In vitro processing of amyloid precursor protein by cathepsin D

The formation of beta A4 amyloid in the brains of individuals with Alzheimer's disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimer's disease.     

Aging impact on amyloid precursor protein neuronal trafficking

Neurons live a lifetime. Neuronal aging may increase the risk of Alzheimer's disease. How does neuronal membrane trafficking maintain synapse function during aging? In the normal aged brain, intraneuronal beta-amyloid (Aβ) accumulates without Alzheimer's disease mutations or risk variants. However, do changes with neuronal aging potentiate Aβ accumulation? We reviewed the membrane trafficking of the amyloid precursor protein in neurons and highlighted its importance in Aβ production. Importantly, we reviewed the evidence supporting the impact of aging on neuronal membrane trafficking, APP processing, and consequently Aβ production. Dissecting the molecular regulators of APP trafficking during neuronal aging is required to identify strategies to delay synaptic decline and protect from Alzheimer's disease.     

Copper promotes the trafficking of the amyloid precursor protein

Accumulation of the amyloid β peptide in the cortical and hippocampal regions of the brain is a major pathological feature of Alzheimer disease. Amyloid β peptide is generated from the sequential protease cleavage of the amyloid precursor protein (APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution, including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis. Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper homeostasis and the role of copper in Alzheimer disease.     

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.     

Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults

To develop a therapeutic intervention for Alzheimer's disease (AD), it is necessary to clarify the mechanisms underlying the pathogenesis of AD, in which senile plaques, neurofibrillary tangles and neuronal loss in the cerebrum are the central abnormalities. A number of studies have focused on the major component of the senile plaques, which is amyloid-beta (Abeta) and its precursor protein APP, and have investigated the roles of these molecules in the onset, progression and inhibition of AD. For multiple reasons, however, their roles in AD, especially in neuronal death, remain elusive and a unified concept for their roles has not yet been established. Recently, it has been found that APP functions normally as a neuronal surface transmembrane protein. In this article, we review the molecular mechanisms of neuronal cell death by these APP-relevant insults and discuss the functions of APP in regard to intracellular signal transducers, including c-Jun N-terminal kinase. We also revise the roles of Abeta in neuronal death and survival.     

A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis

Although there is considerable evidence suggesting that altered metabolism of beta-amyloid precursor protein (APP) and accumulation of its beta-amyloid fragment are key features of Alzheimer's disease (AD), the normal physiological function of APP remains elusive. We investigated the potential role of APP in neurons using the monoclonal antibody 22C11, which binds to the extracellular domain of the human, rat, or mouse APP. Exposure of cortical neurons to 22C11 induced morphological changes including neurite degeneration, nuclear condensation, and internucleosomal DNA cleavage that were consistent with neurons dying by apoptosis. Supporting a role for 22C11-mediated apoptosis occurring by binding to APP were data demonstrating that preincubation of 22C11 with either purified APP or a synthetic peptide (APP(66-81)) that contains the epitope for 22C11 significantly attenuated neuronal damage induced by 22C11. The specificity of 22C11 was further supported by data showing no apparent effects of either mouse IgG or the monoclonal antibody P2-1, which is specific for the aminoterminal end of human but not rat APP. In addition, biochemical features indicative of apoptosis were the formation of 120- and 150-kDa breakdown products of fodrin following treatment of cortical neurons with 22C11. Both the morphological and the biochemical changes induced by 22C11 were prevented following pretreatment of neurons with the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethyl ketone. Prior incubation of cortical neurons with GSH ethyl ester (GEE), a cell-permeable form of GSH, resulted in complete protection from the 22C11 insult, thus implicating an oxidative pathway in 22C11-mediated neuronal degeneration. This was further supported by the observation that prior treatment of neurons with buthionine sulfoximine, an inhibitor of gamma-glutamylcysteinyl synthetase, potentiated the toxic effects of 22C11. Finally, with use of compartmented cultures of hippocampal neurons, it was also demonstrated that selective application of 22C11 caused local neuritic degeneration that was prevented by the addition of GEE to the neuritic compartment. Thus, the binding of a monoclonal antibody to APP initially triggers neurite degeneration that is followed by caspase-dependent apoptosis in neuronal cultures and illustrates a novel property of this protein in neurons that may contribute to the profound neuronal cell death associated with AD.     

