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618 Physiological aspects of β-amyloid precursor protein (β-APP) in brain
S154
FIFTH INTERNATIONAL
CA, 90073; 2Bayer Corp., Pharmaceutical Division, West-Haven, CT, 06516 Metrifonate is a long-acting inhibitor of acetylcholinesterase and has been investigated for the treatment of Alzheimer's Disease (AD) since 1990 (Becker et al). The results of a recently unhlinded dose-finding study, designed to evaluate the safety and efficacy profile of metrifonate will be reported. This double-blind, placebo controlled, multicenter study included 480 patients with probable AD recruited from 28 centers across the US. Metrifonate was administered once daily as a loading dose for 14 days followed by a maintenance dose for twelve weeks. Three dosages of metrifonate were compared to placebo, achieving red blood cell acetylcholinesterase inhibition levels of up to 75 %. Overall, 91% of patients receiving metrifonate completed the study compared to 96% for placebo. Only 22/360 (6%) of the metrifonate treated patients discontinued for adverse events, compared with 5/120 (4%) in the placebo group. The adverse events with a higher frequency in the metrifonate treated group compared to placebo were typical cholinergic symptoms, primarily gastrointestinal in nature. These adverse events were infrequent and were described as mild to moderate in intensity. The group receiving the highest doses of metrifonate demonstrated statistically significant effects compared to placebo on both primary outcome variables i.e. ADAS-Cog and CIBICplus. The overall efficacy and tolerability assessment suggests that metrifonate is beneficial in the treatment of AD.
CONFERENCE
ON ALZHEIMER'S
DISEASE
Univ. Heidelberg, D-69120 Heidelberg, Germany #Dept. Pathology, Univ. Melbourne, Parkville, Victoria, 3052, Australia APP binds zinc(II) and copper(H) at two distinct sites. The zinc binding site has been shown to reside within residues 181-200 and is encoded by exon 5. A Cu(II) binding peptide of APP containing the consensus motif for type II copper binding proteins corresponds to residues 135-155 and is encoded by exon 4. Zn(II) and Cu(II) binding of APP regulate APP conformation, stability and homophilic binding. Whereas Zn(II) is assumed to play a structural role and to be transported by transcytosed APP from the axonal to the dendritic surface of neurons, we find APP or a synthetic peptide representing its Cu(H) binding site to reduce bound Cu(II) to Cu(I). Thus neuronal APP may transport Cu(I) and not Cu(II) in the therefore mentioned transcytotic pathway which also implements APP-mediated transsynaptic Cu(I) transport. The Cuion mediated redox-reaction leads to intramolecular disulfide bond formation of APP. This indicates that free sulfhydryl groups of APP are involved. Addition of either superoxide dismutase or catalase had no significant effect on the kinetics of Cu(II) reduction by the peptide but the unrelated human plasma copper-binding growth factor Gly-His-Lys competes for binding and subsequent reduction. This suggests a fast mechanism for Cu(I) formation following direct binding of Cu(lI) to APP. The reduction of Cu(II) to Cu(I) by APP involves a rapid electron transfer reaction, and the reduced Cu could enhance the production of hydroxyl radicals which could then attack sites near the location of the metal. The implication of oxygen radical-induced neuronal damage raises the possibility that Cu-mediated toxicity contributes to neurodegeneration in Alzheimer's disease.
