Amyloid Precursor Protein Catabolism Research Articles

Impact of Sub-chronic Aluminium-maltolate Exposure on Catabolism of Amyloid Precursor Protein in Rats

ObjectiveTo investigate the impact of sub-chronic Aluminium-maltolate [Al(mal)3] exposure on the catabolism of amyloid precursor protein (APP) in rats. MethodsForty adult male Sprague-Dawley (SD) rats were randomly divided into five groups: the control group, the maltolate group (7.56 mg/kg BW), and the Al(mal)3 groups (0.27, 0.54, and 1.08 mg/kg BW, respectively). Control rats were administered with 0.9% normal saline through intraperitoneal (i.p.) injection. Maltolate and Al(mal)3 were administered to the rats also through i.p. injections. Administration was conducted daily for two months. Rat neural behavior was examined using open field tests (OFT). And the protein expressions and their mRNAs transcription related with APP catabolism were studied using enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR). ResultsThe expressions of APP, β-site APP cleaving enzyme 1 (BACE1) and presenilin-1 (PS1) proteins and their mRNAs transcription increased gradually with the increase of Al(mal)3 doses (P<0.05). The enzyme activity of BACE1 in the 0.54 and 1.08 mg/kg Al(mal)3 groups increased significantly (P<0.05). The expression of β-amyloid protein (Aβ) 1–40 gradually decreased while the protein expression of Aβ1–42 increased gradually with the increase of Al(mal)3 doses (P<0.05). ConclusionResult from our study suggested that one of the possible mechanisms that Al(mal)3 can cause neurotoxicity is that Al(mal)3 can increase the generation of Aβ1–42 by facilitating the expressions of APP, β-, and γ-secretase.

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.

Proteomic analysis of the amyloid precursor protein fragment C99: expression in yeast

The accumulation and aggregation of fragments of amyloid precursor protein (APP) are central to the development of Alzheimer’s disease. The production of the small fragment C99 is thought to form the rate-limiting step in the APP processing pathway, which can lead to the production of the toxic Aβ peptide. It has also been suggested that the proteasome contributes to APP catabolism. While the identities and aggregation propensities of many APP fragments have been studied in vitro, the sequences, structures, and cellular sources of fragments generated in vivo remains poorly elucidated. To better identify the specific APP fragments generated in vivo and to elucidate the role of the proteasome in APP processing, we developed a C99 yeast expression system. Using Zip Tip immunocapture, a specific anti-Aβ antiserum (6E10), and matrix-assisted laser desorption ionization- time of flight mass spectrometry, we identified over one dozen APP-generated peptide fragments in wild-type yeast ( PRE1PRE2) and over three dozen unique fragments in proteasome mutant cells ( pre1- 1pre2-1) expressing C99. Based on the identities of the immunocaptured species, we propose that defects in proteasome function are compensated by other proteases and that the combination of techniques described here will be invaluable to further delineate the APP processing pathway in vivo.

Toward prevention of alzheimers disease – Potential nutraceutical strategies for suppressing the production of amyloid beta peptides

Alzheimers disease (AD) can be viewed as a vicious cycle in which excess production and deposition of amyloid beta (Abeta) peptides promote microglial activation, and the resultant production of inflammatory mediators further boosts Abeta production while inducing death and dysfunction of neurons. Abeta production is mediated by beta- and gamma-secretase activities; it is prevented by alpha-secretase activity, and insulin-degrading enzyme (IDE) catabolizes Abeta. High cellular cholesterol content increases Abeta synthesis by boosting beta-secretase activity; inhibition of cholesterol syntheses and/or stimulation of cholesterol export thus diminishes Abeta production. PPARgamma activity decreases Abeta production by promoting harmless catabolism of amyloid precursor protein while blocking the up-regulatory impact of cytokines on beta-secretase expression. Nitric oxide produced by the healthy cerebral microvasculature can suppress Abeta production by boosting expression of alpha-secretase while suppressing that of beta-secretase; conversely, cerebral ischemia provokes increased APP expression. Good insulin sensitivity and efficient brain insulin function protect by inhibiting gamma-secretase activity and increasing expression of IDE. The DHA provided by fish oil diminishes cerebral Abeta deposition in rodent AD models, for unclear reasons. Various measures which oppose microglial activation can inhibit up-regulation of beta-secretase and gamma-secretase by oxidants and cytokines, respectively. These considerations suggest that a number of nutraceutical or lifestyle measures may have potential for preventing or slowing AD: policosanol; 9-cis-beta-carotene; isomerized hops extract; DHA; measures which promote efficient endothelial NO generation, such as low-salt/potassium-rich diets, exercise training, high-dose folate, and flavanol-rich cocoa; chromium picolinate and cinnamon extract as aids for insulin sensitivity; and various agents which can oppose microglial activation, including vitamin D, genistein, and sesamin. The impact of these measures on Abeta production in rodent models of AD should be evaluated, with the intent of defining practical strategies for AD prevention.