Intracellular pathways of folded and misfolded amyloid precursor protein degradation

A number of studies suggest that early events in the maturation of amyloid precursor protein (APP) are important in determining its entry into one of several alternative processing pathways, one of which leads to the toxic protein beta-amyloid (Abeta). In pulse-labeled APP expressing CHO cells two proteolytic systems can degrade newly translated APP: the proteosome and a cysteine protease. When N-glycosylation was inhibited by tunicamycin, the former system is the dominant mechanism of APP degradation. Without tunicamycin present, the cysteine protease is operational: cysteine protease inhibitors completely inhibit APP turnover in cells in which the secretory pathway is interrupted with brefeldin A or when alpha-secretase and endosomal degradation are also pharmacologically blocked. APP immunoprecipitated from cells extracted under mild conditions and labeled in the presence of tunicamycin exhibited greater sensitivity to endoproteinase glu-C (V8) or lys-C than from cells without drug. The V8 fragment missing in tunicamyin treated cells encompassed the KPI inhibitor insertion site but was distinct from the site of N-glycosylation. It is concluded that a conformational change caused by interrupted N-glycosylation shunts newly translated APP into the proteasomal degradation pathway. Pulse-labeled and chased cells showed an additional V8 fragment that was not present in pulsed-labeled cells and was not due to glycosylation since it was also present in cells labeled in the presence of brefeldin. This latter result indicates that an additional, delayed conformational alteration occurs in the endoplasmic reticulum.     

Expression of beta-amyloid precursor protein in reactive astrocytes following neuronal damage

Although the beta-amyloid peptide is an established core component of neuritic plaques that accumulate in Alzheimer's disease, the mechanisms responsible for its deposition are not well understood. We now report that lesions of rat hippocampal neurons cause a time-dependent, long-lasting elevation of immunoreactivity for the beta-amyloid precursor protein (APP) in neighboring astrocytes, a cell type not normally containing the protein. The increase represents astroglial expression of the protein rather than a scavenging of APP released by damaged neurons. Immunoelectron microscopy confirmed that APP-containing cells are reactive astroglia, both surrounding capillaries and within the neuropil. These results demonstrate that neuronal damage stimulates APP expression in adult brain and suggest that reactive astrocytes may be a source of the beta-amyloid that forms neuropathological plaques in Alzheimer's disease.     

Disruption of axonal transport and neuronal viability by amyloid precursor protein mutations in Drosophila.

We tested the hypothesis that amyloid precursor protein (APP) and its relatives function as vesicular receptor proteins for kinesin-I. Deletion of the Drosophila APP-like gene (Appl) or overexpression of human APP695 or APPL constructs caused axonal transport phenotypes similar to kinesin and dynein mutants. Genetic reduction of kinesin-I expression enhanced while genetic reduction of dynein expression suppressed these phenotypes. Deletion of the C terminus of APP695 or APPL, including the kinesin binding region, disrupted axonal transport of APP695 and APPL and abolished the organelle accumulation phenotype. Neuronal apoptosis was induced only by overexpression of constructs containing both the C-terminal and Abeta regions of APP695. We discuss the possibility that axonal transport disruption may play a role in the neurodegenerative pathology of Alzheimer's disease.     

Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death

Amyloid precursor protein (APP), the precursor of Abeta, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Abeta neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Abeta independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Galphao, but not that of Galphai, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of Go.     

