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424 The regulation of alternative splicing at exon 15 in β-Amyloid precursor protein mRNA
S106
FIFI'H INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE
microscopic examination of the blood vessel shows that 13-amyloid treatment results in significant damage to the endothelium. These observations suggest a normal vasoactive role for 13-amyloid as well as a mechanism by which 13-amyloid may play a role in vascular degeneration and free radical mediated neurodegeneration in AD.
423 13-Amyloid Neurotoxieity in vitro: Examination of Potential Contributions by Oxidative Pathways C.J. Pike* and C.W. Cotman Institute for Brain Aging and Dementia, Department of Psychobiology, University of California, Irvine, CA 92717-4540 U.S.A. Several converging lines of evidence support a prominent role of [3-amyloid protein (AI3)in the pathogenesis of Alzheimer's disease (AD). Consistent with this position, cell culture studies have demonstrated that flbrillar aggregates of AI3 induce apoptotic neuronal degeneration. Although the mechanism by which AI3 triggers neuronal loss in vitro has yet to be clearly elucidated, data from some recent studies suggest potential involvement of oxidative pathways. To evaluate this possibility, we used embryonic rat hippocampal and cortical cultures to compare both the efficacies of neuroprotective agents and the characteristics of cell death in neurons exposed to AI3 peptides and various oxidative insults (e.g., iron, hydrogen peroxide, tert-buytl hydroperoxide). We report that doses of several antioxidants (e.g., trolox, propyl gallate, promethazine) which robustly attenuate various oxidative insults either fall to attenuate Al~-indueed neurotoxicity or provide only mild protection. Further, although TBAR assays of AI~-treated cultures showed some evidence of lipid peroxidation during the course of degeneration, an effective inhibitor of lipid peroxidation (probueol) observed to both inhibit TBAR formation and robustly inhibit iron-mediated neurotoxicity failed to protect neurons against AI3. In addition, TBARs were elevated in a cell free system by iron but not A]3 peptides, indicating that AI~ itself is not a potent free radical generator. However, Al~-induced neurodegeneration was significantly potentiated in cultures pre-treated with subtoxic doses of iron. These initial studies fail to support the position that the primary mechanism of All toxicity in vitro involves damage mediated by free radicals but suggest that ceils under oxidative stress are particularly vulnerable to AI3. Given prior evidence of an oxidative neural environment in AD, the current data suggest that although antioxidant agents may not directly antagonize neurotoxic pathways of AlL their clinical use may delay the onset and or progression of cognitive deficits in AD patients by increasing neuronal resistance to the toxic effects of fibriltar AI3deposits.
425 Alternative splicing of exon 15 regulates APP conformation L. Hesse*, D. Beher, C. L. Masters*, K. Beyreuther and G. Multhaup ZMBH, Univ. Heidelberg, INF 282, D-69120 Heidelberg, Germany *Dept. Pathology, Univ. Melbourne, Parkville, Victoria 3052, Australia Alternative splicing of exons 7 (KPI), 8 (OX-2) and 15 (GAGInhibibitor) generates eight amyloidogenic APP isoforms. Neurons express mainly exon 15-containing APP and non-neuronal cells exon 15-lacking LAPP isoforms. We have expressed in E. coli the two exon 7-containing isoforms with exon 15 (APP 751) and without exon 15 (L-APP 733), purified the proteins and analysed their properties. We report that exon 15 splicing regulates APP conformation. For expression of the two isoforms we used plasmids which encode B1-8 VHCH1-APP751 or -APP733 fusion proteins linked by a recognition sequence for the IgA protease from Neisseria gonorrhoeae. Recombinant APP and L-APP are obtained after solubilisation/denaturation of the E. coli inclusion bodies, IgA-protease treatment and renaturation. N-terminal sequencing of recombinant APP751 and L-APP733 shows that the amino terminus of both APP and L-APP is precisely processed. The monoclonal antibody ALZ90 which recognizes an epitope within exon 15 binds to recombinant APP751 and not to L-APP733. This confirmes the identity of the two isoforms. L-APP733 migrates with an unexpected mobility on SDS gels by exhibiting a higher apparent molecular weight (131kDa) than APP751 (129kDa). Both apparent molecular weights exceed the predicted sizes of 83,2kDa for APP751 and of 81,3kDa for L-APP733 indicating that the molecules retain different secondary structure in the presence of SDS. Our data show that exon 15 regulates the conformation of APP. We suggest that the part of the APP ectodomaln that precedes the transmemhrane domain is stabilized in the absence of exon 15 (L-APP733) inhibiting the proximal 13 secretase but not the distal alpha secretase cleavage. In the presence of exon 15 (APP 751), this region appears to form a more flexible hinge-like structure and therefore to become more accessible to 13secretase cleavage. The latter may explain why neurons which mainly express APP isoforms containing exon 15 release higher amounts of BA4 proteins than non-neuronal cells.
