Biomedical subjects

E H Koo

Publications and source records attributed to E H Koo.

At least 55 records · Page 3Linked to original sources

PubMed1995

Aggregation of secreted amyloid beta-protein into sodium dodecyl sulfate-stable oligomers in cell culture.

Filamentous aggregates of the 40-42-residue amyloid beta-protein (A beta) accumulate progressively in the limbic and cerebral cortex in Alzheimer's disease, where they are intimately associated with neuronal and glial cytopathology. Attempts to model this cytotoxicity in vitro using synthetic peptides have shown that monomeric A beta is relatively inert, whereas aggregated A beta reproducibly exerts a variety of neurotoxic effects. The processes that mediate the conversion of monomeric A beta into a toxic aggregated state are thus of great interest. Previous studies of this conversion have employed high concentrations (10(-5)-10(-3) M) of synthetic A beta peptides under nonbiological conditions. We report here the detection of small amounts (< 10(-9) M) of SDS-stable A beta oligomers in the culture media of Chinese hamster ovary cells expressing endogenous or transfected amyloid beta-protein precursor genes. The identity of these oligomers (primarily dimers and trimers) was established by immunoprecipitation with a panel of A beta antibodies, by electrophoretic comigration with synthetic A beta oligomers, and by amino acid sequencing. The oligomeric A beta species comprised approximately 10-20% of the total immunoprecipitable A beta in these cultures. A truncated A beta species beginning at Arg 5 was enriched in the oligomers, suggesting that amino-terminal heterogeneity can influence A beta oligomerization in this system. Addition of Congo red (10 microM) during metabolic labeling of the cells led to increased monomeric and decreased oligomeric A beta. The ability to detect and quantitate oligomers of secreted A beta peptides in cell culture should facilitate dynamic studies of the critical process of initial A beta aggregation under physiological conditions.

Amyloid beta-Peptides↗

PubMed1995

Polarized sorting of beta-amyloid precursor protein and its proteolytic products in MDCK cells is regulated by two independent signals.

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). We analyzed the mechanisms regulating the polarized basolateral sorting of beta APP and its proteolytic derivatives in MDCK cells. Deletion of the last 32 amino acids (residues 664-695) of the beta APP cytoplasmic tail had 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 altered the distribution of cell surface beta APP so that approximately 40-50% of the molecules were inserted apically. In parallel, A beta was now secreted from both surfaces. Surprisingly, this change in surface beta APP had no influence on the basolateral secretion of APPs and p3. This result suggests that most beta APP molecules which give rise to APPs in MDCK cells are cleaved intracellularly before reaching the surface. Consistent with this conclusion, we readily detected intracellular APPs in carbonate extracts of isolated membrane vesicles. Moreover, ammonium chloride treatment resulted in the equal secretion of APPs into both compartments, as occurs with other non-membranous, basolaterally secreted proteins, but it did 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.

Amino Acid Sequence↗

PubMed1995

Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons.

Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells.

Amyloid beta-Protein Precursor↗

PubMed1995

Cell-surface beta-amyloid precursor protein stimulates neurite outgrowth of hippocampal neurons in an isoform-dependent manner.

beta-Amyloid precursor protein (beta APP) is an integral membrane polypeptide expressed in many neural and non-neural cells. beta APP occurs in part at the cell surface and undergoes proteolytic processing to release the large soluble ectodomain (APPs) and the amyloid beta-peptide (A beta), both of which have apparent trophic activity in vitro. Despite intense interest in beta APP expression and metabolism, there is limited knowledge about the function mediated by beta APP inserted at the cell-surface. We established a coculture system in which beta APP-transfected CHO cells serve as a substrate for the growth of primary rat hippocampal neurons. Compared to nontransfected CHO cells, the increased surface beta APP of the transfectants stimulated short-term neuronal adhesion and longer-term neurite outgrowth, whereas the increased amount of secreted APPs and A beta in conditioned medium produced no such effects when neurons were grown either on untransfected CHO cells or on a polylysine substrate. Moreover, a peptide which has been shown to block the trophic effects of secreted APPs (Ninomiya et al., 1993) failed to interrupt the neurite promoting activity mediated by the surface-expressed beta APP. Surface-expressed beta APP751 or beta APP770 isoforms mediated more neurite outgrowth than did the beta APP695 isoform. Antibody blocking and regional deletion experiments indicated that the mid-region of the beta APP ectodomain (residues 361-648) is involved in promoting neurite outgrowth. We conclude that surface-expressed cellular beta APP has a neurite-promoting function which is distinct from the trophic function of the secreted beta APP derivatives and may have special significance during brain development.