620
SYMPOSIUM: M E C H A N I S M S OF N E U R O D E G E N E R A T I O N II 618 Physiological Aspects of B-Amyloid Precursor Protein (B-APP) in Brain G. Huberl,*J.R. Martin l, Y. Bailly2, J. Mariani3 and B. Brugg4 IPharma Division, Preelinieal CNS Research, F. Hoffmann-La Roche Ltd, 4070 Basel, Switzerland; 2Laboratoire de Neurobiologie Cellulaim CNRS, Strasbourg; 3Universit6 P. & M. Curie, CNRS URA 1488 and 4Htpital de la Salptri~re, INSERM U289, Paris, France. Alzheimer's Disease (AD) is characterized by loss of higher brain functions accompanied by specific pathological brain lesions such as neurofibrillary tangles, I]-amyloid plaques and neuronal degeneration. As gamyloid generation and exlracollular accumulation occur very early in the disease process the fate and role of its precursor proteins, the BAPPs, is important for understanding AD pathogenesis. 13-APPs are developmentally regulated neuronal proteins reaching highest expression in brain during synaptogenesis. They are mainly localized at synaptie sites as shown by immunohistochemistty, and intracerebroventricular injection of anti-l]-APP antibodies produced impaired retention in a passive avoidance task in rats. The potential roles of B-APPs in cognitive functions was further examined in rats raised under either impoverished or enriched environmental conditions. Such treatment is known to affect learning capacity. Quantitative biochemical and immunohistologieal analysis in cortical brain areas of the animals raised under enriched conditions showed a distinct change in B-APP synaptic expression, fl-APP levels increased in parallel with increased synaptophysin expression and correlated with increased ability to learn a new task. These findings suggest a role of fl-APPs in cognitive behavior and emphasize their relevance for higher brain function.
619 Reduction of Copper(II) to Copper(I) by the Amyloid Precursor Protein (APP) of Alzheimer's Disease G. Multhaup*, A. Schlicksupp, L. Hesse, D. Beher, T. Ruppert~, C. L. Masters# and K. Beyreuther ZMBH, Univ. Heidelberg, INF 282, D-69120 Heidelberg; SDept. Virology,
Interaction Between Alzheimer Disease Abnormally Phosphorylated Tau and Microtubule Associated Proteins A. del C. Alonso*, I. Grundke-Iqbal, H.S. Barra and K. Iqbal New York Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA and Dpto de Qufmica Biol6gica, Univ. Nac. de Cdrdoba, Cdrdoba, Argentina Microtubule associated protein (MAP) tan is abnormally hyperphosphorylated in Alzheimer disease (AD) and accumulates in neurons undergoing neurofibrillary degeneration. The AD hyperphosphorylated tau (AD P-tan) binds to normal tan and inhibits the in vitro assembly of microtubules. In the present study the nature of the associations of AD P-tau with normal tan, MAP1 and MAP2 Was investigated. The AD P-tan was found to aggregate with normal tan in solution. The association of normal tau to AD P-tan was apparently by hydrophobic interactions. The binding was increased in the presence of - 200 mM NaCI, by increasing temperature from 4°C to 37 ° C, and in slightly basic pH. The presence of 0.4% Triton X~I00, a non-ionic detergent, abolished the binding of tan to AD P-tan. The aggregation of n~rmal tan with AD P-tan was not saturable and resulted in large tffngles of 3.3 - 0.7 nm diameter filaments. These tangles, which were not detected in identically treated normal tau or AD P-tau alone, were made up of filaments several microns in length and were labeled with tau antibodies. Dephosphorylation with alkaline phosphatase treatment abolished the ability of AD P-tau to aggregate with normal tan and prevented tangle formation. AD P-tau dissasembled microtubules assembled from normal tan and tubulin. Like normal tau, MAPI and MAP2 also were found to associate to AD P-tau. However, the affinity of tau-AD P-tan association was higher than that of the high mol. weight (HMW) MAPs-AD P-tan because normal tan inhibited the latter binding. The association of HMW-MAPs with AD P-tan did not result in the formation of filaments/tangles. These studies suggest that the abnormally phosphorylated tan can sequester both normal tan and HMW MAPs but under physiological conditions can form tangles of filaments only with normal tau. (Supported by NIH grants TW00507, AG08592, AG08076, NS18105 and Zenith Award from Alzheimer's Association, USA and by CONICOR and SECyT (UNC) grants from C6rdoba, Argentina)
