Appican, the proteoglycan form of the amyloid precursor protein, contains chondroitin sulfate E in the repeating disaccharide region and 4-O-sulfated galactose in the linkage region.

Chondroitin sulfate (CS)-D and CS-E, which are characterized by oversulfated disaccharide units, have been shown to regulate neuronal adhesion, cell migration, and neurite outgrowth. CS proteoglycans (CSPGs) consist of a core protein to which one or more CS chains are attached via a serine residue. Although several brain CSPGs, including mouse DSD-1-PG/phosphacan, have been found to contain the oversulfated D disaccharide motif, no brain CSPG has been reported to contain the oversulfated E motif. Here we analyzed the CS chain of appican, the CSPG form of the Alzheimer's amyloid precursor protein. Appican is expressed almost exclusively by astrocytes and has been reported to have brain- and astrocyte-specific functions including stimulation of both neural cell adhesion and neurite outgrowth. The present findings show that the CS chain of appican has a molecular mass of 25-50 kDa. This chain contains a significant fraction (14.3%) of the oversulfated E motif GlcUA beta 1-3GalNAc(4,6-O-disulfate). The rest of the chain consists of GlcUA beta 1-3GalNAc(4-O-sulfate) (81.2%) and minor fractions of GlcUA beta 1-3GalNAc and GlcUA beta 1-3GalNAc(6-O-sulfate). We also show that the CS chain of appican contains in its linkage region the 4-O-sulfated Gal structure. Thus, appican is the first example of a specific brain CSPG that contains the E disaccharide unit in its sugar backbone and the 4-O-sulfated Gal residue in its linkage region. The presence of the E unit is consistent with and may explain the neurotrophic activities of appican.

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.

Presenilin-1 expression in Pick's disease.

Recent studies have reported that neuronal populations expressing low levels of presenilin-1 (PS-1) display increased vulnerability in late-onset sporadic Alzheimer's disease (AD). To examine whether this phenomenon also occurs in other neurodegenerative diseases, we performed a quantitative immunocytochemical study of PS-1 distribution in the cerebral cortex of Pick's disease (PiD) cases and non-demented individuals. In PiD cases, the percentage of PS-1-containing, Pick body (PB)-free neurons was significantly elevated only in cortical areas showing neuronal loss. In these areas, PS-1 levels, measured by immunoblotting, were often higher in PiD compared to non-demented cases. Moreover, PS-1 immunoreactivity was significantly reduced in PB-containing neurons. These data suggest that as previously shown in AD, low cellular expression of PS-1 may be associated with increased neuronal loss and cellular degeneration.

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts.

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.

Enrichment of presenilin 1 peptides in neuronal large dense-core and somatodendritic clathrin-coated vesicles.

Presenilin 1 is an integral membrane protein specifically cleaved to yield an N-terminal and a C-terminal fragment, both membrane-associated. More than 40 presenilin 1 mutations have been linked to early-onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function (s) of the wild-type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear-core synaptic vesicles, the large dense-core vesicles, and the somatodendritic and nerve terminal clathrin-coated vesicles. Both the N- and C-terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense-core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense-core vesicle-mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin-coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear-core synaptic vesicle or in the nerve terminal clathrin-coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full-length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full-length protein may not have a vesicular function.

Overexpression in neurons of human presenilin-1 or a presenilin-1 familial Alzheimer disease mutant does not enhance apoptosis.

Programmed cell death, or apoptosis, has been implicated in Alzheimer's disease (AD). DNA damage was assessed in primary cortical neurons infected with herpes simplex virus (HSV) vectors expressing the familial Alzheimer's disease (FAD) gene presenilin-1 (PS-1) or an FAD mutant of this gene, A246E. After infection, immunoreactivity for PS-1 was shown to be enhanced in infected cells. The infected cells exhibited no cytotoxicity, as evaluated by trypan blue exclusion and mitochondrial function assays. Quantitative analysis of cells that were immunohistochemically labeled using a Klenow DNA fragmentation assay or the TUNEL method revealed no enhancement of apoptosis in PS-1-infected cells. This result was confirmed using assays for chromatin condensation and for DNA fragmentation. Expression of PS-1 protected against induction of apoptosis in the cortical neurons by etoposide or staurosporine. The specificity of this phenotype was demonstrated by the fact that cortical cultures infected with recombinant HSV vectors expressing the amyloid precursor protein (APP-695) showed, in contrast, a significant increase in the number of apoptotic cells and an increase in DNA fragmentation for all parameters tested. Our results indicate that overexpression of wild-type or A246E mutant PS-1 does not enhance apoptosis in postmitotic cortical cells and suggest that the previously reported enhancement of apoptosis by presenilins may be dependent on cell type.