Amino Acid Sequence↗

PubMed1994

Secreted beta-amyloid precursor protein stimulates mitogen-activated protein kinase and enhances tau phosphorylation.

Biological effects related to cell growth, as well as a role in the pathogenesis of Alzheimer disease, have been ascribed to the beta-amyloid precursor protein (beta-APP). Little is known, however, about the intracellular cascades that mediate these effects. We report that the secreted form of beta-APP potently stimulates mitogen-activated protein kinases (MAPKs). Brief exposure of PC-12 pheochromocytoma cells to beta-APP secreted by transfected Chinese hamster ovary cells stimulated the 43-kDa form of MAPK by > 10-fold. Induction of a dominant inhibitory form of ras in a PC12-derived cell line prevented the stimulation of MAPK by secreted beta-APP, demonstrating the dependence of the effect upon p21ras. Because the microtubule-associated protein tau is hyperphosphorylated in Alzheimer disease, we sought and found a 2-fold enhancement in tau phosphorylation associated with the beta-APP-induced MAPK stimulation. In the ras dominant inhibitory cell line, beta-APP failed to enhance phosphorylation of tau. The data presented here provide a link between secreted beta-APP and the phosphorylation state of tau.

Amyloid beta-Protein Precursor↗

PubMed1994

Evidence that production and release of amyloid beta-protein involves the endocytic pathway.

Amyloid beta-protein (A beta), the 40-43-amino acid polypeptide that is the principal constituent of senile plaques found in Alzheimer's disease, is constitutively produced and released into medium of cultured cells by an unclear mechanism. In this study, we report that one route of A beta generation involves the internalization of cell surface amyloid precursor protein (beta PP) via the coated pit-mediated endocytic pathway. Radiolabeled A beta can be recovered in medium following selective cell surface radioiodination, indicating that cell surface beta PP is a direct precursor to A beta. In addition, deletion of the cytoplasmic domains of beta PP or depletion of potassium in medium, both of which resulted in reduced beta PP internalization, significantly diminished A beta release. Moreover, pulse-chase experiments after surface radioiodination showed that the kinetics of beta PP secretion and A beta release was different, with the latter occurring at a significantly slower rate. We therefore hypothesize that the internalization of cell surface beta PP via coated pit-mediated endocytosis is one pathway leading to A beta generation and release into medium.

Amyloid beta-Peptides↗

PubMed1994

Polarized secretion of beta-amyloid precursor protein and amyloid beta-peptide in MDCK cells.

The beta-amyloid precursor protein (beta APP) is a widely expressed integral membrane protein that is proteolytically processed to yield several secreted derivatives, including soluble APP (APPs), the 4-kDa amyloid beta-peptide (A beta), and a related 3-kDa peptide (p3). To understand beta APP trafficking and processing, we analyzed the sorting of beta APP in Madin-Darby canine kidney (MDCK) cells, an epithelial cell known to possess physiologically distinct apical and basolateral plasma membranes. Processing of beta APP resulted in highly polarized secretion of APPs. More than 90% of APPs was detected in the basolateral compartment, and less than 10% was found in the apical compartment. This was associated with a preferential localization of beta APP on the basolateral cell surface. Activation of protein kinase C, which is known to enhance the secretion of APPs, did not change the polarity of APPs release but significantly increased the amount secreted. A beta and p3 peptides were also secreted predominantly basolaterally. In addition, MDCK cells secreted a truncated form of A beta beginning at Arg-5. These data show that the proteolytic processing products of beta APP undergo polarized secretion. Moreover, the results suggest that the amyloidogenic A beta peptide is generated following the polarized sorting of beta APP. The polarized basolateral secretion of A beta in these epithelial cells provides a potential mechanism for the accumulation of A beta in the abluminal basement membrane of brain microvessels during Alzheimer disease.