621
The Regulation of Phosphorylation of Tau in SYSY Neuroblastoma Cells T. Tanaka', IC Iqbal, E. Trenkner and I. Grundke-Iqbal New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA Previously we have reported that a large percentage of tau in the SH-SY5Y human neuroblastoma cells is hyperphnsphorylated (Tanaka et aL FEBS Lett. 360:5-9, 1995). In the present study, the regulation of phnsphorylation of tau was investigated. Undifferentiated SY5Y cells were treated with inhibitors of protein phosphatases (PP) -1, -2A, and -2B. Time and concentration dependent toxicity was observed with PP-1 and -2A
FIFTH INTERNATIONAL
CA, 90073; 2Bayer Corp., Pharmaceutical Division, West-Haven, CT, 06516 Metrifonate is a long-acting inhibitor of acetylcholinesterase and has been investigated for the treatment of Alzheimer's Disease (AD) since 1990 (Becker et al). The results of a recently unhlinded dose-finding study, designed to evaluate the safety and efficacy profile of metrifonate will be reported. This double-blind, placebo controlled, multicenter study included 480 patients with probable AD recruited from 28 centers across the US. Metrifonate was administered once daily as a loading dose for 14 days followed by a maintenance dose for twelve weeks. Three dosages of metrifonate were compared to placebo, achieving red blood cell acetylcholinesterase inhibition levels of up to 75 %. Overall, 91% of patients receiving metrifonate completed the study compared to 96% for placebo. Only 22/360 (6%) of the metrifonate treated patients discontinued for adverse events, compared with 5/120 (4%) in the placebo group. The adverse events with a higher frequency in the metrifonate treated group compared to placebo were typical cholinergic symptoms, primarily gastrointestinal in nature. These adverse events were infrequent and were described as mild to moderate in intensity. The group receiving the highest doses of metrifonate demonstrated statistically significant effects compared to placebo on both primary outcome variables i.e. ADAS-Cog and CIBICplus. The overall efficacy and tolerability assessment suggests that metrifonate is beneficial in the treatment of AD.
CONFERENCE
ON ALZHEIMER'S
DISEASE
Univ. Heidelberg, D-69120 Heidelberg, Germany #Dept. Pathology, Univ. Melbourne, Parkville, Victoria, 3052, Australia APP binds zinc(II) and copper(H) at two distinct sites. The zinc binding site has been shown to reside within residues 181-200 and is encoded by exon 5. A Cu(II) binding peptide of APP containing the consensus motif for type II copper binding proteins corresponds to residues 135-155 and is encoded by exon 4. Zn(II) and Cu(II) binding of APP regulate APP conformation, stability and homophilic binding. Whereas Zn(II) is assumed to play a structural role and to be transported by transcytosed APP from the axonal to the dendritic surface of neurons, we find APP or a synthetic peptide representing its Cu(H) binding site to reduce bound Cu(II) to Cu(I). Thus neuronal APP may transport Cu(I) and not Cu(II) in the therefore mentioned transcytotic pathway which also implements APP-mediated transsynaptic Cu(I) transport. The Cuion mediated redox-reaction leads to intramolecular disulfide bond formation of APP. This indicates that free sulfhydryl groups of APP are involved. Addition of either superoxide dismutase or catalase had no significant effect on the kinetics of Cu(II) reduction by the peptide but the unrelated human plasma copper-binding growth factor Gly-His-Lys competes for binding and subsequent reduction. This suggests a fast mechanism for Cu(I) formation following direct binding of Cu(lI) to APP. The reduction of Cu(II) to Cu(I) by APP involves a rapid electron transfer reaction, and the reduced Cu could enhance the production of hydroxyl radicals which could then attack sites near the location of the metal. The implication of oxygen radical-induced neuronal damage raises the possibility that Cu-mediated toxicity contributes to neurodegeneration in Alzheimer's disease.
