- Email: [email protected]
525 Acetylcholinesterase promotes Alzheimer's amyloid-β fibril formation
FIFTH INTERNATIONAL
CONFERENCE
ON ALZHEIMER'S
Therefore we considered alternative mechanisms for a y-cleavage such as threoninolysis, a chemical mechanism that depends on Thr(43) o f A 4 C T and which is compatible with a cleavage within the membrane. Intermolecular threoninolysis would generate short 13A4, intramolecular threoninolysis would lead to long ~A4. A similar protein cleavage exists for different proenzymes (serinolysis). To investigate this mechanism, we changed by site directed mutagenesis Thr(43) o f A4CT to Ser and Ala and stably transfected the constructs as SPA4CT (APPsignal-peptide-LeuGIu-A4CT) in COS7 cells. With monoclonal antibodies specific for the Cterminus o f long and short 13A4 we immunoprecipitated metabolically labeled 13A4 and P3 from the medium and quantified the bands b y phosphoimaging. We found that, compared with wtSPA4CT, these mutations alter the ratio of long/short 13A4 but have no influence on the amount of released ~A4 and the ratio o f ~A4/P3. We can therefore rule out threoninolysis as a putative mechanism for y-cleavage. These results further show that beside the FAD linked mutations at Val(46) o f A 4 C T also other mutations near the C-terminus of ~A4 are able to influence the y-cleavage site. This suggests that also the lipid composition o f membranes may influence the specificity o f the y-cleavage.
525 Aeetylcholinesterase Promotes Alzheimer's Amyloid-[~ Fibril Formation N.C. Inestrasa* and A. Alvarez Department Cell & Molecular Biology, Catholic University of Chile, P.O. Box 114-D, FAX 56-2-6862717, E-mail: [email protected], Santiago, Chile Brain acetylcholinesterase (ACHE) consistently co-localizes with extracellular amyloid deposits of Alzheimer's disease (AD), such deposits are composed primarily of amyloid-[3-peptide (A[~). A central event in AD is the conformational change from normally circulating soluble A[~ into amyloid fibrils, in the form of senile plaques. We have investigated whether AChE affects the known spontaneous in vitro formation of amyloid fibrils by A[~ homologous synthetic peptides. We report here that bovine brain ACHE, as well as the human and mouse recombinant enzyme directly promotes amyloid fibril formation. AChE accelerates fibril formation of both wild-type A[3t.4o and of an A[3 analogue (A[~ValI8_.>AIa)which alone produces few amyloid-like
fibrils. No effect was observed with the Dutch variant A~Glu22...>GIn. A[~ can adopt two different conformational states in solution (amyloidogenic and nonamyloidogenic conformer) which have distinct abilities to form amyloid fibrils. We show that AChE binds only the non-amyloidogenic conformer of A .~d acceIerates amyloid formation by the same pepti,-'le. The ac,'ion of AChE was independent of the subunit array of the enzyme, it was not affected by edrophonium, an active site inhibitor, it was blocked both by a nonselective bisquaternary inhibitor (decamethonium) and by a peripheral anionic binding site ligand (propidium). We have compared the ability to induce amyloid deposits between AChE and others Al3-associated proteins, such as cqantichymotrypsin and apolipoproteins E (apoE), apoE3 and apoE4. Results indicates that AChE seems to be the strongest amyloid-promoting factor among them. Our in vitro data suggest that ACHE, in addition to its role in cholinergic synapsis may function by accelerating AI3 formation and could play a role during amyloid deposition in AD. Therefore ACHE may acts as a molecular link between the degeneration of AChE-containing presynaptic nerve terminals and the formation of senile plaques in specific regions of AD brain. (Supported by FONDECYT and Presidential Chair to NCI and by FONDECYT to AA)
DISEASE
S 131
that AB(1-40) formed fibrils at a rate amenable to detailed quantitative monitoring by QLS and with reproducible kinetics. These fibrils were highly similar spectroscopically and morphologically to those isolated from amyloid plaques. We monitored quantitatively the temporal change in the hydrodynamic radius (Rh) of AB during the entire process of fibril growth, from the initial nucleation event through the cessation of fibril elongation. Over an AB concentration range of -0.02-2.0 mM, we observed a temporal increase in Rh and an accompanying proportionate increase in the intensity of the scattered light, results characteristic of a process of linear fibril growth. Interestingly, the kinetic evolution of fibrils exhibited two distinct patterns, depending on A8 eaneentration. Above -0.t raM, the initial rate of elongation and the final size of fibrils were independent of A8 concentration. Below 0.I mM, the initial elongation rate was proportional to the peptide concentration and the resulting fibrils were significantly longer than those formed at higher concentration. These observations were consistent with a model of AB fibrillogenesis which included the following key steps: (i) peptide micelles form above a certain critical Aft concentration; (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds); and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data in the context of this model enabled us to determine the critical concentration (for micelle formation), the sizes of Ag micelles and fibril nuclei, and the rates of fibril nucleation (from micelles) and fibril elongation. Our QLS paradigm represents a powerful means for the quantitative analysis of A8 fibrillogenesis and of the effects of potential therapeutic agents on the process. We are now utilizing the approach to study fibrillogenesis of mutant A8 molecules, including those bearing the Flemish and Dutch mutations (Ala21Gly and Glu22Gln, respectively). In addition, we are examining the effects of potential therapeutic agents on fibriUogenesis.
