Immature Amyloid Precursor Protein Research Articles

APP Maturation and Intracellular Localization Are Controlled by a Specific Inhibitor of 37/67 kDa Laminin-1 Receptor in Neuronal Cells.

Amyloid precursor protein (APP) is processed along both the nonamyloidogenic pathway preventing amyloid beta peptide (Aβ) production and the amyloidogenic pathway, generating Aβ, whose accumulation characterizes Alzheimer’s disease. Items of evidence report that the intracellular trafficking plays a key role in the generation of Aβ and that the 37/67 kDa LR (laminin receptor), acting as a receptor for Aβ, may mediate Aβ-pathogenicity. Moreover, findings indicating interaction between the receptor and the key enzymes involved in the amyloidogenic pathway suggest a strong link between 37/67 kDa LR and APP processing. We show herein that the specific 37/67 kDa LR inhibitor, NSC48478, is able to reversibly affect the maturation of APP in a pH-dependent manner, resulting in the partial accumulation of the immature APP isoforms (unglycosylated/acetylated forms) in the endoplasmic reticulum (ER) and in transferrin-positive recycling endosomes, indicating alteration of the APP intracellular trafficking. These effects reveal NSC48478 inhibitor as a novel small molecule to be tested in disease conditions, mediated by the 37/67 kDa LR and accompanied by inactivation of ERK1/2 (extracellular signal-regulated kinases) signalling and activation of Akt (serine/threonine protein kinase) with consequent inhibition of GSK3β.

Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo.

Retention in endoplasmic reticulum 1 (RER1) modulates amyloid-β (Aβ) production by altering trafficking of γ-secretase and amyloid precursor protein (APP).

Aβ production is influenced by intracellular trafficking of secretases and amyloid precursor protein (APP). Retention in endoplasmic reticulum 1 (RER1) regulates the trafficking of γ-secretase and APP, thereby influences Aβ production. RER1, an ER retention/retrieval factor for γ-secretase and APP, modulates Aβ production. RER1 and its influence on γ-secretase and APP may be implicated for a safe strategy to target Aβ production. The presence of neuritic plaques containing aggregated amyloid-β (Aβ) peptides in the brain parenchyma is a pathological hallmark of Alzheimer disease (AD). Aβ is generated by sequential cleavage of the amyloid β precursor protein (APP) by β- and γ-secretase, respectively. As APP processing to Aβ requires transport through the secretory pathway, trafficking of the substrate and access to the secretases are key factors that can influence Aβ production (Thinakaran, G., and Koo, E. H. (2008) Amyloid precursor protein trafficking, processing, and function. J. Biol. Chem. 283, 29615-29619). Here, we report that retention in endoplasmic reticulum 1 (RER1) associates with γ-secretase in early secretory compartments and regulates the intracellular trafficking of γ-secretase. RER1 overexpression decreases both γ-secretase localization on the cell surface and Aβ secretion and conversely RER1 knockdown increases the level of cell surface γ-secretase and increases Aβ secretion. Furthermore, we find that increased RER1 levels decrease mature APP and increase immature APP, resulting in less surface accumulation of APP. These data show that RER1 influences the trafficking and localization of both γ-secretase and APP, thereby regulating the production and secretion of Aβ peptides.

Inhibition of acyl‐coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway

Amyloid beta-peptide (Abeta) has a central role in the pathogenesis of Alzheimer's disease (AD). Cellular cholesterol homeostasis regulates endoproteolytic generation of Abeta from the amyloid precursor protein (APP). Previous studies have identified acyl-coenzyme A: cholesterol acyltransferase (ACAT), an enzyme that regulates subcellular cholesterol distribution, as a potential therapeutic target for AD. Inhibition of ACAT activity decreases Abeta generation in cell- and animal-based models of AD through an unknown mechanism. Here we show that ACAT inhibition retains a fraction of APP molecules in the early secretory pathway, limiting the availability of APP for secretase-mediated proteolytic processing. ACAT inhibitors delayed the trafficking of immature APP molecules from the endoplasmic reticulum (ER) as shown by metabolic labeling and live-cell imaging. This resulted in partial ER retention of APP and enhanced ER-associated degradation of APP by the proteasome, without activation of the unfolded protein response pathway. The ratio of mature APP to immature APP was reduced in brains of mice treated with ACAT inhibitors, and strongly correlated with reduced brain APP-C99 and cerebrospinal fluid Abeta levels in individual animals. Our results identify a novel ACAT-dependent mechanism that regulates secretory trafficking of APP, likely contributing to decreased Abeta generation in vivo.

