Defective neurite extension is caused by a mutation in amyloid β/A4 (Aβ) protein precursor found in Familial Alzheimer's Disease

Clonal central nervous system neuronal cells, B103, do not synthesize detectable endogenous APP or APLP. B103 cells transfected with both wild-type (B103/APP) and mutant APP construct (B103/APPΔNL) secreted comparable amounts of soluble forms of APP (sAPP). B103/APP cells produced sAPP and cleaved at amyloid β/A4 (Aβ) 16, the α-secretase site, and B103/APPΔNL cells produced sAPPβ cleaved at Aβ 1, the β-secretase site. B103/APPΔNL cells developed fewer neurites than B103/APP cells in a serum-free defined medium. Neurite numbers of parent B103 cells were increased by the 50% conditioned medium (CM) from B103/APP cells but reduced by the CM from B103/APPΔNL cells. Chemically synthesized Aβ at concentration levels higher than 1 nM reduced numbers of neurites from B103 or B103/APPΔNL cells. However, Aβ at 1–100 nM could not reduce the neurite number of B103/APP cells. The protective activity against Aβ's deleterious effect to reduce neurite numbers was attributed to sAPPα in the CM. Although sAPPα could block the effect of Aβ, sAPPβ could not do so under the identical condition, suggesting the importance of the C-terminal 15-amino acid sequence in sAPPα. Nevertheless, sAPPα's protective activity required the N-terminal sequence around RERMS, previously identified to be the active domain of sAPPβ. The overall effect of APP mutation which overproduced Aβ and sAPPβ and underproduced sAPPα was a marked decline in the neurotrophic effect of APP. We suggest that the disruption of balance between the detrimental effect of Aβ and the trophic effect of sAPP may be important in the pathogenesis of AD caused by this pathogenic APP mutation © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 469–480, 1997     

Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen-Activated Protein Kinase Cascade

Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β-peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen-activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12-M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC-independent pathway, increased sAPP secretion mainly through a MAP kinase-independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.     

Functions of Aβ, sAPPα and sAPPβ : similarities and differences

Amyloid peptide (Aβ) is derived from the cleavage of amyloid precursor protein (APP), which also generates the soluble peptide APPβ (sAPPβ). An antagonist and major APP metabolic pathway involves cleavage by alpha secretase, which releases sAPPα. Although soluble Aβ oligomers are neurotoxic, Aβ monomers share similar properties with sAPPα. These include neurotrophic and neuroprotective effects, as well as stimulation of neural-progenitor proliferation. The properties of Aβ monomers and the neurotrophic capacity of sAPPβ to stimulate axonal outgrowth suggest that Aβ production is not deleterious per se. Consequently, therapeutic strategies for Alzheimer's disease that are targeted at Aβ-cleaving enzymes should modulate rather than inhibit Aβ generation. These strategies should focus on the factors that induce the conversion of Aβ monomers into toxic soluble oligomers. Another interesting therapeutic approach is to focus on the mechanisms of the different properties of sAPPα. Indeed, increasing sAPPα levels could shift proliferating cells towards tumorigenesis. In contrast to its neuroprotective effects, sAPPα is also able to activate microglia, leading to neurotoxicity. Understanding the mechanisms that underlie the different properties of sAPPα could therefore lead to the development of therapeutic strategies against Alzheimer's disease, which could be curative as well as preventive.     

Novel processing of beta-amyloid precursor protein catalyzed by membrane type 1 matrix metalloproteinase releases a fragment lacking the inhibitor domain against gelatinase A