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.

Presenilin-1-immunoreactive neurons are preserved in late-onset Alzheimer's disease.

Recent studies have suggested that missense mutations in the presenilin-1 gene are causally related to the majority of familial early-onset Alzheimer's disease (AD). To examine the possible involvement of presenilin-1 in late-onset sporadic AD, a quantitative analysis of its distribution in the cerebral cortex of nondemented and AD patients was performed using immunocytochemistry. Stereological analyses revealed that AD brains showed a marked neuronal loss in the CA1 field of the hippocampus and hilus of the dentate gyrus, subiculum, and entorhinal cortex. In these areas, however, the fraction of neurofibrillary tangle (NFT)-free neurons showing presenilin-1 immunoreactivity was increased compared with nondemented controls. In contrast, cortical areas, which displayed no neuronal loss, did not show any significant increase in the fraction of presenilin-1-positive neurons. Moreover, presenilin-1 immunoreactivity was reduced in NFT-containing neurons. Thus, in AD, the fraction of NFT-free neurons that contained presenilin-1 varied from 0.48 to 0.77, whereas the fraction of NFT-containing neurons that were presenilin-1 positive varied from 0.1 to 0.24. Together, these observations indicate that presenilin-1 may have a neuroprotective role and that in AD low cellular expression of this protein may be associated with increased neuronal loss and NFT formation.

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.

Identification and neuron specific expression of the S182/presenilin I protein in human and rodent brains.

Many individuals with familial Alzheimer disease (FAD) have mutations in a gene termed S182 or presenilin I (PS-I). Currently, the PS-I gene product has not been identified and its function remains unknown. Here we report that affinity purified antibodies against the predicted amino acid sequence of the PS-I gene product detected in homogenates of human, mouse, and rat brains a single antigen of approximately 48 kDa. This antigen was also present in immortalized human and mouse neuronal cell cultures. Brain tissue fractionation showed that all PS-I antigen was found in the membrane fraction. In stained tissue sections of mouse central nervous system (CNS), PS-I antigen was found only in neurons throughout brain and spinal cord and was located within cell bodies, axons, and dendrites. Remarkably the relative partition among these three compartments varied dramatically. A striking feature of PS-I expression was its intense concentration in some (but not all) dendrites, at levels substantially above those in the parent perikarya. In most of the cerebrum, PS-I staining in axons was very weak or undetectable. By contrast, many axons in portions of the brainstem and in the spinal cord showed marked PS-I immunoreactivity. Similarly, staining of sections from human temporal cortex showed that PS-I was present mainly in neuronal cell bodies and dendrites. These data show that in the CNS, PS-I is expressed mainly in neurons and suggests that this protein may perform a neuron specific function. The pattern of PS-I expression in the CNS would suggest that the premature neurodegeneration associated with PS-I mutations involves a primary neuronal process rather than a secondary effect of PS-I produced in non-neuronal cells.

Expression of the chondroitin sulfate proteoglycans of amyloid precursor (appican) and amyloid precursor-like protein 2.

The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific antibodies suggested that a CS chain is attached within or proximal to the A beta sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of approximately 100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined.(ABSTRACT TRUNCATED AT 250 WORDS)

The Alzheimer amyloid precursor proteoglycan (appican) is present in brain and is produced by astrocytes but not by neurons in primary neural cultures.

Recent studies showed that the Alzheimer amyloid precursor (APP) occurs as the core protein of a chondroitin sulfate proteoglycan (appican) in C6 glioma cells. In the present study we show that appican is present in both human and rat brain tissue. Cortical rat brain cell cultures were used to identify appican-producing cells. Soluble secreted and cell-associated appican was produced by mixed glial cultures but not by primary neuronal cultures. Among the three major glial cell types, astrocytes produced high levels of appican, while oligodendrocytes failed to produce any. Only low levels of this molecule were occasionally detected in microglial cultures. Expression of appican in astrocyte cultures was regulated by the composition of the growth media. N2a neuroblastoma cells also produced appican; however, treatment with dibutyryl cAMP which promotes neuronal differentiation in these cells inhibited its production without inhibiting synthesis of APP. In contrast to the restricted expression of appican, APP was present in all cultures, and its production was independent of appican synthesis. Neuronal cultures produced mainly APP695 while glial cultures produced the Kunitz type protease inhibitor containing APP. The astrocyte-specific expression of appican suggests a function distinct from the function of APP. Brain appicans may play a role in the development of Alzheimer disease neuropathology.