527 Modulation of AB and Mutant Aggregation In Vitro by Zinc and p i t C.S. Atwood*, R.D. Muir, X. Huang, N.M.E. Bacarra, L. Margol#, C.G. Glabe#, R.E. Tanzi, and A.I. Busht. Genetics and Aging Unit and 1"Department of Psychiatry, Massachusetts General Hospital, Boston, MA; #Molecular Biology and Biochemistry, UC Irvine. AB, a soluble 4.3 kDa peptide found in cerebrospinal fluid, is the principal constituent of amyloid deposits in Alzheimer's disease (AD), found as interstitial plaques and cerebrovascular angiopathy. We are currently appraising candidate neurochemical factors for their ability to account for amyloid precipitation. Previously, we showed that A81.40 specifically and saturably binds zinc and, at physiological concentrations, rapidly induces amyloid formation (Bush et al., Science, 265, 1464; 1994). A point mutation in AB, (Glu to Gin) at position 22 of A8 ("Q22"), causes hereditai'y cerebral hemorrhage with angiopathy-Dutch type (HCHWA-D), where the amyloid deposits principally in the cerebrovascular media in an aggressive, early-onset disease. Using a novel filtration assay, we can quantitate the effects of neurochemical factors on the solubility of these synthetic peptides by colorimetric determination of the protein concentration in the filtered fraction. ABI-40 (2.5 ~tM) begins to precipitate as the pH of a solution is lowered (<6.7). This effect is strongly potentiated by Zn2+ (30 [aM) between pH 6.0-7.4. Q22 begins to precipitate at a higher pH (<7.4) and, in contrast to the wild-type peptide, its solubility at pH 7.4 is unaffected by Zn2+ (<100 I.tM). Competitive binding analysis confirms that Zn2+ binding is abolished in this mutant form of A8. These data confirm that zinc-induced amyloid formation must occur via interaction with the low-affinity zinc binding site, since high-affinity, histidinemediated, zinc binding is abolished below pH 7.0 (Bush et aL, J. Biol. Chem. 269, 12152; 1994). Also, the glutamate at residue 22 might coordinate high affinity zinc binding. The exaggerated susceptibilityof Q22 to acidosis-mediated aggregation may help delineate the pathophysiological sequence of events that distinguish HCHWA-D amyloid pathology from AD amyloid pathology. Lowered cerebral pH is a known complication of aging. The accumulation of Q22 peptide as angiopathy but not interstitial amyloid might indicate that the agerelated lesion of hydrogen ion homeostasis is reflected more in the cerebrovascular wall than in the cortical interstitial space. In sporadic AD, abnormal cerebral zinc homeostasis, also a complication of the disease, may accelerate amyloid deposition in concert with cerebral acidosis but appears unlikely to play a role in inducing the features of HCHWA-D.