In various mammalian cell lines, beta-amyloid precursor protein (APP) is proteolytically processed to release its NH(2)-terminal extracellular domain as a soluble APP (sAPP) that contains the inhibitor domain against gelatinase A. To investigate roles of sAPP in the regulation of gelatinase A activity, we examined the correlation between the activation of progelatinase A and processing of APP. We found that stimulation of HT1080 fibrosarcoma cells with concanavalin A led to an activation of endogenous progelatinase A and to a novel processing of APP, which releases a COOH-terminally truncated form of sAPP (sAPPtrc) into the culture medium. Reverse zymographic analysis showed that sAPPtrc lacked an inhibitory activity against gelatinase A. Analyses of production of sAPPtrc in the presence of various metalloproteinase inhibitors showed that membrane type 1 matrix metalloproteinase (MT1-MMP), an activator of progelatinase A, is most likely responsible for the production of sAPPtrc. When the concanavalin A-stimulated HT1080 cells were cultured in the condition that inhibited MT1-MMP activity, sAPP and APP were associated with the extracellular matrix deposited by the cells, whereas these gelatinase A inhibitors in the matrix were displaced by sAPPtrc after exertion of MT1-MMP activity. Taken together, these data support a model in which MT1-MMP-catalyzed release of sAPPtrc leads to reduction of the extracellular matrix-associated gelatinase A inhibitor, sAPP, thus making it feasible for gelatinase A to exert proteolytic activity only near its activator, MT1-MMP.     

Amyloid precursor protein-processing products affect mononuclear phagocyte activation: pathways for sAPP- and Abeta-mediated neurotoxicity

Increasing evidence strongly supports the role of glial immunity in the pathogenesis of Alzheimer's disease (AD). To investigate such events we have developed cell systems mimicking the interactions between beta-amyloid precursor protein (APP)-expressing neurons and brain mononuclear phagocytes (MP; macrophages and microglia). MP were co-cultured with neuronal cells expressing wild type APP or familial AD-linked APP mutants. The latter was derived from recombinant adenoviral constructs. Neuronal APP processing products induced MP activation, reactive oxygen species, and neurotoxic activities. These occurred without the addition of pro-inflammatory cytokines and were reversed by depletion of amyloid beta-peptide (Abeta) and secreted APP (sAPP). Neurotoxic activities were diminished by superoxide dismutase mimetics and NMDA receptor inhibitors. Microglial glutamate secretion was suppressed by the cystine-glutamate antiporter inhibitor and its levels paralleled the depletion of sAPP and Abeta from conditioned media prepared from APP-expressing neurons. The excitotoxins from activated MP were potent enough to evoke recombinant NMDA receptor-mediated inward currents expressed in vitro in the Xenopus oocytes. These results demonstrate that neuronal APP-processing products can induce oxidative neurotoxicity through microglial activation.     

Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing

The amyloid precursor protein (APP) is a key protein involved in the development of Alzheimer's disease. We previously identified a signal transduction secretory pathway in which the small G protein Rac sets downstream of the cAMP/Epac/Rap1 signalling cascade regulating the alpha cleavage of APP [Maillet, M. et al. (2003) Crosstalk between Rap and Rac regulates secretion of sAPP alpha. Nat. Cell Biol. 5, 633-639]. We now report that Rap1 can physically and specifically associate with the guanine nucleotide exchange factor (GEF) STEF through its TSS region. A deleted TSS domain of STEF cells fails to activate Rac1 and dramatically decreases secretion of the non-amyloidogenic soluble form of APP (sAPP alpha) induced by the cAMP-binding protein Epac. Altogether, our data show that upon Epac activation, Rap1 recruits STEF through its TSS region and activates Rac1, which mediates APP processing.     

Polarized transport of Alzheimer amyloid precursor protein is mediated by adaptor protein complex AP1-1B

Alzheimer amyloid precursor protein (APP) is the precursor for the Abeta peptide involved in pathogenesis of Alzheimer's disease. The soluble ectodomain fragment of APP (sAPP) functions as a growth factor for epithelial cells, suggesting an important function for APP outside neuronal tissue. Previous studies have shown that in polarized epithelial cells, APP is targeted to the basolateral domain. Tyr653 within the cytoplasmic tail of APP mediates the basolateral targeting of APP, but the sorting machinery that binds to this residue has largely remained unknown. In this study, we analyzed the role of adaptor complexes in the polarized sorting of APP. We show that the medium subunit mu1B of the epithelia-specific adaptor protein (AP)-1B binds onto the cytoplasmic tail of APP in a Tyr653-dependent way. Moreover, ectopic expression of mu1B in cells lacking AP-1B resulted in correction of apical missorting of wild-type but not Tyr653Ala APP. Basolateral secretion of sAPP was found to be independent of Tyr653. We propose a model for polarized targeting of APP according to which sorting of APP to basolateral domain is dependent on binding of AP-1B on Tyr653 in basolateral endosomes. This model is in accordance with the current understanding of sorting mechanisms mediating polarized targeting of membrane proteins.     