Age-dependent degradation of amyloid precursor protein in the post-mortem mouse brain cortex

We have examined the degradation of amyloid precursor protein (APP) in the brain cortex of adult (24±2) and old (58±2) mice at different post-mortem time intervals (0, 1.5, 3, 6, 12 and 24 h). The brain cortex extract was prepared and processed for immunoblotting using antibodies against N-terminal 47–62 amino acids (Asp29) and central 301–316 amino acids containing Kunitz protease inhibitor (KPI) domain (Asp45) of APP. Asp29 (N-terminal) recognizes two bands of 140 and 112 kDa. The amount of 140 kDa is relatively higher in adult than old. The level of 112 kDa is 1.6 times lower in adult than old. It shows no remarkable change with varying post-mortem time. On the other hand, Asp45 (KPI) detects two bands of 110 and 116 kDa. While 116 kDa disappears rapidly after death of the animal, 110 kDa shows no remarkable change with different post-mortem periods. Further incubation of the disrupted tissue at 4 °C for 24 h and immunoblot analysis with Asp29 (N-terminal) shows 112 kDa in both ages but 58.5 kDa in adult and 70 kDa in old only. Analysis with Asp45 (KPI) shows only 54 kDa which increases after 3 h in adult but decreases significantly after 1.5 h and becomes undetectable at 24 h in old. Thus the present findings indicate that APP is degraded in a precise pattern and it depends on cellular intactness, post-mortem period and age of the animal.     

Inhibiting amyloid precursor protein C-terminal cleavage promotes an interaction with presenilin 1

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein, which is central to the pathogenesis of Alzheimer's disease. It has been demonstrated that PS1 regulates the gamma-secretase proteolysis of the amyloid precursor protein (APP) C-terminal fragment (APP-C100), which is the final step in amyloid-beta protein production. The mechanism and detailed pathway of this PS1 activity has yet to be fully resolved, but it may be due to a presenilin-controlled trafficking of the APP fragment or possibly an inherent PS1 proteolytic activity. We have investigated the possibility of a direct interaction of PS1 and the APP-C100 within the high molecular mass presenilin complex. However, the APP-C100 is rapidly degraded, and if it forms, then any PS1.APP complex is likely to be very transitory. To circumvent this problem, we have utilized the protease inhibitor N-acetyl-leucyl-norleucinal (LLnL) and the lysosomotropic agent NH(4)Cl, which inhibits the turnover of the APP-C100. Under these conditions, levels of the fragment increased appreciably, and as shown by glycerol gradient analysis, the APP-C100 shifted to a higher molecular mass complex that overlapped with PS1. Immunoprecipitation studies demonstrated that a significant population of the APP-C100 co-precipitated with PS1. These findings suggest that PS1 may mediate the shuttling of APP fragments and/or facilitate their presentation for gamma-secretase cleavage through a direct interaction.     

Depletion of cathepsin D by transglutaminase 2 through protein cross-linking promotes cell survival

Transglutaminase 2 (TGase 2) promotes nuclear factor-κB (NF-κB) activity through depletion of the inhibitory subunit of NF-κB (I-κBα) via protein cross-linking, leading to resolution of inflammation. Increased expression of TGase 2 contributes to inflammatory disease pathogenesis via constitutive NF-κB activation. Conversely, TGase 2 inhibition often reverses inflammation in animal models. The role of TGase 2 in apoptosis remains less clear, as both pro- and anti-apoptotic functions of TGase 2 have been demonstrated under different experimental conditions. Apoptosis is intact in a TGase 2 knock out mouse (TGase2^?/?), which is phenotypically normal. However, upon exposure to tumor necrosis factor (TNF)-α-induced apoptotic stress, mouse embryonic fibroblasts (MEFs) from TGase2^?/? mice were more sensitive to cell death than MEFs from wild-type (TGase 2^+/+) mice. In the current study, to explore the role of TGase 2 in apoptosis, TGase 2-binding proteins were identified by LC/MS. TGase 2 was found to associate with cathepsin D (CTSD). Binding of TGase 2 to CTSD resulted in the depletion of CTSD via cross-linking in vitro as well as in MEFs, leading to decreased levels of apoptosis. Furthermore, cytoplasmic CTSD levels were higher in MEFs from TGase 2^?/? mice than in those from TGase 2^+/+ mice, as were caspase 3 activation and poly (ADP-ribose) polymerase (PARP) processes. These results suggest that TGase 2, while not previously implicated as a major regulatory factor in apoptosis, may regulate the balance between cell survival and cell death through the modulation of CTSD levels.     