424 The Regulation of Alternative Splicing at Exon 15 in B - A m y l o i d Precursor Protein mRNA S. Sudohl, 2, H. Kawakami2, Y. Mimori2, S. Nakamura2, K. Yanagisawat, K. Kitani I, and A. Igata 1 1Division of Dementia Research, National Institute for Longevity Science (NILS), 36-3 G-engo, Morioka-cho, Obu, Aichi 474, Japan. 2Third Department of Internal of Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan. The alternative splicing at exon 15 in 13-amyloid precursor protein (APP) gene produces two isoforms. Leukocyte derived APP (L-APP) isoform, which lacks exon 15 ubiquitously express in almost all tissues except neurons. The exon 15-encoded lesion is located close to the NH2 terminus cleavage site of BIA4 protein, suggesting that the alteration in the secondary protein structure might alter the metabolism of amyloidogenic B/A4 protein. However, little is known about the regulation of L-APP isoform. We examined the regulation of L-APP isoforms both in vitro and in lymphocyte from the patient with Alzheimer's disease (AD). We found that serum deprivation increased total APP mRNA levels approximately 4-fold over controls by Northern blot analysis. Using this system, we examined whether the increased levels of APP mRNA induced by serum deprivation were accompanied by alterations in the alternative splicing pattern of the alternative splice site at exon 15 by quantitative reverse transcription polymerase chain reaction (RT-PCR). After serum deprivation, the relative amount of L-APP significantly increased in a time dependent manner (19.4 ± 0.3% at 0 h; 34.8 ± 0.7% at 30 h, p < 0~01; and 39.6% • 0.2% at 48 h,p < 0.001 compared to 0 h). We next examined the proportion of L-APP isoform in lymphocyte from 26 probable AD patients and from 20 normal controls. The proportion of LAPP was 43 ± 3% in AD group and 50 ± 5% in normal control group. There was a significant decrease of the proportion of L-APP in AD group compared with normal control group (p < 0.01). In conclusion, our in vitro data provide evidence that the alternative splicing of exon 15 is "plastic", and in vivo data suggest that L-APP isoforms might play a crucial role in the pathogenesis of AD.
426 APP shares glycan modifications characteristic of cell adhesion molecules D. Beher*, G.C. Peraus, M. Schachner#, C.L. Masters*, K. Beyreuther, G. Multhaup ZMBH, Univ. Heidelberg, INF 282, D-69120 Heidelberg, Germany #ETH, Hgnggerberg, CH-8093 Ztirich, Switzerland *Dept. Pathology, Univ. Melbourne, Parkville, Victoria, 3052, Australia Amyloid precursor proteins (APP) are N- and O-glycosylated, tyrosine sulfated transmembrane proteins. Alternative processing of APP generates the 13A4 peptide which is a major component of amyloid plaques in brains of patients with Alzheimer's disease. Normal processing produces secretory APP forms by cleveage at lysine residue 16 of the 13A4 region, which prevents the formation of 13A4. The finding that APP binds to molecules of the extracellular matrix like heparansulfate proteoglycanes, laminin and collagens suggests that APP may function in cell-matrix interaction and/or regulation and 13A4deposists interfere with these functions. To further investigate this functional aspect we analyzed APP for the presence of a carbohydrate epitope recognized by the monoclonal antibody HNK-I. This epitope is characterized by the presence of a 3'-sulfated glucuronic acid and a common modification of neuronal cell adhesion molecules like N-CAM. By surface plasmon resonance (SPR) and Westernblot analysis we show that APP purified from human brain contains this sulfated carbohydrate modification on the N-glycan. Using SH-SY5Y cells stably transfected with a N-terminal e-myc tagged APP695 isoform, metabolic sulfate labeling of APP followed by immunoprecipitation, removal of the N-glyean and subsequent quantification demonstrated that the N-linked oligosaccharide is sulfated. This modification seems to occur in SY5Y cells in a different way, because no HNK-1 immunoreactivity was detectable by Western-blot analysis. To gain a better insight into the structures of the APP glycan chains a variety of lectins were tested by SPR-analysis for binding to APP purified from human brain. The results show that the N-glycan core contains an alpha (1-6) linked fucose residue. APP-linked carbohydrates are also characterized by terminal alpha (2-6) and alpha (2-3) sialic acids. This corroborates the suggestion for a putative function for APP in cell-cell interactions because sialic acids of selectins are known to be directly involved in these processes.
