PrP in scrapie and β/A4 in Alzheimer's disease show similar patterns of deposition in the brain
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S106 THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE amyloid DA4 protein of Alzheimer’s disease at N-terminal position, form highly insoluble aggregates if expressed in the rabbit reticulocyte translation system (1). Here we report that aggregation of the APP fragment A4CT depends on additional factors. In contrast to the reticulocyte expression system, expression of A4CT in HeLa, PC12 and COS cells results in only stable monomeric forms. We have identified the factors in Ihe rabbit reticulocyte system which are responsible for the transformation of soluble A4CT into insoluble and aggregating A4CT. Monomeric MCT expressed in eucariotic cells can be transformed into aggregating MCT by addition of these factors. We have also been able to show that these factors influence the amyloidogenic properties of amyloid OA4. Our results suggest that the factors which are necessary to generate the amyloidogenicity of A4CT and BA4 molecules also play a key role in amyloid formation in Alzheimer’s disease. References: (1) Dyrks, T.. Weidemann, A., Multhaup. G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Miiller-Hill, B., Masters, C.L. and Beyreuther, K. (1988) Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer’ s disease. EMBO J. 7: 949-951 Acknowledgements: Supported by the DFG (SFB 258 and 317), BMR, MetropolitanLifeFoundalion andbytheNHCandthe MRC of Australia and the Victorian Health Promotion Foundation. 417 CHARACTERIZATION OF “SECRETASE”, ALZHEIMER AMYLOID PROTEIN PRECURSOR SECRETING ENZYME, K. Maruyama, M. Usami, W. Yamao-Harigaya, F. Kametani, and K. Yoshikawa. Department of Molecular Biology, Tokyo Institute of Psychiatry (formerly, Psychiatric Research Institute of Tokyo), Kamikitazawa, Setagaya, Tokyo 156, Japan The formation of senile plaque is now thought to be one of the most promising causes of Alzheimer’s disease (AD). The main component of senile plaque is 42 amino-acid O/A4 protein (OP), which is derived from amyloid protein precursor (APP). In normal condition APP is cleaved at the internal site of OP, preventing the formation of OP, by a hypothetical proteinase “secretase”. The activity of “secretase” was characterized in culture cells by the expression of mutated APPs. Metho&-The mutated cDNAs of APP were expressed transiently in COS-1 cells (derived from monkey kidney fibroblast). The expressed APPs were detected by immunoblotting with anti-C antibody raised against the synthesized peptide GIy756-Asn770 of APP. The stable transformants of embryonal carcinoma cell line P19 were obtained by transfection of wild APP cDNA. Results-The wild APP expressed in COS-1 cells was detected as 110 kD and 9 kD bands. The N-terminal sequence of 9 kD fragment started as Leu-Val-Phe-Phe, the same results as that of Esch, F.S. et al (1990 Science 248, 1122-l 124). The APP expressed in PlS cells was processed in a similar way to that expressed in COS- 1 cells. The point mutations reported in hereditary cerebral hemorrhage with amyloidosis (Dutch): Glu693 to Gln and familial AD: Val717 to Ile did not affect the processing of APP. The mutations around the cleavage site of “secretase” (Gln686-Lys687-Leu6@J) did not affect the processing, either. The changes of Lys724-Lys725-Lys726 greatly reduced the secretion of APP. Q_nclusiorl-“Secretase” is not a sequence-specific proteinase. lt may recognize the conformation of APP and probably cleaves APP according to the length from cellular membrane. F-This work was supported by an award from the Sandoz Foundation for Gerontological Research and the Foundation For Total Health Promotion (to KM.) 418 MEMBRANE SURFACE PHENOMENA IN CELLS OVEREXPRESSING B/A4 AMYLDID DNA, G.E. Maestre, B.A. Tate, R.E. Majocha, C.A. Marotta. Harvard Medical School and Massachusetts General Hospital, Boston MA 02114 USA, and Instituto de Investigaciones Biologicas, Universidad de1 Zulia, Apdo . 