ß amyloid protein precursor-like
Progressive cerebral deposition of the amyloid (A beta) beta-protein is an early and invariant feature of Alzheimer's disease. A beta is derived by proteolysis from the membrane-spanning beta-amyloid precursor protein (beta APP). beta APP is processed into various secreted products, including soluble beta APP (APPs), the 4-kD A beta peptide, and a related 3-kD peptide (p3). The mechanisms regulating the polarized basolateral sorting of beta APP and its proteolytic derivatives were examined in MDCK cells. Deletion of the last 32 amino acids (residues 664-695) of the beta APP cytoplasmic tail has no influence on either the constitutive approximately 90% level of basolateral sorting of surface beta APP, or the strong basolateral secretion of APPs, A beta, and p3. However, deleting the last 42 amino acids (residues 654-695) or changing tyrosine 653 to alanine alters the distribution of cell surface beta APP so that approximately 40%-50% of the molecules are inserted apically. In parallel, A beta is now secreted from both surfaces. Surprisingly, this change in surface beta APP has no influence on the basolateral secretion of APPs and p3. This result suggests that most beta APP molecules that give rise to APPs in MDCK cells are cleaved intracellularly before reaching the surface. Consistent with this conclusion, intracellular APPs are readily detected in carbonate extracts of isolated membrane vesicles. Moreover, ammonium chloride treatment results in the equal secretion of APPs into both compartments, as occurs with other non-membranous, basolaterally secreted proteins, but it does not influence the polarity of cell surface beta APP. These results demonstrate that in epithelial cells two independent mechanisms mediate the polarized trafficking of beta APP holoprotein and its major secreted derivative (APPs) and that A beta peptides are derived in part from beta APP holoprotein targeted to the cell surface by a signal that includes tyrosine 653 (Haass, 1996).
Embryonic cortical neurons in culture contain transmembrane amyloid precursor protein (APP) capable of associating with the detergent-insoluble cytoskeleton through interactions requiring the presence of its C-terminal. These transmembrane APPs are not detectable at the surface of living cells. When neurons are fixed with paraformaldehyde alone, APP is mainly visualized close to the membrane of the axon and cell body of 40% of neurons, with virtually no dendritic staining. Membrane permeabilization with detergent or methanol extends APP immunostaining to 100% of the cells and to all compartments, including the dendrites. Taken together, these results suggest that APP in embryonic neurons is present in two compartments, one more readily detectable in some axons and cell bodies and the other distributed throughout all neurons. The axonal and somatic pool of APP detectable after paraformaldehyde fixation alone is highly and rapidly augmented after exposure to calcium ionophores. It has been proposed that calcium entry increases the amount of axonal APP close to the cell surface, but that the stabilization of the protein at the cell surface and its subsequent secretion require further physiological stimuli (Allinquant, 1994).
Beta-amyloid precursor protein is a transmembrane protein that can be processed to release a large secretory product or processed in the endosomal/lysosomal pathway without secretion. Previous studies have shown that from the cell surface, beta-amyloid precursor protein may be released after cleavage or internalized without cleavage, the latter in a pathway that both produces amyloid beta-protein and also targets some molecules to the lysosomal compartment. Analysis of beta-amyloid precursor protein trafficking is confounded by the concomitant secretion and internalization of molecules from the cell surface. To address this issue, an assay was developed, based on the binding of radioiodinated monoclonal antibody, to measure the release and internalization of cell surface beta-amyloid precursor protein in transfected cells. With this approach, it has been shown that surface beta-amyloid precursor protein is either rapidly released or internalized, such that the duration at the cell surface is very short. Approximately 30% of cell surface beta-amyloid precursor protein molecules are released. Following internalization, a fraction of molecules are recycled while the majority of molecules are rapidly sorted to the lysosomal compartment for degradation. When the C terminus of beta-amyloid precursor protein is deleted, secretion is increased by approximately 2.5-fold as compared to wild-type molecules. There is concomitant decrease in internalization in these mutant molecules as well as prolongation of the resident time on the cell surface. This observation is consistent with recent evidence that signals within the cytoplasmic domain mediate beta-amyloid precursor protein internalization (Koo, 1996).
The principal component of Alzheimer's amyloid plaques, Abeta, derives from proteolytic processing of the Alzheimer's amyloid protein precursor (APP). FE65 is a brain-enriched protein that binds to APP. Although several laboratories have characterized the APP-FE65 interaction in vitro, the possible relevance of this interaction to Alzheimer's disease has remained unclear. APP and FE65 co-localize in the endoplasmic reticulum/Golgi and possibly in endosomes. Moreover, FE65 increases translocation of APP to the cell surface, as well as both alphaAPPs and Abeta secretion. The dramatic (4-fold) FE65-dependent increase in Abeta secretion suggests that agents that inhibit the interaction of FE65 with APP might reduce Abeta secretion in the brain and therefore be useful for preventing or slowing amyloid plaque formation (Sabo, 1999).
