β-Amyloid containing peptides produced in newborn rat cerebral cortex primary cultures
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SlO4 THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE 408 DIFFUSE PLAQUES AND CEREBROVASCULAR AMYLOIDOSIS ARE ACCOMPANIED BY ENTORHINAL PERINEURONAL AND HIPPOCAMPAL TERMINAL ZONE DEPOSITION OF AMYLOID IN THE AGING DOG. EVIDENCE FOR A NEURONAL ORIGIN OF AMYLOID, A.P. Osmand and R.C. Switzer. Departments of Medicine and Pathology, University of Tennessee Medical Center. Knoxville, Tennessee, USA 37920. Several investigators have described the accumulation in the brains of aging carnivores of both diffuse plaques and cerebrovascular arnyloid, in dogs during the second decade of life. These deposits have been shown to bs immunoreactive for the D/A4 protein characteristic of cerebral amyloid in Alzheimefs disease (AD) and thus provide an animal model for the amyloidosis of AD. Using sensitive immunohistochemical and argyrophilic methods we have studied the distribution of amyloid in coronal sections of brain tissue from four aged dogs (11, 14, 15 and 16 yrs). Numerous diffuse plaques and widespread cerebfovascular amyloid deposits were seen in the cortex in the three older dogs, with small numbers found in the youngest animal. Antisera to paired helical filaments were found to be immunoreactive only with scattered spherotial structures, with no evidence for the presence of neurofibrillary inclusions. The hippocampus and entorhinal cortex lacked amyloid plaques, in marked contrast to the distribution of these lesions typically seen in Alzheimer’s disease. However, in the two oMest animals a striking deposition of diffuse amyloid was seen in the molecular layers of the dentate gyrus and the hippocampus, and, in addition, numerous pyramidal neurons within the entorhinal cortex were seen to have perfkatyonal amyloid deposits on their surfaces. Amyloid deposits in these areas were both argyrophilic and immunoreactive with antisera to O-protein. Neurons in the entorhinal region provide extensive cortical input to the terminal zones of the hippocampal formation. It is probable that those cells in the entorbinal cortex that accumulate surface deposits of amyloid also give rise to amyloid in their terminal zones. We propose that such neurons may be particularly sensitive to the age-associated processes that pomote amyioid fonation. The aging dog should provide a predictable model for investigations both into the development and expression of cerebral O-protein amyloidosis and into its prevention. Supported by grants from the American Health Assistance Foundation and the Robert H. and Monica Cole Foundation. 409 EFFECT OF a2-MACROGLOBULIN AND OTHER PROTEINASE INHIBITORS ON THE CLEAVAGE OF AMYLOIDPRECURSOR PROTEIN.B. De Stroqxr and F. Van Leuven. Center for Human Genetics, University of Leuven,Leuven.Belgium. The formatlon d amyioid predpltates In the braln k the central event In the pathogeneskof Alzhelmer’s Dkease. Sincethe amyidd sequence Is deaved during the n& processing of the amyfold precursor protein(APP),it has to be fxWdated that amyloid in Alzheimerpknts Is dertued from precursor protein that escaped this cleavagestep. To investigate the fate d undeaved amylold precursor protein In neural cells. we have attemptedto inhibit the cleavage step In neuro 2a neuroblastoms cell cdturas by protease lnhlbitors d all major classes. (Phenantrdine, 1mM; alanti- chymotrypsln, 50 ag/m4: trypsinlnhibltw, 100 pg/ml; aprotlnin 10 &ml; TLCK. loOpg/ml; ES4. lOpg/ml; Leupeptln. lag/ml; pepstatln A lpg/rd; Bestatln 5@g/ml; icdoacetamkie, 2oOpM; u2macroglobulln, loorg/rrJ). Protelnase lnt&itorswereadded to the culture medium and the accumulation d metabdkally labeled.soluble (deaved) APP In the medium during a two hour Intervalwas fdlcwed by double immune predpltatlonv&h a polydonal anti-APPantibody. None d theproteinase inhibitors displayed any effect In thk assay. Since Gantar et al. (FEBS 282, 127.131, 1991) have found that a2-macroglobtiln, a broad spectrumproteinase Inhibitor of human serum, can lnhlbil the daavage d APP in SH-SY5Y neuroblastoma cells using a similar assay, we studied the effect d ~2. macroglobulin in moredatall.Neither In the culture supematants no in the cell extracts, alterations In the metabolism c4 APP were observed after the addition d a2- rnacroglobulln at 1 mg/ml for up to 24 hours. Since the addition d protelnase lnhlbltom In the extracellular medium had no effecton the deavage of APP.we speculate that the normal deavage step occurs not at the cdl surfacebul In an lntracdlular compartment. Prellmlnary data support the hypothesis that the cleavage step occur In an Intracellular acM compartment. 