Amino Acid Sequence↗

PubMed1993

Protein phosphorylation inhibits production of Alzheimer amyloid beta/A4 peptide.

The major component of amyloid plaque cores and cerebrovascular amyloid deposits found in Alzheimer disease is the beta/A4 peptide, which is derived from the Alzheimer amyloid protein precursor (APP). Recent evidence suggests that abnormalities in beta/A4 peptide production or beta/A4 peptide aggregation may underlie cerebral amyloidosis. In the present study, treatment of cells with phorbol dibutyrate, which activates protein kinase C, and/or okadaic acid, which inhibits protein phosphatases 1 and 2A, reduced beta/A4 peptide production by 50-80%. These effects were observed with APP695 and APP751 expressed in stably transfected CHO cells, as well as with endogenous APP in human glioma (Hs 683) cells. Phorbol dibutyrate also decreased beta/A4 peptide production in cells expressing various mutant forms of APP associated with familial Alzheimer disease, one of which was reported to manifest greatly increased beta/A4 peptide production in cultured cells. Mastoparan and mastoparan X, compounds which can activate phospholipase C and hence protein kinase C, also decreased beta/A4 peptide production in CHO cells stably transfected with APP695. A model is presented in which decreases in beta/A4 peptide production can be achieved by accelerating the metabolism of APP through a nonamyloidgenic secretory pathway.

Amino Acid Sequence↗

PubMed1993

Amyloid beta-protein as a substrate interacts with extracellular matrix to promote neurite outgrowth.

Progressive deposition of amyloid beta-protein (A beta) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic A beta to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble A beta may not accumulate in significant quantities in brain, we asked whether immobilized A beta peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of beta-amyloid. We detected no detrimental effects of A beta substrate on neurite outgrowth. Rather, A beta in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble A beta in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of A beta production by cultured neurons, our data suggest that A beta plays a normal physiological role in brain by complexing with the extracellular matrix.

Alzheimer Disease↗

PubMed1993

beta-Amyloid protein is higher in Alzheimer's disease brains: description of a quantitative biochemical assay.

Deposition of beta-amyloid protein (A beta) in senile plaques and in the walls of cerebral vessels is a pathologic hallmark of Alzheimer's disease (AD). The current diagnostic criteria for AD requires the presence of neurofibrillary tangles and a minimum number of senile plaques in cortex. Senile plaques are readily visualized by silver staining or immunocytochemistry using antibodies raised to A beta. Available histochemical and immunocytochemical methods are sensitive but the results may occasionally be variable and sampling from many brain regions is difficult and impractical. This study describes a simple biochemical method for quantifying the A beta load in unfixed brain homogenates. The immunoassay recognizes all forms of A beta deposits (neuritic and diffuse plaques, and cerebrovascular amyloid) and has a sensitivity and specificity comparable to immunocytochemistry. In direct comparisons, results from the dot blot method correspond well with both Western blot analysis of partially purified A beta and plaque counting by immunocytochemistry. In a retrospective series of 39 postmortem AD and control cases, the amount of A beta in brain by dot blot immunoreactivity effectively separated the two groups. Therefore, this method provides a rapid, sensitive, and accurate quantitation of A beta in postmortem brain tissue and represents an alternative approach for studying A beta deposition in aging and AD.

Aged↗

PubMed1993

Identification and transport of full-length amyloid precursor proteins in rat peripheral nervous system.