Sortilin-related Receptor with A-type Repeats (SORLA) Affects the Amyloid Precursor Protein-dependent Stimulation of ERK Signaling and Adult Neurogenesis

Sortilin-related receptor with A-type repeats (SORLA) is a sorting receptor that impairs processing of amyloid precursor protein (APP) to soluble (s) APP and to the amyloid beta-peptide in cultured neurons and is poorly expressed in patients with Alzheimer disease (AD). Here, we evaluated the consequences of Sorla gene defects on brain anatomy and function using mouse models of receptor deficiency. In line with a protective role for SORLA in APP metabolism, lack of the receptor results in increased amyloidogenic processing of endogenous APP and in aggravated plaque deposition when introduced into PDAPP mice expressing mutant human APP. Surprisingly, increased levels of sAPP caused by receptor deficiency correlate with pro-found stimulation of neuronal ERK signaling and with enhanced neurogenesis, providing in vivo support for neurotrophic functions of sAPP. Our data document a role for SORLA not only in control of plaque burden but also in APP-dependent neuronal signaling and suggest a molecular explanation for increased neurogenesis observed in some AD patients.

SorLA/LR11 Regulates Processing of Amyloid Precursor Protein via Interaction with Adaptors GGA and PACS-1

SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation.

An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β-peptide generation

Aging of the brain is characterized by marked changes in the expression levels of the neurotrophin receptors, TrkA and p75(NTR). An expression pattern in which TrkA predominates in younger animals switches to one in which p75(NTR) predominates in older animals. This TrkA-to-p75(NTR) switch is accompanied by activation of the second messenger ceramide, stabilization of beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1), and increased production of amyloid beta-peptide (Abeta). Here, we show that the insulin-like growth factor-1 receptor (IGF1-R), the common regulator of lifespan and age-related events in many different organisms, is responsible for the TrkA-to-p75(NTR) switch in both human neuroblastoma cell lines and primary neurons from mouse brain. The signaling pathway that controls the level of TrkA and p75(NTR) downstream of the IGF1-R requires IRS2, PIP3/Akt, and is under the control of PTEN and p44, the short isoform of p53. We also show that hyperactivation of IGF1-R signaling in p44 transgenic animals, which show an accelerated form of aging, is characterized by early TrkA-to-p75(NTR) switch and increased production of Abeta in the brain.

Down-regulation of Endogenous Amyloid Precursor Protein Processing due to Cellular Aging

Processing of amyloid precursor protein (APP) is a well acknowledged central pathogenic mechanism in Alzheimer disease. However, influences of age-associated cellular alterations on the biochemistry of APP processing have not been studied in molecular detail so far. Here, we report that processing of endogenous APP is down-regulated during the aging of normal human fibroblasts (IMR-90). The generation of intracellular APP cleavage products C99, C83, and AICD gradually declines with increasing life span and is accompanied by a reduced secretion of soluble APP (sAPP) and sAPPalpha. Further, the maturation of APP was reduced in senescent cells, which has been shown to be directly mediated by age-associated increased cellular cholesterol levels. Of the APP processing secretases, protein levels of constituents of the gamma-secretase complex, presenilin-1 (PS1) and nicastrin, were progressively reduced during aging, resulting in a progressive decrease in gamma-secretase enzymatic activity. ADAM10 (a disintegrin and metalloprotease 10) and BACE (beta-site APP-cleaving enzyme) protein levels exhibited no age-associated regulation, but interestingly, BACE enzymatic activity was increased in aged cells. PS1 and BACE are located in detergent-resistant membranes (DRMs), well structured membrane microdomains exhibiting high levels of cholesterol, and caveolin-1. Although total levels of both structural components of DRMs were up-regulated in aged cells, their particular DRM association was decreased. This age-dependent membrane modification was associated with an altered distribution of PS1 and BACE between DRM and non-DRM fractions, very likely affecting their APP processing potential. In conclusion, we have found a significant modulation of endogenous APP processing and maturation in human fibroblasts caused by age-associated alterations in cellular biochemistry.