A possible role for the Alzheimer amyloid precursor protein in the regulation of epidermal basal cell proliferation

The regulation of epidermal growth involves a number of ions, growth factors and cytokines and possibly additional but as yet unknown factors. Here we report on the potential role of the secretory N-terminal domain (sAPP) of the Alzheimer amyloid precursor protein (APP) in the regulation of keratinocyte proliferation. In human skin APP was detectable predominantly in the basal cell layer of the epidermis whereas the immunocytochemical signal in the underlying mesenchymal tissue was very low. Cultured normal human keratinocytes expressed the three APP isoforms 695, 751 and 770 with highest values for the isoforms 751 and 770. HaCaT cells, a spontaneously immortalized human keratinocyte cell line, exhibited almost identical patterns in the expression of the APP isoforms and in the release of endogenous sAPP. In HaCaT cells, recombinant sAPP (sAPPrec) was found to compete with endogenous sAPP for the same binding sites. Binding of sAPPrec was specific and occurred in microdomains of approximately 0.1 to approximately 0.3 microm in diameter. At 10 nM, sAPPrec binding induced a 2- to 4-fold increase in the rate of cell growth. sAPP concentrations in the conditioned media were found to reach 5-20 nM which is in the mitogenic range of sAPPrec. The proliferative effect of sAPP was inhibited by approximately 50% when antisense oligonucleotides directed against the APP mRNA were applied. The predominant expression of     

Secreted type of amyloid precursor protein induces glial differentiation by stimulating the BMP/Smad signaling pathway

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases leading to dementia. Although cytotoxicity of amyloid β peptides has been intensively studied within pathophysiology of AD, the physiological function of amyloid precursor protein (APP) still remains unclarified. We have shown previously that secreted APPα (sAPPα) is associated with glial differentiation of neural stem cells. To elucidate specific mechanisms underlying sAPPα-induced gliogenesis, we examined the potential involvement of bone morphogenic proteins (BMPs). BMPs are one of the factors involved in glial differentiation of neural progenitor cells. When expressions of BMP-2, -4, and -7 were examined, upregulation of BMP-4 expression was solely observed as a result of treatment with sAPPα in a time and dose-dependent manner. Furthermore, the treatment of sAPPα promoted phosphorylation of Smad1/5/8, a downstream signaling mediator of BMP receptors. Interestingly, N-terminal domain of APP (1–205) was sufficient to elevate BMP4 expression, resulting in an increase of glial fibrillary acidic protein (GFAP) expression and phosphorylation of Smad1/5/8. However, the application of APP neutralizing antibody and anti-BMP4 antibody significantly suppressed expression of BMP-4 as well as phosphorylation of Smad1/5/8. Thus, our results indicate that sAPPα-induced gliogenesis is in part mediated by the BMP-4 signaling pathway. We also observed upregulation of BMP-4 and phosphorylation of Smad1/5/8 in APP transgenic mice. It is imperative to unravel the mechanisms underlying the role of BMP-4 during APPα-induced glial differentiation in hope of providing novel prevention or treatment for AD.     

APP independent and dependent effects on neurite outgrowth are modulated by the receptor associated protein (RAP)

Amyloid precursor protein (APP) and its secreted form, sAPP, contribute to the development of neurons in hippocampus, a brain region critical for learning and memory. Full‐length APP binds the low‐density lipoprotein receptor‐related protein (LRP), which stimulates APP endocytosis. LRP also contributes to neurite growth. Furthermore, the receptor associated protein (RAP) binds LRP in a manner that blocks APP–LRP interactions. To elucidate APP contributions to neurite growth for full‐length APP and sAPP, we cultured wild type (WT) and APP knockout (KO) neurons in sAPPα and/or RAP and measured neurite outgrowth at 1 day in vitro. Our data reveal that WT neurons had less axonal outgrowth including less axon branching. RAP treatment potentiated the inhibitory effects of APP. KO neurons had significantly more outgrowth and branching, especially in response to RAP, effects which were also associated with ERK2 activation. Our results affirm a major inhibitory role by full‐length APP on all aspects of axonal and dendritic outgrowth, and show that RAP–LRP binding stimulated axon growth independently of APP. These findings support a major role for APP as an inhibitor of neurite growth and reveal novel signaling functions for LRP that may be disrupted by Alzheimer's pathology or therapies aimed at APP processing.     

