Serum Starvation Induces BACE1 Processing and Secretion.

BACKGROUND: ß-secretase (BACE1) is a type 1 transmembrane protein implicated in Alzheimer's Disease (AD) pathogenesis. Cleavage of Amyloid Precursor Protein (APP), initiated by BACE1 and followed by γ-secretase, leads to the formation of toxic Aß peptides. Increased levels of BACE1 have been detected in the CSF of AD patients compared to age-matched healthy controls indicating that neurodegenerative conditions induce shedding of BACE1. OBJECTIVE: To mimic such conditions, we examined whether serum deprivation stimulates proteolysis-dependent secretion of BACE1. METHOD: Detection of BACE1 secretion in BACE1 overexpressing cells or ADAM10/ADAM17 knockout fibroblasts cultured under serum deprivation conditions, using western blot analysis. RESULTS: We found that serum deprivation of U251 neuroblastoma or HEK293T cells overexpressing BACE1 stimulated secretion of BACE1. Using ADAM10/ADAM17 knockout fibroblasts and inhibitors of both ADAM10 and ADAM17, we obtained data indicating that these proteases are involved in serum-starvation induced shedding of BACE1. This is unexpected since BACE1 is localized mainly in lipid rafts while ADAM10 is localized mainly in nonlipid raft domains. We hypothesized that serum deprivation results in alterations in the lipid composition of the membrane which can alter the localization of ADAM10 and BACE1. In support, we obtained results indicating that extraction of membrane cholesterol following incubation with methyl ß cyclodextrin potentiated the effect of serum deprivation. Secreted BACE1 was also found to be enzymatically active towards immunoprecipiated full length APP. CONCLUSION: Serum starvation induces ADAM10-mediated BACE1 secretion.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Cellular processing and proteoglycan nature of amyloid precursor proteins.

Amyloid beta protein (beta/A4 or A beta), the main proteinaceous component of the amyloid depositions of the Alzheimer's brain, derives from the proteolytic processing of the amyloid precursor protein (APP). Cleavage of the amyloid precursor by at least two distinct secretase activities produces soluble secreted APP. The major secretase cleavage (site I) takes place between A beta 16 and 17, while the minor cleavage (site II) takes place after A beta Lys 28 and may produce potentially amyloidogenic secreted APP. Full-length cellular APP is cleaved by secretase intracellularly in the Trans-Golgi Network (TGN) or in post-Golgi vesicles. The resultant soluble APP is transported to the plasma membrane and exocytosed. The biological activity of the APP is still not completely understood, although it seems to act as a cell adhesion molecule. Recent studies have shown that in glioma cells, most of the soluble secreted APP occurs as a chondroitin sulfate proteoglycan (CSPG). In addition, full length APP CSPG has been detected in neuroblastoma and fibroblast cells as well as on the surface of glioma cells, and in human brain. These results suggest that the proteoglycan nature of the APP proteins may be important for their biological function.

A novel Polish presenilin-1 mutation (P117L) is associated with familial Alzheimer's disease and leads to death as early as the age of 28 years.

The majority of early-onset familial Alzheimer's disease (FAD) is associated with mutations in the presenilin-1 (PS1) gene. We describe a novel Polish PS1 mutation of Pro117Leu, associated with the earliest average age of onset and death so far reported in a PS-linked, FAD kindred. Human kidney 293 and mouse neuroblastoma N2a cells were stably transfected with wild-type and PS1 P117L. There was a significant increase in the amyloid beta42/40 ratio in the N2a P117L PS1 transfected cells compared with N2a transfected with wild-type PS1. What role PS has in the pathogenesis of AD remains to be determined, however, the severity of the clinical picture associated with this PS1 mutation stresses the importance of presenilin.

Chondroitin sulfate proteoglycan form of cellular and cell-surface Alzheimer amyloid precursor.