Spatial Segregation of γ-Secretase and Substrates in DistinctMembraneDomains

Gamma-secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional gamma-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and gamma-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking gamma-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic immunoisolation studies indicate recruitment of APP CTFs into cholesterol- and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active gamma-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WX-soluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that gamma-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of gamma-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates.

Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5

The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP β (sAPP β) and β-amyloid peptide (Aβ), the major component of senile plaques in patient brains inflicted with Alzheimer’s disease (AD), or soluble APP α (sAPP α) and a peptide smaller than Aβ. One of the protein kinases known to phosphorylate APP Thr668 is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative effects of p35 and p25 on APP Thr668 phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP Thr668 phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP Thr668 phosphorylation, specifically, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these differential effects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of Aβ, sAPP β, as well as sAPP α. These results confirm the involvement of Cdk5 in APP processing, and suggest that p35- and p25-mediated Cdk5 activities lead to discrete APP phosphorylation.

Differential Roles of JIP Scaffold Proteins in the Modulation of Amyloid Precursor Protein Metabolism

We previously found that the JNK-interacting proteins JIP1b and JIP2 associate with the cytoplasmic domain of the Alzheimer's amyloid precursor protein (APP) (Taru, H., Iijima, K., Hase, M., Kirino, Y., Yagi, Y., and Suzuki, T. (2002) J. Biol. Chem. 277, 20070-20078). This interaction involves the carboxyl-terminal phosphotyrosine interaction (PI) domain of JIP1b or JIP2 and the GYENPTY motif in the APP cytoplasmic domain. The expression of JIP1b stabilizes immature APP and suppresses the production of a secreted large extracellular amino-terminal domain of APP, the generation of a cleaved intracellular carboxyl-terminal fragment of APP, and the secretion of beta-amyloid 40 and 42. Deletion of the PI domain or alteration of PI amino acid residues prevents JIP1b from interacting with APP and affecting its metabolism, but deletion of the JNK-binding domain of JIP1b has no effect. JIP2, a weaker APP-binding protein, does not influence the processing of APP, although it is known that both JIP1b and JIP2 equally regulate the JNK signaling cascade. The present results suggest that JIP1b can directly modulate APP metabolism by interacting with the APP cytoplasmic domain, independent of its regulation of the JNK signaling cascade.

Pulse-Chase Experiments Revealed β-Secretase Cleavage from Immature Full-Length Amyloid Precursor Protein Harboring the Swedish Mutation: Implications for Distinct Pathways

The molecular mechanisms of the nonamyloidogenic and the amyloidogenic pathways of the amyloid precursor protein (APP) are unknown, but proteolysis of APP is essential for the generation of beta-amyloid. To study the time-course of C-terminal fragment generation by alpha- and beta-secretase, we expressed the APP751 isoform with the Swedish mutation in the human neuroblastoma cell line SY5Y as previously described (Urmoneit et al., 1995). We show in pulse-chase experiments that the C-terminal fragments, CT, generated by alpha-secretase and A4CT, generated by beta-secretase, could be generated from immature full-length APP before O-glycosylation is completed. Thus beta A4 may be generated from immature APP that has not passed through the trans-Golgi-network (TGN), which presents experimental evidence for the intracellular localization of beta-secretase activity in an earlier Golgi complex.