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.     

Acidic-rich region of amyloid precursor protein induces glial cell apoptosis

Amyloid precursor protein (APP) has several caspase cleavage sites in its C-terminal cytoplasmic domain and N-terminal extracellular domain. Caspase cleavages of APP at its cytosolic tail may result in releasing the domain and inducing cell death. During apoptosis, the N-terminal domain may also be processed at amino acids 197 and 219 by caspases leading to unmasking of an acidic-rich region (AR). In this study, AR-exposing APP was shown to inhibit cell growth after transfection into RBA-1 astrocytes and BV-2 microglial cells. The recombinant AR from residue 220 to 288 of APP (APP220-288) was produced and its biological activities were analyzed. APP220-288 induced morphological changes, cell death, and DNA fragmentation in BV-2 and RBA-1 cells. However, AR was determined to have no apparent effects in suspension cells, erythroleukemia K562 cells, and Jurkat T cells. The cytotoxicity was depending on negative charge cluster and the apoptotic activity of AR was attributed to the inhibition of cell adhesion. In BV-2 microglial cells, AR significantly stimulated Fas expression, although expressions of the pro-inflammatory cytokine genes were not detected. APP220-288 also induced nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. These findings indicate that the acidic-rich domain of APP may have apoptotic activity due to inhibition of cell adhesion and induction of iNOS and Fas expressions. Moreover, unmasking the apoptosis-induced AR may activate and exacerbate glial cells which in turn lead to further progression of the death program.     

Autophagy protein Ulk1 promotes mitochondrial apoptosis through reactive oxygen species

Regardless of rapid progression in the field of autophagy, it remains a challenging task to understand the cross talk with apoptosis. In this study, we overexpressed Ulk1 in HeLa cells and evaluated the apoptosis-inducing potential of the Ulk1 gene in the presence of cisplatin. The gain of function of Ulk1 gene showed a decline in cell viability and colony formation in HeLa cells. The Ulk1-overexpressing cells showed higher apoptotic attributes by an increase in the percentage of annexin V, escalated expression of Bax/Bcl2 ratio, and caspase-9, -3/7 activities. Further, reactive oxygen species (ROS) generation was found to be much higher in HeLa-Ulk1 than in the mock group. Scavenging the ROS by N-acetyl-L-cysteine increased cell viability and colony number as well as mitochondrial membrane potential (MMP). Our data showed that Ulk1 on entering into mitochondria inhibits the manganese dismutase activity and intensifies the mitochondrial superoxide level. The Ulk1-triggered autophagy (particularly mitophagy) resulted in a fall in ATP; thus the nonmitophagic mitochondria overwork the electron-transport cycle to replenish energy demand and are inadvertently involved in ROS overproduction that led to apoptosis. In this present investigation, our results decipher a previously unrecognized perspective of apoptosis induction by a key autophagy protein Ulk1 that may contribute to identification of its tumor-suppressor properties through dissecting the connection among cellular bioenergetics, ROS, and MMP.     

Heparin promotes ß-secretase cleavage of the Alzheimer''s amyloid precursor protein

In Alzheimer's disease, abnormal processing of the amyloid precursor protein (APP) is thought to play an important role in amyloid deposition. We investigated the effect of heparin, a highly sulfated glycosaminoglycan related to heparan sulfate, on the secretion of the ß-secretase cleavage product of APP (sAPPß) in a human neuroblastoma cell line. Heparin induced an increase in the secretion of total APP, and an even greater relative increase in the secretion of sAPPß. The effect on sAPPß was specific to heparin. These data support the hypothesis that highly sulfated heparan sulfate proteoglycans may promote amyloidogenic pathways of APP metabolism.