526 Maracaibo, Venezuela. The consequences of amyloid CveraCCUUdatiOn were investigated using PC12 cells permanently transfected with DNA corresponding to the B/A4-C terminal region of the amyloid precursor protein (APP) . Transfected cell lines and controls were examined at both the light and transmission electron microscopy (TEM) levels. Many morphological parameters were similar between the B/A4-positive and control cell lines including number and size of mitochondria, number of lipofuscin granules and ratio of nucleus to cytoplasm. However, B/A4- positive cells exhibited membrane extensions significantly more than the control cell lines. The extensions were identified as microvilli, blebs and ruffles by TEM. In addition, ruffling activity was assessed in live cells by phase contrast microscopy and sequential photography. After application of monoclonal antibodies to B/A4, the transfected cell lines exhibited increased antigen along the length of the plasma membrane and the membrane extension. Finally, the quantitative assays indicated that cell spreading and substrate adhesion were increased in the amyloidotic cell lines. Membrane extensions may be related to phenomena such as neurosecretion, cell adhesion and cell-cell interaction. They may also be involved in the delivery of the O/A4 peptide to the external surface of the cell of origin and release to extracellular sites. Similar surface features of AD neurons, should they occur, may indicate the applicability of the proposed mechanism to the pathogenesis of the disorder. Supported by NIH AG02126, Metropolitan Life Foundation and the Axelrod Family Fund. GEM supported by Universidad de1 Zulia and Fundayacucho. 419 PrP IN SCRAPIE AND B/A4 IN ALZEBIIIBR'S DISEASE SEOW SItlILAR PATTERNS OF DEPOSITION IN TAB BRAIN H.E. Bruce’, P.A. McBride',H. Jeffrey',J.M. Rozcmuller3 and P. Eikelenboom’. ‘APRC 6 URC Neuropathogenesis Unit, Edinburgh, UK: 'Central Veterinary Laboratory, Edinburgh, UK; ‘Depts of Neuropathology b Psychiatry, Free University, Amsterdam, The Netherlands. Immunostaining of brain sections for PrP in the case of experimental scrapie in mice and B/A4 in the case of Alzheiner’ s disease has revealed several striking similarities in the pattern of deposition of these proteins. Host obviously, both PrP and B/A4 accumulate focally to form amyloid plaque cores; amyloid plaques in scrapie and Alzheimer’ s disease have long been known to have the same basic structure, consisting of an amyloid core surrounded by degenerating neurites, microglia and astrocytic processes. Bowever, the more usual pattern in scrapie is a diffuse or granular accumulation of PrP in the neuropil which is precisely targeted to particular groups of neurons according to the strain of scrapie. In some scrapie models diffuse, streaky PrP deposits are seen in the molecular layer of the cerebellum. The characteristic banding of these lesions suggests that they are associated vith Purkinje cell dendrites. In Alzheimer’s disease diffuse B/A4 plaques vith a similar appearance to the scrapie lesions are also often seen in the molecular layer of the cerebellum. In some other parts of the brain in scrapie, it is clear that PrP accumulates a.t the cell surface of certain individual neurons and their processes. A closely similar pattern of deposition of B/A4 has been observed around individual neurons in Alzheimer’ s disease. These result? suggest that similar mechanisms are involved in the development of diffuse changes in scrapie and Alzheimer’ s disease, as veil as in the development of amyloid plaques. 420 SCRAPIB-INDUCED OBESITY IN HAMSTERS: ASSOCIATION WITH PITUITARY AND HYF’OTH-C LESIONS. X. Ye.,Q3 R.I. Carp,‘ .= Y. Yu,) R. Koziel~ki,~ P. Kozlo~ki~. ‘Graduate School of the City University of New York, 2CSI/lBR Center for Developmental Neuroscience, ‘New York State InstituteforBasic ResearchinDevelopmentalDisabilities,10SOForestHillRoad, Staten Island, New York, 10314.