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(beta) (sAPP(beta)) and beta-amyloid peptide (Abeta), the major component of senile plaques in patient brains inflicted with Alzheimer's disease (AD), or soluble APP(alpha) (sAPP(alpha)) and a peptide smaller than Abeta. 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 Abeta, sAPP(beta), as well as sAPP(alpha). These results confirm the involvement of Cdk5 in APP processing, and suggest that p35- and p25-mediated Cdk5 activities lead to discrete APP phosphorylation.     

Expression and potential function of beta-amyloid precursor proteins during cutaneous wound repair

sAPP, the secretory domain of the beta-amyloid precursor protein (APP), exerts a growth promoting and motogenic activity on keratinocytes. Here we report on the expression of APP and its homologue, the amyloid precursor like protein 2 (APLP2), during cutaneous wound repair using a full-thickness excisional wound healing model in mice. In unwounded skin APP was predominantly expressed in the basal cell layer. During wound healing increased suprabasal expression of APP was observed in all cell layers of the hyperproliferative epithelium at the wound margin. APP mRNA was increased up to 2.3-fold, whereas the APLP2 mRNA was decreased. Immunocytochemically, all proliferation competent keratinocytes of the normal as well as the wound site epidermis showed increased expression of APP but not of APLP2. Using culture models of keratinocyte differentiation the release of sAPP was found to be significantly higher in proliferating cells, i.e., when cultured at subconfluency or at low [Ca(2+)], than in quiescent, partially differentiated keratinocytes cultured at confluency or at high [Ca(2+)]. Our results suggest that sAPP secretion is presumably also increased in proliferation competent keratinocytes of the wound margin and that sAPP due to its growth promoting and motogenic function might participate in the control of epidermal wound repair.     

Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone

The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. As neurogenesis is an ongoing process in the adult mammalian brain, we have investigated a role for sAPP in adult neurogenesis. We show that the subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site and that binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system.     

Rivastigmine lowers Aβ and increases sAPPα levels, which parallel elevated synaptic markers and metabolic activity in degenerating primary rat neurons

Overproduction of amyloid-β (Aβ) protein in the brain has been hypothesized as the primary toxic insult that, via numerous mechanisms, produces cognitive deficits in Alzheimer's disease (AD). Cholinesterase inhibition is a primary strategy for treatment of AD, and specific compounds of this class have previously been demonstrated to influence Aβ precursor protein (APP) processing and Aβ production. However, little information is available on the effects of rivastigmine, a dual acetylcholinesterase and butyrylcholinesterase inhibitor, on APP processing. As this drug is currently used to treat AD, characterization of its various activities is important to optimize its clinical utility. We have previously shown that rivastigmine can preserve or enhance neuronal and synaptic terminal markers in degenerating primary embryonic cerebrocortical cultures. Given previous reports on the effects of APP and Aβ on synapses, regulation of APP processing represents a plausible mechanism for the synaptic effects of rivastigmine. To test this hypothesis, we treated degenerating primary cultures with rivastigmine and measured secreted APP (sAPP) and Aβ. Rivastigmine treatment increased metabolic activity in these cultured cells, and elevated APP secretion. Analysis of the two major forms of APP secreted by these cultures, attributed to neurons or glia based on molecular weight showed that rivastigmine treatment significantly increased neuronal relative to glial secreted APP. Furthermore, rivastigmine treatment increased α-secretase cleaved sAPPα and decreased Aβ secretion, suggesting a therapeutic mechanism wherein rivastigmine alters the relative activities of the secretase pathways. Assessment of sAPP levels in rodent CSF following once daily rivastigmine administration for 21 days confirmed that elevated levels of APP in cell culture translated in vivo. Taken together, rivastigmine treatment enhances neuronal sAPP and shifts APP processing toward the α-secretase pathway in degenerating neuronal cultures, which mirrors the trend of synaptic proteins, and metabolic activity.     