Phosphorylation of amyloid precursor carboxy-terminal fragments enhances their processing by a gamma-secretase-dependent mechanism

In Alzheimer's disease, the complex catabolism of amyloid precursor protein (APP) leads to the production of amyloid-beta (Abeta) peptide, the major component of amyloid deposits. APP is cleaved by beta- and alpha-secretases to generate APP carboxy-terminal fragments (CTFs). Abeta peptide and amyloid intracellular domain are resulting from the cleavage of APP-CTFs by the gamma-secretase. In the present study, we hypothesize that post-translational modification of APP-CTFs could modulate their processing by the gamma-secretase. Inhibition of the gamma-secretase was shown to increase the total amount of APP-CTFs. Moreover, we showed that this increase was more marked among the phosphorylated variants and directly related to the activity of the gamma-secretase, as shown by kinetics analyses. Phosphorylated CTFs were shown to associate to presenilin 1, a major protein of the gamma-secretase complex. The phosphorylation of CTFs at the threonine 668 resulting of the c-Jun N-terminal kinase activation was shown to enhance their degradation by the gamma-secretase. Altogether, our results demonstrated that phosphorylated CTFs can be the substrates of the gamma-secretase and that an increase in the phosphorylation of APP-CTFs facilitates their processing by gamma-secretase.

Amyloid precursor protein mRNA levels in Alzheimer's disease brain

Insoluble β-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K +, APP 751, APP 770, APRP 365 and APRP 563), and those without (K −, APP 695 and APP 714,). Given the hypothesis that Aβ is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan™ real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI+ to KPI− mRNA isoforms of APP. Three separate probes, designed to recognise only KPI+ mRNA species, gave increases of between 28% and 50% in AD brains relative to controls ( p=0.002). There was no change in the mRNA levels of KPI-(APP 695) ( p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI+species increased specifically in the AD brains.

Cellular Signaling Mechanisms for Muscarinic Acetylcholine Receptors

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.

Cellular Signaling Mechanisms for Muscarinic Acetylcholine Receptors

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.

The processing and biological function of the human amyloid precursor protein (APP): lessons from different cellular models.

One of the major neuropathological hallmarks of Alzheimer's disease is the presence of senile plaques in vulnerable regions of CNS. These plaques are formed of aggregated amyloid peptide. Amyloid peptide is released by the cleavage of its precursor (APP). The establishment of cell lines expressing human APP allowed to characterize both amyloidogenic and non-amyloidogneic pathways of APP catabolism and to identify some of the proteins involved in this processing (known as secretases). This led to a better comprehension of amyloid peptide production, which needs to be further characterized since gamma-secretase is as yet not identified; moreover, we still lack a clear overview of the interactions between APP and other proteins promoting Alzheimer's disease (tau, presinilinsellipsis). An important limitation of these cell lines for studying the mechanisms involved in Alzheimer's disease is supported by the observation that human APP expression does not modify transfected cells survival. The infection of primary neuronal cultures with full-length human APP indicates that APP expression induces neuronal apoptosis by itself; this neurotoxicity does not rely on extracellular production of APP derivatives (secreted APP, amyloid peptide). It is now essential to understand, in neuronal models, the production, localization and involvement of amyloid peptide in neurodegenerative processes.