Amyloid deposits are a characteristic feature of the senile plaques identified in the brains of aged primates, individuals with Down's syndrome, and cases of Alzheimer's disease. The beta-amyloid protein (A beta), the principal component of amyloid, is a 4 kDa peptide derived from larger amyloid precursor protein(s) (APP). Four mRNAs, generated by alternative splicing of pre-mRNA derived from a single gene, encode A beta-containing membrane glycoproteins termed APP-695, -714, -751, and -770; the latter two isoforms contain a domain homologous to Kunitz protease inhibitors (KPI). The present study uses in vitro and in vivo strategies to examine the expression of APP in neurons of the dorsal root ganglia and the nature of APP transported in sciatic nerves of rats. Using quantitative in situ hybridization and semiquantitative PCR analysis, we document that mRNAs encoding APP-695 are expressed preferentially over transcripts that encode KPI-containing isoforms in rat sensory ganglia. Furthermore, we provide compelling evidence that APP-695 is the predominant isoform synthesized in sensory neurons of the rat PNS and that full-length APP-695 and, to a lesser extent, APP-751/770 are rapidly transported anterogradely in axons.

Amino Acid Sequence↗

PubMed1992

Increased expression of beta-amyloid precursor protein during neuronal differentiation is not accompanied by secretory cleavage.

Despite increasing evidence for a pathogenetic role for the beta-amyloid precursor protein (beta APP) in Alzheimer disease, the physiological function of the protein remains unclear. The expression of the neural-specific isoform containing 695 amino acids, beta APP695, is consistent with a role for the protein in neuronal development. In this study, we analyzed the expression of beta APP during the retinoic acid-induced neuronal differentiation of P19 murine embryonal carcinoma cells. Northern blot and RNase protection analyses show a selective increase in beta APP695 expression, concomitant with the morphologic differentiation of P19-derived neurons. Moreover, the time course of increase observed for the beta APP695 mRNA is paralleled by other neuronal-specific transcripts. A similar increase in beta APP695 is observed at the protein level. Furthermore, we show that levels of beta APP695 protein progressively increase during the in vitro differentiation of primary hippocampal neurons. The finding that beta APP695 increases selectively and progressively during neuronal differentiation in two different cell culture systems suggests that this isoform has an important cellular function during this process in the brain. Unlike beta APP in most peripheral cell types, the increased levels of beta APP found in terminally differentiated neuronal cells are not processed in significant amounts by secretory cleavage. Thus, differentiation of neurons is accompanied by increased beta APP695 expression and membrane retention of the protein as intact, full-length molecules that could serve as potential substrates for amyloidogenesis.

Amyloid beta-Protein Precursor↗

PubMed1992

Amyloid beta-peptide is produced by cultured cells during normal metabolism.

Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.

Amyloid beta-Peptides↗

PubMed1992

A calcium-stimulated serine protease from monkey brain degrades the beta-amyloid precursor protein.

Amyloid deposition, a histopathological feature of Alzheimer's disease brain, may be the underlying cause of this disease. The isolation of enzymes involved in both the normal and aberrant or alternative processing of the beta-amyloid precursor protein may lead to an understanding of how beta-protein, the major component of amyloid deposits, is formed in the brain parenchyma and vasculature of Alzheimer's disease patients and aged humans. As the same kind of deposits is also found in aged primates, the use of primates will undoubtedly help to understand the mechanisms of amyloid deposition, both spatially and temporally. Here we report the partial purification from adult monkey brain of a calcium-activated serine protease that is immunoreactive with antibodies against cathepsin G and is potentially involved in the abnormal degradation of the beta-amyloid precursor protein. Moreover, immunoreactivity with cathepsin G antibodies was localised to astrocytes in both adult and aged monkey cortex, suggesting that our protease may be expressed in astrocytes.

Aging↗

PubMed1992

Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments.

Progressive cerebral deposition of the amyloid beta-peptide is an early and invariant feature of Alzheimer's disease. The beta-peptide is released by proteolytic cleavages from the beta-amyloid precursor protein (beta APP), a membrane-spanning glycoprotein expressed in most mammalian cells. Normal secretion of beta APP involves a cleavage in the beta-peptide region, releasing the soluble extramembranous portion and retaining a 10K C-terminal fragment in the membrane. Because this secretory pathway precludes beta-amyloid formation, we searched for an alternative proteolytic processing pathway that can generate beta-peptide-bearing fragments from full-length beta APP. Incubation of living human endothelial cells with a beta APP antibody revealed reinternalization of mature beta APP from the cell surface and its targeting to endosomes/lysosomes. After cell-surface biotinylation, full-length biotinylated beta APP was recovered inside the cells. Purification of lysosomes directly demonstrated the presence of mature beta APP and an extensive array of beta-peptide-containing proteolytic products. Our results define a second processing pathway for beta APP and suggest that it may be responsible for generating amyloid-bearing fragments in Alzheimer's disease.