410 SYNTHETIC HUMAN P-AMYLOID HAS SELECTIVE VULNERABLE EFFECTS ON DIFFERENT TYPES OF NEURONS AND GLIAL CELLS IN IN VITRO CULTURES DERIVED FROM EMBRYONIC RAT BRAIN CORTEX P. Raw, M. Pakaski and *B. Penke. Central Research Laboratory and *Dept of Chemistry, Albert Szent-Gyargyi Medical University, 6720 Szeged, Hungary. In Alzheimer’s disease, the most characteristic neuropathological changes in the brain are the amyloid-containing senile plaques and the neurofibtillary tangles. Recently, the neurotrophic and/or neurotoxic effects of amyloid p protein have been described (Yankner et al., PNAS:250, 279-282, 1990). It has also been demonstrated that tachykinin antagonists rather than tachykinins themselves can produce the same neurotoxic effects as amyloid p protein in tissue cultures. In these investigations, our aim was to study the neurotoxic effects of synthetic human amyloid p protein (pl-42) on different types of neurons and glial cells by using embryonic rat brain mixed neuronal and glial cell cultures. Peptides: Substance-P, [D-Pro’ ,D-Trp’ .9]-substance-P and [D-Pro4,D-Trp7,‘ ,Nle”l- Fragment-Cl 1 were also used to study the trophic and/or toxic effects on the mixed cultures. 18-day-old embryonic rat brain cortical cultures containing different types of neurons and glial cells were cultured for 4 days in vitro (DIV) and thereafter were treated for 3DIV either with different concentrations (0.1, 1.0, 10 and 20 uM) of human synthetic p protein @l-42) or with the peptides (0.1, 1.0 and 10 uM) introduced directly into the incubation medium. The different types of neurons and the different types of glial cells were found to be affected in a dose-dependent manner by the amyloid p protein. The most sensitive neurons were small round and bipolar types, while the most resistant were the large multipolar neurons and the type 1 aseocytes. It is concluded that the vulnerability to synthetic peptides of neurons and glial cells cultured in viao differ. Supported by the OTKA (2723) and by the E’IT (T-123). 411 O-AMYLOID CONTAINING PEPTIDES PRODUCED IN NEWBORN RAT CEREBRAL CORTEX PRIMARY CULTURES. A. LeBlanc, D. LaSala, R. Xue, P. Gambetti. Institute of Pathology,CWRU. Cleveland, Ohio 44106. The composition of newborn rat cerebral cortex primary cultures changes with time in vitro. Cultures of 7 days in vitro (DIV) are composed mostly of type I astrocytes, microglial cells and large neurons. Between 14-32 DIV, clusters of small cells appear and are closely associated with large neurons. These cells stain intensely with hematoxilin, with the glial cell marker, A2B5, and the carbohydrate epitope common to cell adhesion molecules, HNK-1, but do not stain with the astrocyte marker, glial fibrillary acidic protein (GFAP-), oligodendrocyte markers, galactocerebroside (GalCer) or myelin associated glycoprotein (MAG). On the basis of these findings, these cluster-forming cells are tentatively identified as immature neurons or glial precursors. Regardless of the time in vitro, the rat cerebral cortex primary cultures express the same high levels of KPI-containing APP mRNAs and proteins. lmmunoblot analyses of cellular proteins revealed that the 7 DIV cultures which do not contain the clusters of small cells, express full-length forms of APP and a series of five smaller C-terminal peptides. These smaller C-terminal peptides include the f3-amyloid domain as evidenced by their immunoreactivity to l3-amyloid antibodies and by their size. By 21-32 DIV. an additional full length APP form is detected and four of the small C-terminal APP peptides have decreased significantly. Moreover, a peptide which migrates at a slightly slower rate than synthetic B-amyloid (l-40) peptide is detected by a monoclonal antibody to O-amyloid. lmmunocytochemistry of 21 DIV cultures has revealed cell surface immunoreactivity to B-amyloid peptide antibodies around the clusters of small cells suggesting that the small B-amyloid containing peptides are located in these clusters. Further characterization of the B-amyloid containing peptides is underway but these experiments suggest that Primary cultures of the newborn rat cerebral cortex are capable of Producing amyloidogenic peptides. Supported by NIH NIA ROl AGNS 08155, Britton fund and NIH NIA AGOB992-01. 412 Alteratlon In the level of APP by chemical treatment In cultutred cells: a probe to study the possible celluar function of APP. Ryuichi Fukuyama, Krish Chandrasekaran. Zygmunt Galdzicld, James Stall and Stanley I. Rapoport; Laboratory of Neuroaciences, National Institute on Aging, NIH, Bethesda, MD 20892, U.S.A.