Transcript of PrP in scrapie and β/A4 in Alzheimer's disease show similar patterns of deposition in the brain
S106 THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE
amyloid DA4 protein of Alzheimer’s disease at N-terminal position, form highly insoluble aggregates if expressed in the rabbit reticulocyte translation system (1). Here we report that aggregation of the APP fragment A4CT depends on additional factors. In contrast to the reticulocyte expression system, expression of A4CT in HeLa, PC12 and COS cells results in only stable monomeric forms.
We have identified the factors in Ihe rabbit reticulocyte system which are responsible for the transformation of soluble A4CT into insoluble and aggregating A4CT. Monomeric MCT expressed in eucariotic cells can be transformed into aggregating MCT by addition of these factors. We have also been able to show that these factors influence the amyloidogenic properties of amyloid OA4.
Our results suggest that the factors which are necessary to generate the amyloidogenicity of A4CT and BA4 molecules also play a key role in amyloid formation in Alzheimer’s disease.
References: (1) Dyrks, T.. Weidemann, A., Multhaup. G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Miiller-Hill, B., Masters, C.L. and Beyreuther, K. (1988) Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer’s disease. EMBO J. 7: 949-951
Acknowledgements: Supported by the DFG (SFB 258 and 317), BMR, MetropolitanLifeFoundalion andbytheNHCandthe MRC of Australia and the Victorian Health Promotion Foundation.
417 CHARACTERIZATION OF “SECRETASE”, ALZHEIMER AMYLOID PROTEIN PRECURSOR SECRETING ENZYME, K. Maruyama, M. Usami, W. Yamao-Harigaya, F. Kametani, and K. Yoshikawa. Department of Molecular Biology, Tokyo Institute of Psychiatry (formerly, Psychiatric Research Institute of Tokyo), Kamikitazawa, Setagaya, Tokyo 156, Japan The formation of senile plaque is now thought to be one of the most promising causes of Alzheimer’s disease (AD). The main component of senile plaque is 42 amino-acid O/A4 protein (OP), which is derived from amyloid protein precursor (APP). In normal condition APP is cleaved at the internal site of OP, preventing the formation of OP, by a hypothetical proteinase “secretase”. The activity of “secretase” was characterized in culture cells by the expression of mutated APPs.
Metho&-The mutated cDNAs of APP were expressed transiently in COS-1 cells (derived from monkey kidney fibroblast). The expressed APPs were detected by immunoblotting with anti-C antibody raised against the synthesized peptide GIy756-Asn770 of APP. The stable transformants of embryonal carcinoma cell line P19 were obtained by transfection of wild APP cDNA.
Results-The wild APP expressed in COS-1 cells was detected as 110 kD and 9 kD bands. The N-terminal sequence of 9 kD fragment started as Leu-Val-Phe-Phe, the same results as that of Esch, F.S. et al (1990 Science 248, 1122-l 124). The APP expressed in PlS cells was processed in a similar way to that expressed in COS- 1 cells. The point mutations reported in hereditary cerebral hemorrhage with amyloidosis (Dutch): Glu693 to Gln and familial AD: Val717 to Ile did not affect the processing of APP. The mutations around the cleavage site of “secretase” (Gln686-Lys687-Leu6@J) did not affect the processing, either. The changes of Lys724-Lys725-Lys726 greatly reduced the secretion of APP.
Q_nclusiorl-“Secretase” is not a sequence-specific proteinase. lt may recognize the conformation of APP and probably cleaves APP according to the length from cellular membrane.
F-This work was supported by an award from the Sandoz Foundation for Gerontological Research and the Foundation For Total Health Promotion (to KM.)
418 MEMBRANE SURFACE PHENOMENA IN CELLS OVEREXPRESSING B/A4 AMYLDID DNA, G.E. Maestre, B.A. Tate, R.E. Majocha, C.A. Marotta. Harvard Medical School and Massachusetts General Hospital, Boston MA 02114 USA, and Instituto de Investigaciones Biologicas, Universidad de1 Zulia, Apdo . 526 Maracaibo, Venezuela.