The transmembrane protein amyloid-beta precursor protein (APP) and the vesicle-associated protein c-Jun NH(2)-terminal kinase-interacting protein-1 (JIP-1; see Drosophila APP-like protein interacting protein 1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. This study tested whether APP and JIP-1 are transported together or separately on different vesicles. It was found that, within the cellular context, JIP-1 preferentially interacts with Thr(668)-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP-JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP (Muresan, 2005).
Considerable circumstantial evidence suggests that Aβ42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Aβ42 for amyloid deposition has never been demonstrated in vivo. This was addressed by developing transgenic models that express Aβ1-40 or Aβ1-42 in the absence of human amyloid β protein precursor (APP) overexpression. Mice expressing high levels of Aβ1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Aβ1-42 accumulate insoluble Aβ1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Aβ deposits. When mice expressing Aβ1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Aβ1-42 is essential for amyloid deposition in the parenchyma and also in vessels (McGowan, 2005).
Much of the data that support a pivotal role for Aβ42 in AD have come from the study of mutations in the APP and presenilin genes that cause early-onset familial forms of AD. The vast majority of these mutations selectively increase the relative levels of Aβ42. However, even in typical late-onset AD there is evidence that Aβ42, a minor Aβ species, usually representing less then 20% of the total Aβ secreted, is both the earliest form and the predominant species deposited in the brain parenchyma. In contrast, Aβ40, the major Aβ peptide secreted by cells, appears to be the predominant species deposited in the amyloid deposits in the cerebral vasculature (congophillic angiopathy, CAA). Transgenic mouse studies using mutant APP and PS transgenes have provided some insights into the effects that altering the ratio of Aβ40 and Aβ42 have on time to onset of deposition, type of deposit (e.g., diffuse versus compact), and extent of CAA. However, such studies have not definitively identified which Aβ species are responsible for seeding amyloid deposition in either the parenchyma or vasculature (McGowan, 2005).
To address this question, transgenic mice were generated that express Aβ1-40 or Aβ1-42 without APP overexpression. For these studies cDNAs were used that express fusion proteins between the BRI protein, involved in amyloid deposition in Familial British (FBD) and Danish Dementia (FDD) and Aβ1-40 (BRI-Aβ40) or Aβ1-42 (BRI-Aβ42). Transfection of BRI-Aβ cDNAs results in high-level expression and secretion of the encoded Aβ peptide through proteolytic cleavage of the fusion protein at a furin cleavage site immediately preceding Aβ. Efficient secretion of Aβ from the BRI fusion protein distinguishes this approach from studies using Aβ minigene constructs that generate high levels of intracellular Aβ and minimal secreted Aβ. The BRI-Aβ transgenic mice generated provide substantial evidence that Aβ1-42 but not Aβ1-40 is sufficient to promote Aβ deposition in mice (McGowan, 2005).
Progressive memory loss and cognitive dysfunction are the hallmark clinical features of Alzheimer's disease (AD). Identifying the molecular triggers for the onset of AD-related cognitive decline presently requires the use of suitable animal models, such as the 3xTg-AD mice, which develop both amyloid and tangle pathology. This study characterizes the onset of learning and memory deficits in this model. Two-month-old, prepathologic mice are cognitively unimpaired. The earliest cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Aβ in the hippocampus and amygdala. Plaque or tangle pathology is not apparent at this age, suggesting that they contribute to cognitive dysfunction at later time points. Clearance of the intraneuronal Aβ pathology by immunotherapy rescues the early cognitive deficits on a hippocampal-dependent task. Reemergence of the Aβ pathology again leads to cognitive deficits. This study strongly implicates intraneuronal Aβ in the onset of cognitive dysfunction (Billings, 2005).
LRP (low-density lipoprotein receptor-related protein) is linked to Alzheimer's disease (AD). Amyloid beta-peptide Abeta40 binds to immobilized LRP clusters II and IV with high affinity (Kd = 0.6-1.2 nM) compared to Abeta42 and mutant Abeta, and LRP-mediated Abeta brain capillary binding, endocytosis, and transcytosis across the mouse blood-brain barrier are substantially reduced by the high beta sheet content in Abeta and deletion of the receptor-associated protein gene. Despite low Abeta production in the brain, transgenic mice expressing low LRP-clearance mutant Abeta develop robust Abeta cerebral accumulations much earlier than Tg-2576 Abeta-overproducing mice (see Hsiao, 1996). While Abeta does not affect LRP internalization and synthesis, it promotes proteasome-dependent LRP degradation in endothelium at concentrations > 1 microM, consistent with reduced brain capillary LRP levels in Abeta-accumulating transgenic mice, AD, and patients with cerebrovascular beta-amyloidosis. Thus, low-affinity LRP/Abeta interaction and/or Abeta-induced LRP loss at the blood-brain barrier mediate brain accumulation of neurotoxic Abeta (Deane, 2004).