Transcript of β-Amyloid containing peptides produced in newborn rat cerebral cortex primary cultures
SlO4 THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE
408 DIFFUSE PLAQUES AND CEREBROVASCULAR AMYLOIDOSIS ARE ACCOMPANIED BY ENTORHINAL PERINEURONAL AND HIPPOCAMPAL TERMINAL ZONE DEPOSITION OF AMYLOID IN THE AGING DOG. EVIDENCE FOR A NEURONAL ORIGIN OF AMYLOID, A.P. Osmand and R.C. Switzer. Departments of Medicine and Pathology, University of Tennessee Medical Center. Knoxville, Tennessee, USA 37920.
Several investigators have described the accumulation in the brains of aging carnivores of both diffuse plaques and cerebrovascular arnyloid, in dogs during the second decade of life. These deposits have been shown to bs immunoreactive for the D/A4 protein characteristic of cerebral amyloid in Alzheimefs disease (AD) and thus provide an animal model for the amyloidosis of AD.
Using sensitive immunohistochemical and argyrophilic methods we have studied the distribution of amyloid in coronal sections of brain tissue from four aged dogs (11, 14, 15 and 16 yrs). Numerous diffuse plaques and widespread cerebfovascular amyloid deposits were seen in the cortex in the three older dogs, with small numbers found in the youngest animal. Antisera to paired helical filaments were found to be immunoreactive only with scattered spherotial structures, with no evidence for the presence of neurofibrillary inclusions.
The hippocampus and entorhinal cortex lacked amyloid plaques, in marked contrast to the distribution of these lesions typically seen in Alzheimer’s disease. However, in the two oMest animals a striking deposition of diffuse amyloid was seen in the molecular layers of the dentate gyrus and the hippocampus, and, in addition, numerous pyramidal neurons within the entorhinal cortex were seen to have perfkatyonal amyloid deposits on their surfaces. Amyloid deposits in these areas were both argyrophilic and immunoreactive with antisera to O-protein. Neurons in the entorhinal region provide extensive cortical input to the terminal zones of the hippocampal formation. It is probable that those cells in the entorbinal cortex that accumulate surface deposits of amyloid also give rise to amyloid in their terminal zones. We propose that such neurons may be particularly sensitive to the age-associated processes that pomote amyioid fonation. The aging dog should provide a predictable model for investigations both into the development and expression of cerebral O-protein amyloidosis and into its prevention.
Supported by grants from the American Health Assistance Foundation and the Robert H. and Monica Cole Foundation.
409 EFFECT OF a2-MACROGLOBULIN AND OTHER PROTEINASE INHIBITORS ON THE CLEAVAGE OF AMYLOID PRECURSOR PROTEIN. B. De Stroqxr and F. Van Leuven. Center for Human Genetics, University of Leuven, Leuven. Belgium. The formatlon d amyioid predpltates In the braln k the central event In the pathogenesk of Alzhelmer’s Dkease. Since the amyidd sequence Is deaved during the n& processing of the amyfold precursor protein (APP), it has to be fxWdated that amyloid in Alzheimer pknts Is dertued from precursor protein that escaped this cleavage step. To investigate the fate d undeaved amylold precursor protein In neural cells. we have attempted to inhibit the cleavage step In neuro 2a neuroblastoms cell cdturas by protease lnhlbitors d all major classes. (Phenantrdine, 1mM; alanti- chymotrypsln, 50 ag/m4: trypsinlnhibltw, 100 pg/ml; aprotlnin 10 &ml; TLCK. loOpg/ml; ES4. lOpg/ml; Leupeptln. lag/ml; pepstatln A lpg/rd; Bestatln 5@g/ml; icdoacetamkie, 2oOpM; u2macroglobulln, loorg/rrJ). Protelnase lnt&itorswere added to the culture medium and the accumulation d metabdkally labeled. soluble (deaved) APP In the medium during a two hour Interval was fdlcwed by double immune predpltatlon v&h a polydonal anti-APP antibody. None d the proteinase inhibitors displayed any effect In thk assay. Since Gantar et al. (FEBS 282, 127.131, 1991) have found that a2-macroglobtiln, a broad spectrum proteinase Inhibitor of human serum, can lnhlbil the daavage d APP in SH-SY5Y neuroblastoma cells using a similar assay, we studied the effect d ~2. macroglobulin in more datall. Neither In the culture supematants no in the cell extracts, alterations In the metabolism c4 APP were observed after the addition d a2- rnacroglobulln at 1 mg/ml for up to 24 hours. Since the addition d protelnase lnhlbltom In the extracellular medium had no effect on the deavage of APP. we speculate that the normal deavage step occurs not at the cdl surface bul In an lntracdlular compartment. Prellmlnary data support the hypothesis that the cleavage step occur In an Intracellular acM compartment.