FIFI'H INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE
microscopic examination of the blood vessel shows that 13-amyloid treatment results in significant damage to the endothelium. These observations suggest a normal vasoactive role for 13-amyloid as well as a mechanism by which 13-amyloid may play a role in vascular degeneration and free radical mediated neurodegeneration in AD.
423 13-Amyloid Neurotoxieity in vitro: Examination of Potential Contributions by Oxidative Pathways C.J. Pike* and C.W. Cotman Institute for Brain Aging and Dementia, Department of Psychobiology, University of California, Irvine, CA 92717-4540 U.S.A. Several converging lines of evidence support a prominent role of [3-amyloid protein (AI3)in the pathogenesis of Alzheimer's disease (AD). Consistent with this position, cell culture studies have demonstrated that flbrillar aggregates of AI3 induce apoptotic neuronal degeneration. Although the mechanism by which AI3 triggers neuronal loss in vitro has yet to be clearly elucidated, data from some recent studies suggest potential involvement of oxidative pathways. To evaluate this possibility, we used embryonic rat hippocampal and cortical cultures to compare both the efficacies of neuroprotective agents and the characteristics of cell death in neurons exposed to AI3 peptides and various oxidative insults (e.g., iron, hydrogen peroxide, tert-buytl hydroperoxide). We report that doses of several antioxidants (e.g., trolox, propyl gallate, promethazine) which robustly attenuate various oxidative insults either fall to attenuate Al~-indueed neurotoxicity or provide only mild protection. Further, although TBAR assays of AI~-treated cultures showed some evidence of lipid peroxidation during the course of degeneration, an effective inhibitor of lipid peroxidation (probueol) observed to both inhibit TBAR formation and robustly inhibit iron-mediated neurotoxicity failed to protect neurons against AI3. In addition, TBARs were elevated in a cell free system by iron but not A]3 peptides, indicating that AI~ itself is not a potent free radical generator. However, Al~-induced neurodegeneration was significantly potentiated in cultures pre-treated with subtoxic doses of iron. These initial studies fail to support the position that the primary mechanism of All toxicity in vitro involves damage mediated by free radicals but suggest that ceils under oxidative stress are particularly vulnerable to AI3. Given prior evidence of an oxidative neural environment in AD, the current data suggest that although antioxidant agents may not directly antagonize neurotoxic pathways of AlL their clinical use may delay the onset and or progression of cognitive deficits in AD patients by increasing neuronal resistance to the toxic effects of fibriltar AI3deposits.
425 Alternative splicing of exon 15 regulates APP conformation L. Hesse*, D. Beher, C. L. Masters*, K. Beyreuther and G. Multhaup ZMBH, Univ. Heidelberg, INF 282, D-69120 Heidelberg, Germany *Dept. Pathology, Univ. Melbourne, Parkville, Victoria 3052, Australia Alternative splicing of exons 7 (KPI), 8 (OX-2) and 15 (GAGInhibibitor) generates eight amyloidogenic APP isoforms. Neurons express mainly exon 15-containing APP and non-neuronal cells exon 15-lacking LAPP isoforms. We have expressed in E. coli the two exon 7-containing isoforms with exon 15 (APP 751) and without exon 15 (L-APP 733), purified the proteins and analysed their properties. We report that exon 15 splicing regulates APP conformation. For expression of the two isoforms we used plasmids which encode B1-8 VHCH1-APP751 or -APP733 fusion proteins linked by a recognition sequence for the IgA protease from Neisseria gonorrhoeae. Recombinant APP and L-APP are obtained after solubilisation/denaturation of the E. coli inclusion bodies, IgA-protease treatment and renaturation. N-terminal sequencing of recombinant APP751 and L-APP733 shows that the amino terminus of both APP and L-APP is precisely processed. The monoclonal antibody ALZ90 which recognizes an epitope within exon 15 binds to recombinant APP751 and not to L-APP733. This confirmes the identity of the two isoforms. L-APP733 migrates with an unexpected mobility on SDS gels by exhibiting a higher apparent molecular weight (131kDa) than APP751 (129kDa). Both apparent molecular weights exceed the predicted sizes of 83,2kDa for APP751 and of 81,3kDa for L-APP733 indicating that the molecules retain different secondary structure in the presence of SDS. Our data show that exon 15 regulates the conformation of APP. We suggest that the part of the APP ectodomaln that precedes the transmemhrane domain is stabilized in the absence of exon 15 (L-APP733) inhibiting the proximal 13 secretase but not the distal alpha secretase cleavage. In the presence of exon 15 (APP 751), this region appears to form a more flexible hinge-like structure and therefore to become more accessible to 13secretase cleavage. The latter may explain why neurons which mainly express APP isoforms containing exon 15 release higher amounts of BA4 proteins than non-neuronal cells.