The Role of Presenilin-1 in the γ-Secretase Cleavage of the Amyloid Precursor Protein of Alzheimer's Disease

Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the gamma-secretase cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a gamma-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous gamma-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Abeta secretion. Deletion of the cytoplasmic sequence of APP (APPDeltaC) inhibits both APP endocytosis and Abeta production. When APPDeltaC and PS1 are coexpressed in CHO cells, Abeta is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Abeta without endocytosis of APP by CHO cells.

Intracellular Accumulation of Insoluble, Newly Synthesized Aβn-42 in Amyloid Precursor Protein-transfected Cells That Have Been Treated with Aβ1–42

Our early study indicates that intracellular Abeta1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Abeta1-42 also leads to the accumulation of newly synthesized intracellular Abeta. Here we describe that newly synthesized Abeta, especially Abetan-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Abeta1-42 treatment. These results suggest that intracellular Abeta may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Abeta1-40, the solid-phase intracellular pathway preferentially produces Abetan-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Abeta also share the same types of Abeta structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.

Intracellular Aβ1-42 Aggregates Stimulate the Accumulation of Stable, Insoluble Amyloidogenic Fragments of the Amyloid Precursor Protein in Transfected Cells

We have analyzed the effect of internalized amyloid beta-protein (A beta) 1-42 aggregates on the metabolism of the amyloid precursor protein (APP) in stably transfected 293 cells. The amount of potentially amyloidogenic fragments of APP immunoprecipitated by anti-carboxyl-terminal APP and anti-A beta antibodies is dramatically enhanced by the treatment of the cells with A beta 1-42, which is resistant to degradation, but not A beta 1-28, which does not accumulate in cells. This accumulation of amyloidogenic carboxyl-terminal fragments is specific, since there is relatively little effect of A beta 1-42 on the amount of the nonamyloidogenic alpha-secretase carboxyl-terminal fragment. The amyloidogenic fragments accumulate in the same nonionic detergent-insoluble fraction of the cell that contains the internalized A beta 1-42. Western analysis indicates that a subset of the amyloidogenic fragments react with antibodies that recognize a conformation of A beta that is specifically associated with aggregated forms of A beta, suggesting that the adoption of this aggregation-related conformation may be an early event which precedes the final processing that produces A beta. Pulse-chase analysis of the [35S]Met-labeled 16-kDa amyloidogenic fragment indicates that it is relatively stable in A beta 1-42-treated cells, with a half-life of approximately 50 h. This fragment is degraded with a half-life of 30 min in control cells treated with A beta 1-28. In contrast, the turnover of the nonamyloidogenic alpha-secretase product is not significantly altered by the presence of A beta 1-42. The continuous uptake of A beta 1-42 from the medium is not required for the stimulation of amyloidogenic fragment accumulation, suggesting that the presence of intracellular A beta 1-42 aggregates establishes a new pathway for APP catabolism in cells which leads to the long term stability of the fragments. If these amyloidogenic fragments of APP ultimately give rise to A beta, then the production of A beta may be an autocatalytic, "runaway" process in cells containing A beta 1-42 nuclei. It is conceivable that the accumulation of insoluble APP and amyloidogenic fragments of APP in response to A beta 1-42 aggregates may mimic the pathophysiology of dystrophic neurites, where the accumulation of intracellular APP and APP fragments has been documented by immunohistochemistry.

Catabolism of amyloid precursor protein leads to beta-amyloid formation : J. Higaki, Z. Zhong, D. Quon and B. Cordell. Scios Nova Inc. Mountain View, California 94043 USA

Catabolism of amyloid precursor protein leads to beta-amyloid formation : J. Higaki, Z. Zhong, D. Quon and B. Cordell. Scios Nova Inc. Mountain View, California 94043 USA

Evidence that β-Amyloid Protein in Alzheimer's Disease Is Not Derived by Normal Processing

The beta-amyloid protein (beta/A4), derived from a larger amyloid precursor protein (APP), is the principal component of senile plaques in Alzheimer's disease. APP is an integral membrane glycoprotein and is secreted as a carboxyl-terminal truncated molecule. APP cleavage, which is a membrane-associated event, occurred at a site located within the beta/A4 region. This suggests that an intact amyloidogenic beta/A4 fragment is not generated during normal APP catabolism. Therefore, an early event in amyloid formation may involve altered APP processing that results in the release and subsequent deposition of intact beta/A4.