFs were co-localized by immunolabeling to vesicular compartments, including the early endosome and the trans-Golgi network. These results demonstrate a relationship between endosomal pathway activity, betaCTF generation, and Abeta production. Our findings in this model system suggest that the endosomal pathology seen at the earliest stage of sporadic AD may contribute to APP proteolysis along a beta-amyloidogenic pathway.     

A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins

We demonstrate the existence of a large endoplasmic reticulum (ER)-localized multiprotein complex that is comprised of the molecular chaperones BiP; GRP94; CaBP1; protein disulfide isomerase (PDI); ERdj3, a recently identified ER Hsp40 cochaperone; cyclophilin B; ERp72; GRP170; UDP-glucosyltransferase; and SDF2-L1. This complex is associated with unassembled, incompletely folded immunoglobulin heavy chains. Except for ERdj3, and to a lesser extent PDI, this complex also forms in the absence of nascent protein synthesis and is found in a variety of cell types. Cross-linking studies reveal that the majority of these chaperones are included in the complex. Our data suggest that this subset of ER chaperones forms an ER network that can bind to unfolded protein substrates instead of existing as free pools that assembled onto substrate proteins. It is noticeable that most of the components of the calnexin/calreticulin system, which include some of the most abundant chaperones inside the ER, are either not detected in this complex or only very poorly represented. This study demonstrates an organization of ER chaperones and folding enzymes that has not been previously appreciated and suggests a spatial separation of the two chaperone systems that may account for the temporal interactions observed in other studies.     

Expression of beta-amyloid precursor protein-CD3gamma chimeras to demonstrate the selective generation of amyloid beta(1-40) and amyloid beta(1-42) peptides within secretory and endocytic compartments.

Amyloid beta-protein (Abeta) is the main constituent of amyloid fibrils found in senile plaques and cerebral vessels in Alzheimer's disease (AD) and is derived by proteolysis from the beta-amyloid precursor protein (APP). We have analyzed the amyloidogenic processing of APP using chimeric proteins stably transfected in Chinese hamster ovary cells. The extracellular and transmembrane domains of APP were fused to the cytoplasmic region derived from the CD3 gamma chain of the T cell antigen receptor (CD3gamma). CD3gamma contains an endoplasmic reticulum (ER) retention motif (RKK), in the absence of which the protein is targeted to lysosomes without going through the cell surface (Letourneur, F., and Klausner, R.D. (1992) Cell 69, 1143-1157). We used the wild-type sequence of CD3gamma to create an APP chimera predicted to remain in the ER (gammaAPP(ER)). Deletion of the RKK motif at the C terminus directed the protein directly to the lysosomes (gammaAPP(LYS)). A third chimera was created by removing both lysosomal targeting signals in addition to RKK (gammaAPP(DeltaDelta)). This last construct does not contain known targeting signals and consequently accumulates at the cell surface. We show by immunofluorescence and by biochemical methods that all three APP chimeras localize to the predicted compartments within the cell, thus providing a useful model to study the processing of APP. We found that Abeta(1-40) is generated in the early secretory and endocytic pathways, whereas Abeta(1-42) is made mainly in the secretory pathway. More importantly, we provide evidence that, unlike in neuronal models, both ER/intermediate compartment- and endocytic-derived Abeta forms can enter the secretable pool. Finally, we directly demonstrate that lysosomal processing is not involved in the generation or secretion of either Abeta(1-40) or Abeta(1-42).     

Amyloid beta-induced changes in nitric oxide production and mitochondrial activity lead to apoptosis

Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid beta (Abeta) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial Abeta levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular Abeta production by a functional gamma-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of Abeta in these processes. Extracellular treatment of PC12 cells with comparable Abeta concentrations only leads to weak changes, demonstrating the important role of intracellular Abeta. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable Abeta levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased Abeta levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death.     

A distinct ER/IC gamma-secretase competes with the proteasome for cleavage of APP

The deposition of amyloid-beta peptides (Abeta) in senile plaques (SPs) is a central pathological feature of Alzheimer's disease (AD). Since SPs are composed predominantly of Abeta1-42, which is more amyloidogenic in vitro, the enzymes involved in generating Abeta1-42 may be particularly important to the pathogenesis of AD. In contrast to Abeta1-40, which is generated in the trans-Golgi network and other cytoplasmic organelles, intracellular Abeta1-42 is produced in the endoplasmic reticulum/intermediate compartment (ER/IC), where it accumulates in a stable insoluble pool. Since this pool of insoluble Abeta1-42 may play a critical role in AD amyloidogenesis, we sought to determine how the production of intracellular Abeta is regulated. Surprisingly, the production of insoluble intracellular Abeta1-42 was increased by a putative gamma-secretase inhibitor as well as by an inhibitor of the proteasome. We further demonstrate that this increased generation of Abeta1-42 in the ER/IC is due to a reduction in the turnover of Abeta-containing APP C-terminal fragments. We conclude that the proteasome is a novel site for degradation of ER/IC-generated APP fragments. Proteasome inhibitors may augment the availability of APP C-terminal fragments for gamma-secretase cleavage and thereby increase production of Abeta1-42 in the ER/IC. Based on the organelle-specific differences in the generation of Abeta by gamma-secretase, we conclude that intracellular ER/IC-generated Abeta1-42 and secreted Abeta1-40 are produced by different gamma-secretases. Further, the fact that a putative gamma-secretase inhibitor had opposite effects on the production of secreted and intracellular Abeta may have important implications for AD drug design.