424 The Regulation of Alternative Splicing at Exon 15 in B - A m y l o i d Precursor Protein mRNA S. Sudohl, 2, H. Kawakami2, Y. Mimori2, S. Nakamura2, K. Yanagisawat, K. Kitani I, and A. Igata 1 1Division of Dementia Research, National Institute for Longevity Science (NILS), 36-3 G-engo, Morioka-cho, Obu, Aichi 474, Japan. 2Third Department of Internal of Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan. The alternative splicing at exon 15 in 13-amyloid precursor protein (APP) gene produces two isoforms. Leukocyte derived APP (L-APP) isoform, which lacks exon 15 ubiquitously express in almost all tissues except neurons. The exon 15-encoded lesion is located close to the NH2 terminus cleavage site of BIA4 protein, suggesting that the alteration in the secondary protein structure might alter the metabolism of amyloidogenic B/A4 protein. However, little is known about the regulation of L-APP isoform. We examined the regulation of L-APP isoforms both in vitro and in lymphocyte from the patient with Alzheimer's disease (AD). We found that serum deprivation increased total APP mRNA levels approximately 4-fold over controls by Northern blot analysis. Using this system, we examined whether the increased levels of APP mRNA induced by serum deprivation were accompanied by alterations in the alternative splicing pattern of the alternative splice site at exon 15 by quantitative reverse transcription polymerase chain reaction (RT-PCR). After serum deprivation, the relative amount of L-APP significantly increased in a time dependent manner (19.4 ± 0.3% at 0 h; 34.8 ± 0.7% at 30 h, p < 0~01; and 39.6% • 0.2% at 48 h,p < 0.001 compared to 0 h). We next examined the proportion of L-APP isoform in lymphocyte from 26 probable AD patients and from 20 normal controls. The proportion of LAPP was 43 ± 3% in AD group and 50 ± 5% in normal control group. There was a significant decrease of the proportion of L-APP in AD group compared with normal control group (p < 0.01). In conclusion, our in vitro data provide evidence that the alternative splicing of exon 15 is "plastic", and in vivo data suggest that L-APP isoforms might play a crucial role in the pathogenesis of AD.
426 APP shares glycan modifications characteristic of cell adhesion molecules D. Beher*, G.C. Peraus, M. Schachner#, C.L. Masters*, K. Beyreuther, G. Multhaup ZMBH, Univ. Heidelberg, INF 282, D-69120 Heidelberg, Germany #ETH, Hgnggerberg, CH-8093 Ztirich, Switzerland *Dept. Pathology, Univ. Melbourne, Parkville, Victoria, 3052, Australia Amyloid precursor proteins (APP) are N- and O-glycosylated, tyrosine sulfated transmembrane proteins. Alternative processing of APP generates the 13A4 peptide which is a major component of amyloid plaques in brains of patients with Alzheimer's disease. Normal processing produces secretory APP forms by cleveage at lysine residue 16 of the 13A4 region, which prevents the formation of 13A4. The finding that APP binds to molecules of the extracellular matrix like heparansulfate proteoglycanes, laminin and collagens suggests that APP may function in cell-matrix interaction and/or regulation and 13A4deposists interfere with these functions. To further investigate this functional aspect we analyzed APP for the presence of a carbohydrate epitope recognized by the monoclonal antibody HNK-I. This epitope is characterized by the presence of a 3'-sulfated glucuronic acid and a common modification of neuronal cell adhesion molecules like N-CAM. By surface plasmon resonance (SPR) and Westernblot analysis we show that APP purified from human brain contains this sulfated carbohydrate modification on the N-glycan. Using SH-SY5Y cells stably transfected with a N-terminal e-myc tagged APP695 isoform, metabolic sulfate labeling of APP followed by immunoprecipitation, removal of the N-glyean and subsequent quantification demonstrated that the N-linked oligosaccharide is sulfated. This modification seems to occur in SY5Y cells in a different way, because no HNK-1 immunoreactivity was detectable by Western-blot analysis. To gain a better insight into the structures of the APP glycan chains a variety of lectins were tested by SPR-analysis for binding to APP purified from human brain. The results show that the N-glycan core contains an alpha (1-6) linked fucose residue. APP-linked carbohydrates are also characterized by terminal alpha (2-6) and alpha (2-3) sialic acids. This corroborates the suggestion for a putative function for APP in cell-cell interactions because sialic acids of selectins are known to be directly involved in these processes.