The consequences of amyloid CveraCCUUdatiOn were investigated using PC12 cells permanently transfected with DNA corresponding to the B/A4-C terminal region of the amyloid precursor protein (APP) . Transfected cell lines and controls were examined at both the light and transmission electron microscopy (TEM) levels. Many morphological parameters were similar between the B/A4-positive and control cell lines including number and size of mitochondria, number of lipofuscin granules and ratio of nucleus to cytoplasm. However, B/A4- positive cells exhibited membrane extensions significantly more than the control cell lines. The extensions were identified as microvilli, blebs and ruffles by TEM. In addition, ruffling activity was assessed in live cells by phase contrast microscopy and sequential photography. After application of monoclonal antibodies to B/A4, the transfected cell lines exhibited increased antigen along the length of the plasma membrane and the membrane extension. Finally, the quantitative assays indicated that cell spreading and substrate adhesion were increased in the amyloidotic cell lines.
Membrane extensions may be related to phenomena such as neurosecretion, cell adhesion and cell-cell interaction. They may also be involved in the delivery of the O/A4 peptide to the external surface of the cell of origin and release to extracellular sites. Similar surface features of AD neurons, should they occur, may indicate the applicability of the proposed mechanism to the pathogenesis of the disorder. Supported by NIH AG02126, Metropolitan Life Foundation and the Axelrod Family Fund. GEM supported by Universidad de1 Zulia and Fundayacucho.
419 PrP IN SCRAPIE AND B/A4 IN ALZEBIIIBR'S DISEASE SEOW SItlILAR PATTERNS OF DEPOSITION IN TAB BRAIN H.E. Bruce’, P.A. McBride', H. Jeffrey', J.M. Rozcmuller3 and P. Eikelenboom’. ‘APRC 6 URC Neuropathogenesis Unit, Edinburgh, UK: 'Central Veterinary Laboratory, Edinburgh, UK; ‘Depts of Neuropathology b Psychiatry, Free University, Amsterdam, The Netherlands.
Immunostaining of brain sections for PrP in the case of experimental scrapie in mice and B/A4 in the case of Alzheiner’s disease has revealed several striking similarities in the pattern of deposition of these proteins. Host obviously, both PrP and B/A4 accumulate focally to form amyloid plaque cores; amyloid plaques in scrapie and Alzheimer’s disease have long been known to have the same basic structure, consisting of an amyloid core surrounded by degenerating neurites, microglia and astrocytic processes. Bowever, the more usual pattern in scrapie is a diffuse or granular accumulation of PrP in the neuropil which is precisely targeted to particular groups of neurons according to the strain of scrapie. In some scrapie models diffuse, streaky PrP deposits are seen in the molecular layer of the cerebellum. The characteristic banding of these lesions suggests that they are associated vith Purkinje cell dendrites. In Alzheimer’s disease diffuse B/A4 plaques vith a similar appearance to the scrapie lesions are also often seen in the molecular layer of the cerebellum. In some other parts of the brain in scrapie, it is clear that PrP accumulates a.t the cell surface of certain individual neurons and their processes. A closely similar pattern of deposition of B/A4 has been observed around individual neurons in Alzheimer’s disease. These result? suggest that similar mechanisms are involved in the development of diffuse changes in scrapie and Alzheimer’s disease, as veil as in the development of amyloid plaques.
420 SCRAPIB-INDUCED OBESITY IN HAMSTERS: ASSOCIATION WITH PITUITARY AND HYF’OTH-C LESIONS. X. Ye.,Q3 R.I. Carp,‘.= Y. Yu,) R. Koziel~ki,~ P. Kozlo~ki~. ‘Graduate School of the City University of New York, 2CSI/lBR Center for Developmental Neuroscience, ‘New York State InstituteforBasic ResearchinDevelopmentalDisabilities,10SOForestHillRoad, Staten Island, New York, 10314.