410 SYNTHETIC HUMAN P-AMYLOID HAS SELECTIVE VULNERABLE EFFECTS ON DIFFERENT TYPES OF NEURONS AND GLIAL CELLS IN IN VITRO CULTURES DERIVED FROM EMBRYONIC RAT BRAIN CORTEX
P. Raw, M. Pakaski and *B. Penke. Central Research Laboratory and *Dept of Chemistry, Albert Szent-Gyargyi Medical University, 6720 Szeged, Hungary.
In Alzheimer’s disease, the most characteristic neuropathological changes in the brain are the amyloid-containing senile plaques and the neurofibtillary tangles. Recently, the neurotrophic and/or neurotoxic effects of amyloid p protein have been described (Yankner et al., PNAS:250, 279-282, 1990). It has also been demonstrated that tachykinin antagonists rather than tachykinins themselves can produce the same neurotoxic effects as amyloid p protein in tissue cultures. In these investigations, our aim was to study the neurotoxic effects of synthetic human amyloid p protein (pl-42) on different types of neurons and glial cells by using embryonic rat brain mixed neuronal and glial cell cultures. Peptides: Substance-P, [D-Pro’,D-Trp’.9]-substance-P and [D-Pro4,D-Trp7,‘,Nle”l- Fragment-Cl 1 were also used to study the trophic and/or toxic effects on the mixed cultures. 18-day-old embryonic rat brain cortical cultures containing different types of neurons and glial cells were cultured for 4 days in vitro (DIV) and thereafter were treated for 3DIV either with different concentrations (0.1, 1.0, 10 and 20 uM) of human synthetic p protein @l-42) or with the peptides (0.1, 1.0 and 10 uM) introduced directly into the incubation medium. The different types of neurons and the different types of glial cells were found to be affected in a dose-dependent manner by the amyloid p protein. The most sensitive neurons were small round and bipolar types, while the most resistant were the large multipolar neurons and the type 1 aseocytes. It is concluded that the vulnerability to synthetic peptides of neurons and glial cells cultured in viao differ. Supported by the OTKA (2723) and by the E’IT (T-123).
411 O-AMYLOID CONTAINING PEPTIDES PRODUCED IN NEWBORN RAT CEREBRAL CORTEX PRIMARY CULTURES. A. LeBlanc, D. LaSala, R. Xue, P. Gambetti. Institute of Pathology,CWRU. Cleveland, Ohio 44106. The composition of newborn rat cerebral cortex primary cultures changes with time in vitro. Cultures of 7 days in vitro (DIV) are composed mostly of type I astrocytes, microglial cells and large neurons. Between 14-32 DIV, clusters of small cells appear and are closely associated with large neurons. These cells stain intensely with hematoxilin, with the glial cell marker, A2B5, and the carbohydrate epitope common to cell adhesion molecules, HNK-1, but do not stain with the astrocyte marker, glial fibrillary acidic protein (GFAP-), oligodendrocyte markers, galactocerebroside (GalCer) or myelin associated glycoprotein (MAG). On the basis of these findings, these cluster-forming cells are tentatively identified as immature neurons or glial precursors. Regardless of the time in vitro, the rat cerebral cortex primary cultures express the same high levels of KPI-containing APP mRNAs and proteins. lmmunoblot analyses of cellular proteins revealed that the 7 DIV cultures which do not contain the clusters of small cells, express full-length forms of APP and a series of five smaller C-terminal peptides. These smaller C-terminal peptides include the f3-amyloid domain as evidenced by their immunoreactivity to l3-amyloid antibodies and by their size. By 21-32 DIV. an additional full length APP form is detected and four of the small C-terminal APP peptides have decreased significantly. Moreover, a peptide which migrates at a slightly slower rate than synthetic B-amyloid (l-40) peptide is detected by a monoclonal antibody to O-amyloid. lmmunocytochemistry of 21 DIV cultures has revealed cell surface immunoreactivity to B-amyloid peptide antibodies around the clusters of small cells suggesting that the small B-amyloid containing peptides are located in these clusters. Further characterization of the B-amyloid containing peptides is underway but these experiments suggest that Primary cultures of the newborn rat cerebral cortex are capable of Producing amyloidogenic peptides. Supported by NIH NIA ROl AGNS 08155, Britton fund and NIH NIA AGOB992-01.
412 Alteratlon In the level of APP by chemical treatment In cultutred cells: a probe to study the possible celluar function of APP. Ryuichi Fukuyama, Krish Chandrasekaran. Zygmunt Galdzicld, James Stall and Stanley I. Rapoport; Laboratory of Neuroaciences, National Institute on Aging, NIH, Bethesda, MD 20892, U.S.A.