620
SYMPOSIUM: M E C H A N I S M S OF N E U R O D E G E N E R A T I O N II 618 Physiological Aspects of B-Amyloid Precursor Protein (B-APP) in Brain G. Huberl,*J.R. Martin l, Y. Bailly2, J. Mariani3 and B. Brugg4 IPharma Division, Preelinieal CNS Research, F. Hoffmann-La Roche Ltd, 4070 Basel, Switzerland; 2Laboratoire de Neurobiologie Cellulaim CNRS, Strasbourg; 3Universit6 P. & M. Curie, CNRS URA 1488 and 4Htpital de la Salptri~re, INSERM U289, Paris, France. Alzheimer's Disease (AD) is characterized by loss of higher brain functions accompanied by specific pathological brain lesions such as neurofibrillary tangles, I]-amyloid plaques and neuronal degeneration. As gamyloid generation and exlracollular accumulation occur very early in the disease process the fate and role of its precursor proteins, the BAPPs, is important for understanding AD pathogenesis. 13-APPs are developmentally regulated neuronal proteins reaching highest expression in brain during synaptogenesis. They are mainly localized at synaptie sites as shown by immunohistochemistty, and intracerebroventricular injection of anti-l]-APP antibodies produced impaired retention in a passive avoidance task in rats. The potential roles of B-APPs in cognitive functions was further examined in rats raised under either impoverished or enriched environmental conditions. Such treatment is known to affect learning capacity. Quantitative biochemical and immunohistologieal analysis in cortical brain areas of the animals raised under enriched conditions showed a distinct change in B-APP synaptic expression, fl-APP levels increased in parallel with increased synaptophysin expression and correlated with increased ability to learn a new task. These findings suggest a role of fl-APPs in cognitive behavior and emphasize their relevance for higher brain function.
619 Reduction of Copper(II) to Copper(I) by the Amyloid Precursor Protein (APP) of Alzheimer's Disease G. Multhaup*, A. Schlicksupp, L. Hesse, D. Beher, T. Ruppert~, C. L. Masters# and K. Beyreuther ZMBH, Univ. Heidelberg, INF 282, D-69120 Heidelberg; SDept. Virology,
Interaction Between Alzheimer Disease Abnormally Phosphorylated Tau and Microtubule Associated Proteins A. del C. Alonso*, I. Grundke-Iqbal, H.S. Barra and K. Iqbal New York Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA and Dpto de Qufmica Biol6gica, Univ. Nac. de Cdrdoba, Cdrdoba, Argentina Microtubule associated protein (MAP) tan is abnormally hyperphosphorylated in Alzheimer disease (AD) and accumulates in neurons undergoing neurofibrillary degeneration. The AD hyperphosphorylated tau (AD P-tan) binds to normal tan and inhibits the in vitro assembly of microtubules. In the present study the nature of the associations of AD P-tau with normal tan, MAP1 and MAP2 Was investigated. The AD P-tan was found to aggregate with normal tan in solution. The association of normal tau to AD P-tan was apparently by hydrophobic interactions. The binding was increased in the presence of - 200 mM NaCI, by increasing temperature from 4°C to 37 ° C, and in slightly basic pH. The presence of 0.4% Triton X~I00, a non-ionic detergent, abolished the binding of tan to AD P-tan. The aggregation of n~rmal tan with AD P-tan was not saturable and resulted in large tffngles of 3.3 - 0.7 nm diameter filaments. These tangles, which were not detected in identically treated normal tau or AD P-tau alone, were made up of filaments several microns in length and were labeled with tau antibodies. Dephosphorylation with alkaline phosphatase treatment abolished the ability of AD P-tau to aggregate with normal tan and prevented tangle formation. AD P-tau dissasembled microtubules assembled from normal tan and tubulin. Like normal tau, MAPI and MAP2 also were found to associate to AD P-tau. However, the affinity of tau-AD P-tan association was higher than that of the high mol. weight (HMW) MAPs-AD P-tan because normal tan inhibited the latter binding. The association of HMW-MAPs with AD P-tan did not result in the formation of filaments/tangles. These studies suggest that the abnormally phosphorylated tan can sequester both normal tan and HMW MAPs but under physiological conditions can form tangles of filaments only with normal tau. (Supported by NIH grants TW00507, AG08592, AG08076, NS18105 and Zenith Award from Alzheimer's Association, USA and by CONICOR and SECyT (UNC) grants from C6rdoba, Argentina)
621
The Regulation of Phosphorylation of Tau in SYSY Neuroblastoma Cells T. Tanaka', IC Iqbal, E. Trenkner and I. Grundke-Iqbal New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA Previously we have reported that a large percentage of tau in the SH-SY5Y human neuroblastoma cells is hyperphnsphorylated (Tanaka et aL FEBS Lett. 360:5-9, 1995). In the present study, the regulation of phnsphorylation of tau was investigated. Undifferentiated SY5Y cells were treated with inhibitors of protein phosphatases (PP) -1, -2A, and -2B. Time and concentration dependent toxicity was observed with PP-1 and -2A