The Cytoplasmic Domain of Alzheimer’s Amyloid Precursor Protein Is Phosphorylated at Thr654, Ser655, and Thr668 in Adult Rat Brain and Cultured Cells

The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform). We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines. We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells. The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.

Iron Levels Modulate α‐Secretase Cleavage of Amyloid Precursor Protein

The amyloid precursor protein (APP) is a membrane-spanning glycoprotein that is the source of beta A4 peptides, which aggregate in Alzheimer's disease to form senile plaques. APP is cleaved within the beta A4 sequence to release a soluble N-terminal derivative (APPsol), which has a wide range of trophic and protective functions. In the current study we have examined the hypothesis that iron availability may modulate expression or processing of APP, whose mRNA contains, based on sequence homology, a putative iron response element (IRE). Radiolabeled APP and its catabolites were precipitated from lysates and conditioned medium of stably transfected HEK 293 cells using antibodies selective for C-terminal, beta A4, and N-terminal domains. The relative abundance of the different APP catabolites under different conditions of iron availability was determined by quantitative densitometry after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show a specific effect on the production of APPsol. Using standard conditions previously established for IRE studies, it was found that iron chelation reduces APPsol production, whereas iron level elevation augments it. No changes were observed in levels of immature and mature APP holoprotein or in the C-terminal alpha-secretase derivative C83, beta A4, and p3 peptides. The specificity for modulatory changes in APPsol suggests that iron acts at the level of alpha-secretase activity. In addition to its modulatory effects, iron at very high levels was found to inhibit maturation of APP and production of its downstream catabolites without blocking formation of immature APP. The data establish a potential physiological role for iron in controlling the processing of APP.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of energy metabolism alters the processing of amyloid precursor protein and induces a potentially amyloidogenic derivative.

The cellular mechanisms which lead to the generation and pathological deposition of beta amyloid in Alzheimer's disease are unknown. In this report we describe the proteolytic processing of the amyloid precursor protein (APP) to an 11.5-kDa COOH-terminal derivative which contains the full-length beta amyloid sequence. This processing step normally occurs at low levels in parallel with APP maturation in the secretory pathway. Inhibition of oxidative energy metabolism by sodium azide or the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone increased the proteolysis of APP to the 11.5-kDa derivative by about 80-fold with accumulation of this APP derivative in the Golgi complex. Agents which inhibit protein transport in the secretory pathway, including monensin and brefeldin A, also increased the production of the 11.5-kDa derivative. Inhibition of APP maturation demonstrated that the 11.5-kDa derivative could be produced by proteolysis of immature APP. These results demonstrate that APP processing to potentially amyloidogenic COOH-terminal derivatives occurs in either the endoplasmic reticulum or Golgi complex and can be modulated by the state of cellular energy metabolism. Deficits in oxidative energy metabolism have recently been found in the cerebral cortex of patients with Alzheimer's disease. These findings raise the possibility that energy-related metabolic stress may lead to altered metabolism of APP and contribute to amyloidosis in Alzheimer's disease.

Inhibitors of endocytosis, endosome fusion, and lysosomal processing inhibit the intracellular proteolysis of the amyloid precursor protein

Degradation of the amyloid precursor protein (APP) by lysosomes has been proposed to be the mechanism for generation of the β A4 polypeptide which is the major constituent of amyloid plaques. In this report, we use inhibitors to elucidate the steps involved in the lysosomal degradation of APP in PC12 cells. Monensin treatment significantly elevated the level of immature APP. Reducing the temperature to 17°C, adding cytochalasin B and colchicine, or exchanging K + for Na + resulted in a substantial accumulation of both mature and immature APP isoforms. The inhibitor of autophagy, 3-methyladenine, had no effect on the level of APP isoforms. These results suggest that changes in ionic balance, membrane fluidity or vesicle fusion may affect APP processing.