Increased secreted amyloid precursor protein-α (sAPPα) in severe autism: proposal of a specific, anabolic pathway and putative biomarker

Autism is a neurodevelopmental disorder characterized by deficits in verbal communication, social interactions, and the presence of repetitive, stereotyped and compulsive behaviors. Excessive early brain growth is found commonly in some patients and may contribute to disease phenotype. Reports of increased levels of brain-derived neurotrophic factor (BDNF) and other neurotrophic-like factors in autistic neonates suggest that enhanced anabolic activity in CNS mediates this overgrowth effect. We have shown previously that in a subset of patients with severe autism and aggression, plasma levels of the secreted amyloid-β (Aβ) precursor protein-alpha form (sAPPα) were significantly elevated relative to controls and patients with mild-to-moderate autism. Here we further tested the hypothesis that levels of sAPPα and sAPPβ (proteolytic cleavage products of APP by α- and β-secretase, respectively) are deranged in autism and may contribute to an anabolic environment leading to brain overgrowth. We measured plasma levels of sAPPα, sAPPβ, Aβ peptides and BDNF by corresponding ELISA in a well characterized set of subjects. We included for analysis 18 control, 6 mild-to-moderate, and 15 severely autistic patient plasma samples. We have observed that sAPPα levels are increased and BDNF levels decreased in the plasma of patients with severe autism as compared to controls. Further, we show that Aβ1-40, Aβ1-42, and sAPPβ levels are significantly decreased in the plasma of patients with severe autism. These findings do not extend to patients with mild-to-moderate autism, providing a biochemical correlate of phenotypic severity. Taken together, this study provides evidence that sAPPα levels are generally elevated in severe autism and suggests that these patients may have aberrant non-amyloidogenic processing of APP.     

Regulation of amyloid precursor protein expression and secretion via activation of ERK1/2 by hepatocyte growth factor in HEK293 cells transfected with APP751

The increased intracellular levels and aberrant processing of the amyloid precursor protein (APP) are associated with beta-amyloid peptide (A beta) production, cerebrovascular amyloid deposition, and amyloid plaque formation. Here we report that APP level, soluble APP (sAPP) secretion, and A beta production in HEK293 cells transfected with either wild-type APP(751) or APP(751) carrying the Swedish mutation are all elevated by hepatocyte growth factor (HGF). We investigated the potential molecular mechanisms underlying the HGF effect. Our data show that HGF stimulated extended activation of extracellular signal-regulated protein kinases (ERK1/2). Pretreatment of cells with inhibitors (UO126 or PD98059) for MEK, the upstream kinase of ERK1/2, abolished ERK1/2 activation evoked by HGF, and abrogated HGF-induced increases in APP levels and sAPP secretion. In addition, transient expression of active MEK1 activated ERK1/2 and increased intracellular APP levels and sAPP secretion. Inhibition of ERK1/2 activity, however, failed to block HGF-stimulated A beta production. Consistently, transient expression of active MEK1 did not increase A beta accumulation. Taken together, these results suggest that: (1) HGF regulates the intracellular levels of APP and the secretion of sAPP and A beta; (2) the modulation of APP levels and sAPP secretion induced by HGF is mediated via the MEK1/ERK1/2 signaling pathway; (3) HGF-stimulated A beta production is independent of ERK activity and, therefore, independent of HGF-evoked elevation of intracellular APP levels.     

In Vivo Turnover of Tau and APP Metabolites in the Brains of Wild-Type and Tg2576 Mice: Greater Stability of sAPP in the β-Amyloid Depositing Mice