526 Kinetic Analysis of AB Fibriilogenesis D. B. Teplow *A, A. Lumakin A#, G. B. Benedek#, D. A. Kirschner+, and D. M. WalshA ADepartments of Neurology, Harvard Medical School and Brigham & Women's Hospital, 221 Longwood Ave. (LMRC-114), Boston, MA 02115-5817 USA; #Department of Physics, and Center for Material Science and Engineering, Massachusetts Institute of Technology; +Biological Sciences, University of Massachusetts, Lowell. Deposition of fibers of the amyloid B-protein (Aft) in the cerebral parenchyma and vasculature is a major neuropatbulogie feature of Alzheimer's disease. Although the end stages of fiber formation have been studied extensively in vitro, the initial steps of fibriUogenesis and the kinetics of the process are poorly understood. We have applied the technique of quasielastic light scattering spectroscopy (QLS) to these questions. We found, at low pH,
528 Forces in the Formation of Amyloid Deposits Lee #, J.P., Zhang #, S.S., Hassell #, D., Casey #, N., Stimson ^, ER, Esler ^, WP, & M a g g i e ^ JE #Department of Chemistry, Boston University, 590 Commonwealth Ave. Boston, M A 02115 USA ^Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, 25 Shattuck Street, Boston, M A 02215 USA
CONFERENCE
ON ALZHEIMER'S
Therefore we considered alternative mechanisms for a y-cleavage such as threoninolysis, a chemical mechanism that depends on Thr(43) o f A 4 C T and which is compatible with a cleavage within the membrane. Intermolecular threoninolysis would generate short 13A4, intramolecular threoninolysis would lead to long ~A4. A similar protein cleavage exists for different proenzymes (serinolysis). To investigate this mechanism, we changed by site directed mutagenesis Thr(43) o f A4CT to Ser and Ala and stably transfected the constructs as SPA4CT (APPsignal-peptide-LeuGIu-A4CT) in COS7 cells. With monoclonal antibodies specific for the Cterminus o f long and short 13A4 we immunoprecipitated metabolically labeled 13A4 and P3 from the medium and quantified the bands b y phosphoimaging. We found that, compared with wtSPA4CT, these mutations alter the ratio of long/short 13A4 but have no influence on the amount of released ~A4 and the ratio o f ~A4/P3. We can therefore rule out threoninolysis as a putative mechanism for y-cleavage. These results further show that beside the FAD linked mutations at Val(46) o f A 4 C T also other mutations near the C-terminus of ~A4 are able to influence the y-cleavage site. This suggests that also the lipid composition o f membranes may influence the specificity o f the y-cleavage.
525 Aeetylcholinesterase Promotes Alzheimer's Amyloid-[~ Fibril Formation N.C. Inestrasa* and A. Alvarez Department Cell & Molecular Biology, Catholic University of Chile, P.O. Box 114-D, FAX 56-2-6862717, E-mail: [email protected], Santiago, Chile Brain acetylcholinesterase (ACHE) consistently co-localizes with extracellular amyloid deposits of Alzheimer's disease (AD), such deposits are composed primarily of amyloid-[3-peptide (A[~). A central event in AD is the conformational change from normally circulating soluble A[~ into amyloid fibrils, in the form of senile plaques. We have investigated whether AChE affects the known spontaneous in vitro formation of amyloid fibrils by A[~ homologous synthetic peptides. We report here that bovine brain ACHE, as well as the human and mouse recombinant enzyme directly promotes amyloid fibril formation. AChE accelerates fibril formation of both wild-type A[3t.4o and of an A[3 analogue (A[~ValI8_.>AIa)which alone produces few amyloid-like
fibrils. No effect was observed with the Dutch variant A~Glu22...>GIn. A[~ can adopt two different conformational states in solution (amyloidogenic and nonamyloidogenic conformer) which have distinct abilities to form amyloid fibrils. We show that AChE binds only the non-amyloidogenic conformer of A .~d acceIerates amyloid formation by the same pepti,-'le. The ac,'ion of AChE was independent of the subunit array of the enzyme, it was not affected by edrophonium, an active site inhibitor, it was blocked both by a nonselective bisquaternary inhibitor (decamethonium) and by a peripheral anionic binding site ligand (propidium). We have compared the ability to induce amyloid deposits between AChE and others Al3-associated proteins, such as cqantichymotrypsin and apolipoproteins E (apoE), apoE3 and apoE4. Results indicates that AChE seems to be the strongest amyloid-promoting factor among them. Our in vitro data suggest that ACHE, in addition to its role in cholinergic synapsis may function by accelerating AI3 formation and could play a role during amyloid deposition in AD. Therefore ACHE may acts as a molecular link between the degeneration of AChE-containing presynaptic nerve terminals and the formation of senile plaques in specific regions of AD brain. (Supported by FONDECYT and Presidential Chair to NCI and by FONDECYT to AA)
DISEASE
S 131
that AB(1-40) formed fibrils at a rate amenable to detailed quantitative monitoring by QLS and with reproducible kinetics. These fibrils were highly similar spectroscopically and morphologically to those isolated from amyloid plaques. We monitored quantitatively the temporal change in the hydrodynamic radius (Rh) of AB during the entire process of fibril growth, from the initial nucleation event through the cessation of fibril elongation. Over an AB concentration range of -0.02-2.0 mM, we observed a temporal increase in Rh and an accompanying proportionate increase in the intensity of the scattered light, results characteristic of a process of linear fibril growth. Interestingly, the kinetic evolution of fibrils exhibited two distinct patterns, depending on A8 eaneentration. Above -0.t raM, the initial rate of elongation and the final size of fibrils were independent of A8 concentration. Below 0.I mM, the initial elongation rate was proportional to the peptide concentration and the resulting fibrils were significantly longer than those formed at higher concentration. These observations were consistent with a model of AB fibrillogenesis which included the following key steps: (i) peptide micelles form above a certain critical Aft concentration; (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds); and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data in the context of this model enabled us to determine the critical concentration (for micelle formation), the sizes of Ag micelles and fibril nuclei, and the rates of fibril nucleation (from micelles) and fibril elongation. Our QLS paradigm represents a powerful means for the quantitative analysis of A8 fibrillogenesis and of the effects of potential therapeutic agents on the process. We are now utilizing the approach to study fibrillogenesis of mutant A8 molecules, including those bearing the Flemish and Dutch mutations (Ala21Gly and Glu22Gln, respectively). In addition, we are examining the effects of potential therapeutic agents on fibriUogenesis.