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.

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.

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.

Evidence for intracellular cleavage of the Alzheimer's amyloid precursor in PC12 cells.

The Alzheimer's amyloid precursor (APP) is cleaved by an unidentified enzyme (APP secretase) to produce soluble APP. Fractionation of PC12 cell homogenates in a detergent-free buffer showed the presence of the Kunitz protease inhibitor (KPI)-containing soluble APP (nexin II) in the particulate fraction. Digitonin or sodium carbonate treatment of this fraction solubilized nexin II suggesting that it is contained in the lumen of vesicles. Nexin II production was not affected by lysosomotropic agents, suggesting that APP secretase is not a lysosomal enzyme. Labelling of cell surface proteins by iodination failed to detect full-length APP on the surface of PC12 cells, suggesting that most of this protein is located intracellularly. Furthermore, pulse-chase experiments showed that nexin II is detected in cell extracts before it appears in the culture medium. Cellular nexin II was detected at zero time of chase after only 5 min of pulse labelling with 35S-sulfate, indicated that APP secretase cleavage takes place immediately after APP is sulfated. Temperature block, pulse-chase, and 35S-sulfate-labelling experiments suggested that APP is cleaved by APP secretase intracellularly in the trans-Golgi network (TGN) or in a post-Golgi compartment.

Chondroitin sulfate proteoglycan form of the Alzheimer's beta-amyloid precursor.

The Alzheimer's amyloid beta protein is derived from a family of membrane glycoproteins termed amyloid precursor proteins (APP). Here we show that APP exists as the core protein of a chondroitin sulfate (CS) proteoglycan, ranging in apparent molecular size from 140 to 250 kDa, secreted by glial cell line C6. After partial purification on ion-exchange and gel chromatography, the secreted APP proteoglycan was recognized on Western blots by several antibodies specific to different regions of APP. Chondroitinase AC or ABC treatment of our samples completely eliminated the high molecular weight proteoglycan with a concomitant increase in the APP protein. This digested product reacted with an anti-stub antibody which recognizes 4-sulfated disaccharide. Sequencing of the N terminus of the core protein of this CS proteoglycan yielded 18 residues identical to the N terminus sequence of the mature APP. Quantitative analysis showed that, in this cell line, about 90% of the secreted nexin II form of APP occurs in the proteoglycan form, suggesting that the CS chains have a role in the biological function of this protein. The close proximity of two consensus CS attachment sites to both the N terminus of the amyloid beta protein and the secretase cleavage site, suggests that the CS chains may affect the proteolysis of APP and production of the amyloid beta protein.

Artificially imposed electrical potentials drive L-glutamate uptake into synaptic vesicles of bovine cerebral cortex.

L-Glutamate is a major excitatory neurotransmitter in the central nervous system. MgATP-dependent glutamate uptake and H(+)-pumping ATPase activity were reported in highly purified synaptic vesicles [Naito & Ueda (1983) J. Biol. Chem. 258, 696-699; Shioi, Naito & Ueda (1989) Biochem. J. 258, 499-504], and it is hypothesized that an electrochemical H+ gradient across the vesicle membrane, the so-called protonmotive force, elicits the neurotransmitter uptake. An inside-positive diffusion potential across the vesicle membrane was established with valinomycin plus Rb+. This artificial electrical potential promoted the uptake of glutamate, but not aspartate, in the synaptic vesicles prepared from bovine cerebral cortex. The uptake was inhibited by the protonmotive-force dissipators carbonyl cyanide p-trifluoro-methoxyphenylhydrazone or nigericin, and was enhanced by concomitant imposition of a pH jump (alkalinization) in the external medium. Subcellular and subvesicular distributions showed the uptake system to be predominantly associated with small synaptic vesicles. The results support the hypothesis that glutamate uptake into synaptic vesicles is coupled with a H+ efflux down the electrochemical potential gradient, which is generated by H(+)-pumping ATPase.