The biological function of the amyloid precursor protein (APP) is still not fully understood. Recently, we reported that secreted truncated APP occurs in a chondroitin sulfate proteoglycan form. Here we present evidence that full length APP-chondroitin sulfate proteoglycan is present on the cell surface of C6 glioma cells. In addition, densitometric quantitation of Western blots showed that approximately 50% of the mature cell-associated full length APP is in the proteoglycan form. These findings suggest that the proteoglycan nature of APP may be important for the implementation of its biological function.

Inhibition of neurite outgrowth by familial Alzheimer's disease-linked presenilin-1 mutations.

Two (P117L; M146L) familial Alzheimer's disease (FAD)-causing presenilin-1 (PS1) mutations have been tested fortheir effect in stably transfected mouse neuroblastoma (N2a) cell lines. The P117L mutation is associated with the earliest onset of AD reported so far (24 years), while the M146L is less pathogenic with the onset at about 43 years. Overexpression of wild-type (wt) PS1 gene was associated with the marked increase in the number and the length of neuritic outgrowths accompanied by accumulation of PS1 immunoreactivity in neurites. The highly pathogenic P117L mutation completely suppressed this effect and the pattern of PS1 immunolabeling resembled a cup structure with all immunoreactivity gathered at one pole of the cell. The effect of less pathogenic M146L mutation was similar, but not as pronounced. These findings suggest that one of the normal functions of PS1 may be the control of neurite outgrowth, and the inhibitory effect of two FAD-linked mutations stresses its importance in the cellular mechanism that leads to the development of Alzheimer's disease (AD).

Heat-shock induces abnormalities in the cellular distribution of amyloid precursor protein (APP) and APP fusion proteins.

The heat shock or stress response may play a role in the pathogenesis of Alzheimer's disease. We conducted experiments to visualize microscopically the distribution of wild type amyloid precursor protein (APP) and the behavior of an APP deletion mutant under stress. This was achieved by heat-shock treatment of cells expressing fusion recombinant APP proteins tagged with secreted placental alkaline phosphatase (SEAP). The fusion proteins were cleaved and secreted in a manner similar to wild type APP in unstressed control cells. SEAP activity was detected by cytochemical methods within the cytoplasm in less than 10% of transfected unstressed cells. Heat shocked cells showed a striking difference from the control cells in that over 90% of the stressed cells displayed strong intracytoplasmic SEAP activity occurring with Golgi-like pattern and/or membranous distribution. The effects of heat shock were not due to a peculiar behavior of the clones and depended on the APP portion of the constructs. This study shows miscompartmentalization of APP under stress. Such cellular changes may bear important implications in the processing of APP.

Kinetics of dopamine and noradrenaline transport in synaptosomes from cerebellum, striatum and frontal cortex of normal and reeler mice.

Recent evidence indicates that the cerebellum has a dopaminergic system. In order to elucidate further the dopaminergic system in the cerebellum, we investigated the transport of dopamine (DA) in synaptosomal preparations of normal and reeler mice. For comparative purposes we also studied DA transport in synaptosomal preparations from striatum and frontal cortex and compared DA transport to noradrenaline (NA) transport. [3H]-DA transport into cerebellar synaptosomes was found to be a Na(+)-dependent, two component system--a high affinity, low capacity and a low affinity, high capacity. In striatum [3H]-DA is transported by a similar high but different low affinity component. Maximal velocities of both transport components in the striatum were higher than the corresponding ones in the cerebellum. In the frontal cortex we also observed two [3H]-DA transport components with affinities significantly lower than those in cerebellum and striatum. [3H]-NA transport into synaptosomes, prepared from the three brain regions studied, showed two transport components with similar Kt and Vmax values, except for the high affinity component in striatum whose affinity is lower. In reeler mice [3H]-DA transport was different from normal only in the cerebellum where the maximal velocity for both transport components was significantly higher (2x). In contrast, no significant difference was observed in the transport of [3H]-NA. The accumulated [3H]-DA from cerebellar slices was found to be releasable by K+ stimulation, in a Ca(++)-dependent manner, and most of the released radioactivity was in the form of [3H]-DA. These results indicate that in the cerebellum there is a low-density dopaminergic system which is distinct from the corresponding noradrenergic system.