The amyloid beta-protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by alpha-secretase to generate an N-terminally secreted fragment, or by beta- and gamma-secretases to generate the beta-amyloid protein (Abeta). The accumulation of Abeta in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP that contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decays in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half-lives for APP in each compartment were 1.5 h and 5.7 h, respectively. A small fraction (10%) of the total APP is secreted into the culture medium where it is degraded with a half-life of 9 h. Studies using specific protease inhibitors have demonstrated that this extracellular breakdown is due to cleavage by a trypsin-like serine protease that was secreted into the culture medium. Significantly, this protease is inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP-degrading protease (Caswell, 1999).
Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that 'protease nexin 2 (PN2)', the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibits the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Moreover, PTP and PN2 form SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP, as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass, were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP is also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrates the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease (Hook, 1999).
The cytosolic domain of the ß-amyloid precursor protein APP interacts with three PTB (phosphotyrosine binding domain)-containing adaptor proteins, Fe65, X11, and mDab1. Through these adaptors, other molecules can be recruited at the cytodomain of APP; one of these is Mena, which binds to the WW domain (a protein module with two conserved tryptophans) of Fe65. The enabled and disabled genes of Drosophila, homologs of the mammalian Mena and mDab1 genes, respectively, are genetic modulators of the phenotype observed in flies null for the Abl tyrosine kinase gene. The involvement of Mena and mDab1 in the APP-centered protein-protein interaction network suggests the possibility that Abl plays a role in APP biology. Fe65, through its WW domain, binds in vitro and in vivo the active form of Abl. Furthermore, in cells expressing the active form of Abl, APP is tyrosine-phosphorylated. Phosphopeptide analysis and site-directed mutagenesis support the hypothesis that Tyr682 of APP695 is the target of this phosphorylation. Co-immunoprecipitation experiments demonstrate that active Abl and tyrosine-phosphorylated APP also form a stable complex, which could result from the interaction of the pYENP motif of the APP cytodomain with the SH2 domain of Abl. These results suggest that Abl, Mena, and mDab1 are involved in a common molecular machinery and that APP can play a role in tyrosine kinase-mediated signaling (Zambrano, 2001).
It is worth noting that the Tyr682 of human APP695 and the YENP motif are both conserved among all the known APPs in primates, rodents, Drosophila, and Caenorhabditis and are present also in the APP-related proteins APLP1 and APLP2. Considering that the overall sequence identity between Drosophila APP (Appl) and the mammalian APPs is less than 30%, the 100% conservation of the cytosolic motif containing the phosphorylated tyrosine suggests that it plays a key functional role. This means that the understanding of the molecular basis of the different phenotypes observed in insects bearing mutations of Drosophila Abl (DAbl) and/or disabled and/or enabled should also take into account the involvement of APP. Appl null flies show behavioral defects that are rescued by human APP, and the possible correlation with the defects caused by DAbl, disabled, and enabled gene mutations is not apparent. However, one could gain better insight by the analysis of the phenotypes of insects bearing combined mutations of Appl with the other three genes. For example, the effects of disabled gene mutation on the Abl -/- flies also could be the consequence of the direct interaction of these two proteins with APP, whereas the amelioration observed in Drosophila Abl-/-;disabled-/- following the mutation of the enabled gene could be also based on the competition between the enabled and DAbl gene products for the binding to Appl through Drosophila Fe65 (Zambrano, 2001).
Although the WW domain of Fe65 interacts in vitro with both c-Abl and Abl-PP, only the complexes between Fe65 and the active form of Abl, and not those with the wild type c-Abl, were found in cell extracts. This effect could be due to a lower amount of c-Abl than Abl-PP available for the formation of the in vivo complexes; or it could be due to a low affinity of c-Abl for the WW domain of Fe65 so that, in vivo, it cannot form a significant number of complexes with Fe65 because of the competition of the other ligands of the WW domain of this protein. Furthermore, active Abl probably has a different conformation from that of c-Abl, thus acquiring a higher affinity for the WW domain. On the contrary, the APP-Abl direct interaction probably requires an active Abl, because the binding is based on a pTyr-SH2 interaction (Zambrano, 2001).
It has been hypothesized often that APP could have some role in signaling, and in a recent review article, Bothwell and Giniger (2000) suggested the possibility that intracellular signaling could be involved in the development of AD. Their hypothesis takes into account the numerous reports on various proteins that could be involved in the pathogenesis of AD and suggests a role for c-Abl as a modulator of APP biology. The results presented here support their hypothesis. A point that deserves attention concerns the possible involvement of p73 in the molecular machinery under examination. In fact, this protein is a key regulator of apoptosis that binds to and is activated by Abl as a response to DNA damage. An isoform of p73 functions as an anti-apoptotic protein in developing neurons, and the role of its phosphorylation by Abl has not been addressed. The finding that active Abl binds to APP suggests an examination of the possible regulatory effects of this binding on the p73 phosphorylation by Abl and the consequences on this regulation of the enhanced APP proteolytic processing characteristic of AD (Zambrano, 2001).
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