527 Modulation of AB and Mutant Aggregation In Vitro by Zinc and p i t C.S. Atwood*, R.D. Muir, X. Huang, N.M.E. Bacarra, L. Margol#, C.G. Glabe#, R.E. Tanzi, and A.I. Busht. Genetics and Aging Unit and 1"Department of Psychiatry, Massachusetts General Hospital, Boston, MA; #Molecular Biology and Biochemistry, UC Irvine. AB, a soluble 4.3 kDa peptide found in cerebrospinal fluid, is the principal constituent of amyloid deposits in Alzheimer's disease (AD), found as interstitial plaques and cerebrovascular angiopathy. We are currently appraising candidate neurochemical factors for their ability to account for amyloid precipitation. Previously, we showed that A81.40 specifically and saturably binds zinc and, at physiological concentrations, rapidly induces amyloid formation (Bush et al., Science, 265, 1464; 1994). A point mutation in AB, (Glu to Gin) at position 22 of A8 ("Q22"), causes hereditai'y cerebral hemorrhage with angiopathy-Dutch type (HCHWA-D), where the amyloid deposits principally in the cerebrovascular media in an aggressive, early-onset disease. Using a novel filtration assay, we can quantitate the effects of neurochemical factors on the solubility of these synthetic peptides by colorimetric determination of the protein concentration in the filtered fraction. ABI-40 (2.5 ~tM) begins to precipitate as the pH of a solution is lowered (<6.7). This effect is strongly potentiated by Zn2+ (30 [aM) between pH 6.0-7.4. Q22 begins to precipitate at a higher pH (<7.4) and, in contrast to the wild-type peptide, its solubility at pH 7.4 is unaffected by Zn2+ (<100 I.tM). Competitive binding analysis confirms that Zn2+ binding is abolished in this mutant form of A8. These data confirm that zinc-induced amyloid formation must occur via interaction with the low-affinity zinc binding site, since high-affinity, histidinemediated, zinc binding is abolished below pH 7.0 (Bush et aL, J. Biol. Chem. 269, 12152; 1994). Also, the glutamate at residue 22 might coordinate high affinity zinc binding. The exaggerated susceptibilityof Q22 to acidosis-mediated aggregation may help delineate the pathophysiological sequence of events that distinguish HCHWA-D amyloid pathology from AD amyloid pathology. Lowered cerebral pH is a known complication of aging. The accumulation of Q22 peptide as angiopathy but not interstitial amyloid might indicate that the agerelated lesion of hydrogen ion homeostasis is reflected more in the cerebrovascular wall than in the cortical interstitial space. In sporadic AD, abnormal cerebral zinc homeostasis, also a complication of the disease, may accelerate amyloid deposition in concert with cerebral acidosis but appears unlikely to play a role in inducing the features of HCHWA-D.
526 Kinetic Analysis of AB Fibriilogenesis D. B. Teplow *A, A. Lumakin A#, G. B. Benedek#, D. A. Kirschner+, and D. M. WalshA ADepartments of Neurology, Harvard Medical School and Brigham & Women's Hospital, 221 Longwood Ave. (LMRC-114), Boston, MA 02115-5817 USA; #Department of Physics, and Center for Material Science and Engineering, Massachusetts Institute of Technology; +Biological Sciences, University of Massachusetts, Lowell. Deposition of fibers of the amyloid B-protein (Aft) in the cerebral parenchyma and vasculature is a major neuropatbulogie feature of Alzheimer's disease. Although the end stages of fiber formation have been studied extensively in vitro, the initial steps of fibriUogenesis and the kinetics of the process are poorly understood. We have applied the technique of quasielastic light scattering spectroscopy (QLS) to these questions. We found, at low pH,
528 Forces in the Formation of Amyloid Deposits Lee #, J.P., Zhang #, S.S., Hassell #, D., Casey #, N., Stimson ^, ER, Esler ^, WP, & M a g g i e ^ JE #Department of Chemistry, Boston University, 590 Commonwealth Ave. Boston, M A 02115 USA ^Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, 25 Shattuck Street, Boston, M A 02215 USA