Mechanism of amyloid beta-peptide (1-42) toxicity in PC12 cells.

Amyloid beta-peptide (A beta) deposition and loss of cholinergic neurons are characteristics of Alzheimer's disease. There is evidence that A beta is neurotoxic. The role of signal transduction pathways on A beta-induced toxicity in PC12 cells was investigated. Our results revealed that A beta-induced arachidonic acid was released in a time-dependent manner. Inhibitors of cyclooxygenase (1 microM indomethacin) and lipooxygenase (100 microM nordihydroguairetic acid) protected PC12 cells against A beta-induced toxicity. These data suggest that A beta toxicity is mediated by activation of the arachidonic acid cascade. Furthermore, protein kinase C activators (phorbol ester and 1-oleyl-2-acetyl-glycerol) and tacrine reversed A beta-induced toxicity. These results suggest that A beta toxicity can be modulated by manipulating signal transduction pathways and may provide the basis for novel therapeutic interventions.

Study of the phorbol ester effect on Alzheimer amyloid precursor processing: sequence requirements and involvement of a cholera toxin sensitive protein.

Phorbol esters (PDBu) stimulate alpha-secretase cleavage and secretion of the Alzheimer amyloid precursor protein (APP). To determine whether any cytoplasmic residues or sequence motifs mediate the PDBu effect on APP processing, this region of APP was altered by point mutations or deletions. To differentiate the mutated APP from the endogenous APP, the APP751 ectodomain between amino acids 1 and 647 was replaced by a human secreted alkaline phosphatase derivative (SEAP). The resultant fusion protein (SEAP-APP751) was cleaved by alpha-secretase at the same site as full-length APP, and its secretion was stimulated by PDBu at a level similar to APP751. However, PDBu-stimulated secretion of the SEAP-APP751 fusion protein reached its maximum level after 30 min of treatment, while secretion of APP751 reached its maximum after 60 min, suggesting that the APP ectodomain affects the kinetics of APP secretion. Mutation of the cytoplasmic serines to alanines had no effect on the PDBu-stimulated secretion of the SEAP-APP, indicating that protein kinase C (PKC) phosphorylation of the cytoplasmic domain of APP is not important for stimulation of APP secretion. Similarly, deletion of the cytoplasmic domain between amino acids 719 and 751 had no effect on the PDBu-stimulated secretion. However, deletion of amino acids 707-751 resulted in a significant increase in the secretory cleavage of the SEAP-APP707 delta C construct, suggesting that the sequence 707-719 is important for the regulated secretion of APP. Cholera toxin, but not pertussis toxin, reduced the PDBu-induced secretion of APP by more than two-fold, suggesting that the PDBu response may be modulated by a cholera toxin sensitive heterotrimeric G-protein.

Appican expression induces morphological changes in C6 glioma cells and promotes adhesion of neural cells to the extracellular matrix.