The metabolism of the amyloid precursor protein (APP) and tau are central to the pathobiology of Alzheimer''s disease (AD). We have examined the in vivo turnover of APP, secreted APP (sAPP), Aβ and tau in the wild-type and Tg2576 mouse brain using cycloheximide to block protein synthesis. In spite of overexpression of APP in the Tg2576 mouse, APP is rapidly degraded, similar to the rapid turnover of the endogenous protein in the wild-type mouse. sAPP is cleared from the brain more slowly, particularly in the Tg2576 model where the half-life of both the endogenous murine and transgene-derived human sAPP is nearly doubled compared to wild-type mice. The important Aβ degrading enzymes neprilysin and IDE were found to be highly stable in the brain, and soluble Aβ40 and Aβ42 levels in both wild-type and Tg2576 mice rapidly declined following the depletion of APP. The cytoskeletal-associated protein tau was found to be highly stable in both wild-type and Tg2576 mice. Our findings unexpectedly show that of these various AD-relevant protein metabolites, sAPP turnover in the brain is the most different when comparing a wild-type mouse and a β-amyloid depositing, APP overexpressing transgenic model. Given the neurotrophic roles attributed to sAPP, the enhanced stability of sAPP in the β-amyloid depositing Tg2576 mice may represent a neuroprotective response.     

Activity of α-Secretase as the Common Final Effector of Protein Kinase C-Dependent and -Independent Modulation of Amyloid Precursor Protein Metabolism

The metabolic fate of the amyloid precursor protein (APP) is one of the key factors in the pathogenesis of Alzheimer's disease (AD). A complex cellular mechanism regulates the rate at which the precursor is cleaved by alpha-secretase and released as soluble protein in the extracellular space. We show here that alpha-secretase constitutes the common final effector of several independent means of stimulation of soluble APP (sAPP) release. The release of sAPP by alpha-secretase resembles that of several other membrane-bound proteins with soluble counterparts, a process that is sensitive to matrix metalloprotease inhibitors. The hydroxamic acid-based compound KD-IX-73-4 inhibits phorbol ester-mediated sAPP release from COS cells with an IC50 of 8 microM, consistent with previous data for the same compound against leukocyte L-selectin shedding. Beyond direct protein kinase C (PKC) activation we show that KD-IX-73-4 inhibits also receptor-mediated sAPP release induced by carbachol in SH-SY5Y cells and by bradykinin in human skin fibroblasts, with the latter being a PKC-independent mechanism. Altogether these data suggest that all pharmacological means of stimulating sAPP release converge to a hydroxamic acid-based inhibitor-sensitive proteolytic enzyme. Moreover, because KD-IX-73-4 was effective in the inhibition of stimulated but not constitutive sAPP release, these data suggest the existence of different enzymes regulating the two metabolic pathways leading to sAPP secretion.     

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.     

Secreted forms of the amyloid-beta precursor protein are ligands for the class A scavenger receptor

Upon activation, platelets secrete a 120-kDa protein that competes for the binding and internalization of acetyl low density lipoproteins (AcLDL) by macrophages. From the amino-terminal amino acid sequence, amino acid composition, and immunoblot analysis, we identified the active factor in platelet secretion products as sAPP, an alpha-secretase cleavage product of the beta-amyloid precursor protein (APP), that contains a Kunitz-type protease inhibitor (KPI) domain. We showed that both sAPP751 (also called Nexin II) and sAPP695, which does not contain a KPI domain, are ligands for the class A scavenger receptor (SR-A). Chinese hamster ovary cells stably transfected to express the SR-A bound and internalized 4-fold more human platelet-derived sAPP than control cells. The binding and internalization of sAPP were inhibited by the SR-A antagonist fucoidin. In addition, sAPP competed as effectively as fucoidin for SR-A-mediated cell association and degradation of (125)I-AcLDL. To determine if the KPI domain is required for the binding of sAPP to the SR-A, APP751 and APP695 were expressed in Chinese hamster ovary cells, and sAPP751 and sAPP695 purified from the medium were tested for their binding to the SR-A. sAPP751 and sAPP695 were equally effective in competing for the cell association of (125)I-AcLDL by SR-A-expressing cells, demonstrating that the KPI domain is not essential for binding. We also found that sAPP751 is present in extracts of atherosclerotic lesions and that sAPP competes for the SR-A-mediated cell association of oxidized low density lipoprotein. Deletion mutagenesis indicated that a negatively charged region of APP (residues 191-264) contributes to binding to the SR-A. These results suggest that the SR-A contributes to the clearance of sAPP and that sAPP competes for the cell association of other SR-A ligands.