Alzheimer Disease↗

PubMed1992

Alzheimer beta/A4-amyloid precursor protein: evidence for putative amyloidogenic fragment.

Recombinant baculovirus was used to overexpress human Alzheimer beta/A4-amyloid precursor protein (APP) in Spodoptera frugiperda (Sf9) cells. Lysates of these cells were then analyzed for the presence of carboxyl-terminal fragments of APP by an immunoblotting assay using either an antibody against the APP cytoplasmic domain (rabbit anti-human 695APP645-694) or an antibody against the amino terminus of beta/A4-amyloid (rabbit anti-human 695APP586-606). Anti-human 695APP645-694 identified APP holoprotein, a 25-kDa species, and a prominent group of carboxyl-terminal fragments of 17, 16, and 14 kDa, whereas anti-human 695APP586-606 identified APP holoprotein and a single prominent low-molecular-mass protein species comigrating with the 17-kDa carboxyl-terminal fragment identified by anti-human 695APP645-694. No immunoreactive species was detected at these molecular mass positions when either antibody was used for analysis of lysates of either uninfected Sf9 cells or Sf9 cells infected with wild-type Autographa californica baculovirus. For each antibody, specific immunoreactivity was abolished by preabsorption with the corresponding peptide immunogen. The incorporation of a beta/A4-amyloid amino-terminal epitope into a 17-kDa fragment of APP suggests that, in the baculoviral overexpression system, the electrophoretic microheterogeneity of APP carboxyl-terminal fragments is due, at least in part, to alternative proteolysis of APP. If such carboxyl-terminal fragments of APP containing an intact beta/A4-amyloid domain are produced in human brain, then they may represent intermediates in the conversion of APP to deposited beta/A4-amyloid.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗

PubMed1991

Amyloid precursor protein in aged nonhuman primates.

In individuals with Alzheimer disease and in aged nonhuman primates, deposits of amyloid occur in senile plaques in brain parenchyma and in the walls of some meningeal and cortical vessels. Amyloid is primarily composed of beta/A4, a 4-kDa peptide derived from the transmembrane form of an amyloid precursor protein (APP). We examined the distribution of beta/A4 and APP (outside the beta/A4 domain) in cerebral cortices of monkeys ranging in age from 4 to 41 years. In all animals, APP immunoreactivity was present in cell bodies, proximal dendrites, and axons of cortical neurons. In aged animals, all of which showed senile plaques, large APP-positive axons were conspicuous, and APP immunoreactivity was present in neurites around beta/A4-immunoreactive plaques. In some plaques, APP-immunoreactive elements were located in proximity to deposits of beta/A4. The presence of APP immunoreactivity in neuronal perikarya, dendrites, axons, and in neurites within beta/A4-containing plaques supports the hypothesis that neurons can serve as one source of amyloid deposited in brain parenchyma.

Aging↗

PubMed1991

Amyloid-related proteins and nerve growth factor in Alzheimer's disease and animal models.

Alzheimer's disease (AD), the most common cause of dementia in adult life, is characterized by the deposition of amyloid in brain parenchyma and the degeneration of specific populations of nerve cells, including cholinergic neurons in the basal forebrain. In this review, we first outline studies of cellular and molecular events that lead to age-associated deposition of amyloid in the brains of nonhuman primates and then describe investigations of the effect of treatment with nerve growth factor (NGF) on experimentally induced abnormalities in cholinergic neurons of the basal forebrain. These studies of amyloidogenesis and the efficacy of trophic factors on specific groups of experimentally damaged neurons provide information about issues central to understanding the pathogenesis and treatment of human degenerative diseases, including AD.

Alzheimer Disease↗