Appicans are secreted or cell-associated brain chondroitin sulfate proteoglycans produced by glia cells and containing Alzheimer amyloid precursor protein (APP) as a core protein. Here, we report that rat C6 glioma cells transfected with appican displayed a dramatic change in their phenotypic appearance compared with untransfected cells or cells transfected with APP. Appican-transfected cells lost the round appearance of the untransfected control C6 cells, acquired a flat morphology, and elaborated more processes than control cells. Untransfected, or APP-transfected C6, cells were completely dissociated from their substrate after 40 min of treatment with cell dissociation solution. Under the same conditions, however, <20% of the appican-transfected C6 cells were dissociated from their substrate, suggesting that the appican-transfected glia cells attach more avidly to their substrate than do untransfected or APP transfected control cells. In contrast, appican-transfected fibroblast cells showed no morphological changes and dissociated from their substrate similarly to untransfected fibroblast cells. Extracellular matrix (ECM) prepared from appican-transfected C6 cell cultures contained high levels of appican and was a significantly better substrate for the attachment of C6 cells than ECM from either untransfected or APP-transfected cultures. Furthermore, cell adhesion to ECM was independent of the level of appican expression of the plated cells. ECM from appican-transfected C6 cultures stimulated adhesion of other neural cells including primary astrocytes, Neuro2a neuroblastoma, and PC12 pheochromocytoma, but not fibroblast cells. Conditioned media from appican-transfected C6 cultures failed to promote cell adhesion. Together, these data suggest that secreted appican incorporates into ECM and promotes adhesion of neural cells. Furthermore, our data suggest that the chondroitin sulfate chain engenders APP with novel biological functions.

Characterization of appican, the chondroitin sulfate proteoglycan form of the Alzheimer amyloid precursor protein.

In this report we focus on the characterization of appican, the chondroitin sulfate proteoglycan form of amyloid precursor protein (APP), and the role that it and other proteoglycans may play in AD. Appican is expressed by certain transformed cell lines of neural origin, namely C6 cells and N2a neuroblastomas. It is detected in both human and rat brain and in primary cultures is expressed by astrocytes, but not neurons. The core protein of appican has been shown to be an alternatively spliced isoform of APP, lacking exon 15 of the APP gene, originally identified in leukocytes (L-APP). Splicing out of exon 15 results in the joining of exons 14 and 16, and formation of an Asp-Xaa-Ser-Gly consensus sequence for chondroitin sulfate chain attachment to serine 619 of L-APP, which lies 16 amino acids upstream of the A beta peptide sequence. Mutation of this serine residue to an alanine prevented chondroitin sulfate chain addition to the core protein. Levels of appican expression could be regulated by growth conditions independently of APP, suggesting that these molecules may serve distinct physiological roles within the cell. Morphological changes were also observed in both astrocytic and transformed cell cultures, that appeared to reflect changes in levels of appican expression. Preliminary data suggest that appican may be a strong cell adhesion molecule. Transfected C6 glioma cells overexpressing appican remained attached to tissue culture dishes markedly better than either C6 cells over-expressing exon-15 containing APP or WT C6 cells. Appican-enriched extracellular matrix (ECM) was also observed to serve as a much better substrate for attachment of N2a neuroblastomas, pheocromocytoma PC12 cells and primary astrocytes compared to APP enriched ECM.

The chondroitin sulfate attachment site of appican is formed by splicing out exon 15 of the amyloid precursor gene.

Appicans are secreted and cell-associated chondroitin sulfate proteoglycans containing Alzheimer amyloid precursor (APP) as their core protein. Appicans are found in brain tissue, and in cell cultures their expression depends on both cell type and growth conditions. Here we report that the core protein of appicans derives from an APP mRNA lacking exon 15. Splicing out of this exon creates a new consensus sequence for the attachment of a chondroitin sulfate chain in the resulting APP product. Transfection of C6 glioma or 293 kidney fibroblast cells with APP cDNAs containing exon 15 produced no appican, while transfection with an APP cDNA lacking this exon induced high levels of appican production. Polymerase chain reactions indicated that appican-producing cells contained an APP mRNA species without exon 15, whereas cells without this mRNA produced no appican. Site-directed mutagenesis combined with immunoreactivity experiments showed that the chondroitin sulfate chain is attached to a serine residue 16 amino acids upstream of the amino terminus of the A beta sequence of APP. The attachment of a glycosaminoglycan chain close to the A beta sequence of APP may affect the proteolytic processing of APP and production of A beta. The proteoglycan nature of APP suggests that addition of the chondroitin sulfate glycosaminoglycan is important for the implementation of the biological function of these proteins.

Evidence that secretase cleavage of cell surface Alzheimer amyloid precursor occurs after normal endocytic internalization.

Three different treatments (methylamine, colchicine, and 18 degrees C temperature block), known to disrupt normal endocytic internalization, significantly reduced the secretory cleavage of cell surface-derived Alzheimer amyloid precursor (APP) in non-transfected C6 cell cultures. Conversely, treatments with methylamine or colchicine had no significant effect on the secretion of total APP. Treatment of these cells with the lysosomotropic amine chloroquine resulted in a significant increase in the levels of both cell surface full-length APP and cell surface-derived secreted nexin II (NXII). Immunofluorescence analysis of C6 glioma cells transfected with APP751 indicated that under normal conditions, cell surface APP was internalized, and within 30 minutes was localized in discrete intracellular vesicles. These vesicles contained the endocytic tracer Texas red-conjugated ovalbumin and probably represented late endosomes or lysosomes. However, treatment of the transfected C6 cultures with methylamine or colchicine prevented localization of cell surface APP in intracellular vesicles, suggesting that these treatments altered the normal intracellular trafficking of cell surface-derived APP. Both the biochemical and immunofluorescence data are compatible with the suggestion that inhibition of normal endocytic internalization reduces the secretory cleavage of cell surface APP. Furthermore, our results suggest that following internalization, cell surface APP is cleaved by secretase(s) and secreted or routed to the lysosomes where it is degraded.

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Intracellular cyclic AMP inhibits constitutive and phorbol ester-stimulated secretory cleavage of amyloid precursor protein.

alpha-Secretase cleaves the full-length Alzheimer's amyloid precursor protein (APP) within the amyloid beta peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP751 to examine whether the alpha-secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester-stimulated secretion of nexin II (NXII), the secreted product of the alpha-secretase cleavage of APP751. At 1 microM, forskolin inhibited secretion of NXII by approximately 50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9-dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the alpha-secretase cleavage of APP751. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.

Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins Abeta1-40 and Abeta1-42, respectively.

The carboxy-terminal ends of the 40- and 42-amino acids amyloid beta-protein (Abeta) may be generated by the action of at least two different proteases termed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Abeta1-40 and Abeta1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of gamma-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two gamma-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Abeta1-40, with a concomitant accumulation of its cellular precursor indicating that gamma(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Abeta1-42. No inhibition of Abeta production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that gamma(40)-secretase is a cysteine protease distinct from calpain, whereas gamma(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the beta-secretase C-terminal APP fragment and a decrease of the alpha-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the alpha-secretase site to the beta-secretase site.

Presenilin-1 binds cytoplasmic epithelial cadherin, inhibits cadherin/p120 association, and regulates stability and function of the cadherin/catenin adhesion complex.

Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, binds directly to epithelial cadherin (E-cadherin). This binding is mediated by the large cytoplasmic loop of PS1 and requires the membrane-proximal cytoplasmic sequence 604-615 of mature E-cadherin. This sequence is also required for E-cadherin binding of protein p120, a known regulator of cadherin-mediated cell adhesion. Using wild-type and PS1 knockout cells, we found that increasing PS1 levels suppresses p120/E-cadherin binding, and increasing p120 levels suppresses PS1/E-cadherin binding. Thus PS1 and p120 bind to and mutually compete for cellular E-cadherin. Furthermore, PS1 stimulates E-cadherin binding to beta- and gamma-catenin, promotes cytoskeletal association of the cadherin/catenin complexes, and increases Ca(2+)-dependent cell-cell aggregation. Remarkably, PS1 familial Alzheimer disease mutant DeltaE9 increased neither the levels of cadherin/catenin complexes nor cell aggregation, suggesting that this familial Alzheimer disease mutation interferes with cadherin-based cell-cell adhesion. These data identify PS1 as an E-cadherin-binding protein and a regulator of E-cadherin function in vivo.