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Gelsolin inhibits the fibrillization of amyloid beta-protein, and also defibrillizes its preformed fibrils
Brain Research 853 Ž2000. 344–351 www.elsevier.comrlocaterbres
Research report
Gelsolin inhibits the fibrillization of amyloid beta-protein, and also defibrillizes its preformed fibrils Indrani Ray, Abha Chauhan, Jerzy Wegiel, Ved P.S. Chauhan
)
New York State Institute for Basic Research in DeÕelopmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314-6399, USA Accepted 2 November 1999
Abstract Amyloid beta-protein ŽAb . is present in soluble form in the plasma and cerebrospinal fluid ŽCSF. of normal people and patients with Alzheimer’s disease ŽAD.. However, in AD patients, Ab gets fibrillized as the main constituent of amyloid plaques in the brain. Soluble synthetic Ab also forms amyloid-like fibrils when it is allowed to age. The mechanism that prevents soluble Ab from fibrillization in biological fluids is not clear. We recently reported that gelsolin, a secretory protein, binds to Ab, and that gelsolinrAb complex is present in the plasma wV.P.S. Chauhan, I. Ray, A. Chauhan, H.M. Wisniewski, Biochem. Biophys. Res. Commun. 258 Ž1999. 241–246.x. We now studied the effect of gelsolin on Ab fibrillization. Congo red staining and electron microscopic examination in negative staining of aged samples of Ab alone and Ab incubated with gelsolin showed that gelsolin inhibits the fibrillization of synthetic Ab 1–40 and Ab 1–42 at gelsolin to Ab molar ratio of 1:40. In addition, gelsolin also defibrillized the preformed fibrils of Ab 1–40 and Ab 1–42 in a time-dependent manner. These results suggest that gelsolin functions as an anti-amyloidogenic protein in the plasma and CSF, where it prevents Ab from fibrillization, and helps to maintain it in the soluble form. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Amyloid beta-protein; Alzheimer’s disease; Gelsolin; Cerebrospinal fluid; Plasma; Fibrillization
1. Introduction Alzheimer’s disease ŽAD., a neurodegenerative disorder among the elderly, is characterized by two pathological hallmarks: fibrillization and deposition of amyloid betaprotein ŽAb . of 39–43 amino acids as amyloid plaques, and the formation of neurofibrillary tangles composed of paired helical filaments wreviewed in Ref. w30xx. AD can be divided into an early-onset form Žonset - 60 years. and the more common late-onset form Žonset ) 60 years.. In both cases, Ab fibrillization has been suggested to be a major event in the pathology of AD. Ab is produced by the proteolytic cleavage of b-amyloid precursor protein Žb-APP., and is present as soluble protein in cerebrospinal fluid ŽCSF. and sera of normal individuals as well as AD patients, and in the conditioned media of many types of cultured cells w8,20,26x.
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Ab exists in solution in a monomeric a-helical conformation. Ab fibril formation is a multi step process that is preceded by oligomerization and aggregation of Ab, and involves conformational change of the peptide from ahelical to cross b-pleated sheet secondary conformation w10x. Several investigators have reported that neurotoxicity of Ab is directly correlated to its degree of fibrillization w16x which in turn is related to the b-sheet structure w21x and the size of Ab peptides w10,31x. Recently, it has been reported that amyloid formation by Ab 1–42 plays a key role in promoting AD, specifically genetically linked early onset AD w9x. The genes responsible for the genetic cases have mutations in the b-APP, presenilin ŽPS. 1 and PS 2. Mutations in PS 1 and PS 2 result in the selective processing of b-APP to produce preferentially Ab 1–42 rather than Ab 1–40. Ab 1–42 has been reported to be more fibrillogenic and cytotoxic than shorter Abs w31x. The molecular mechanisms by which the normally produced soluble Ab forms amyloid fibrils are not understood well. We have reported earlier that the fibrillization of Ab 1–40rAb 1–42 is inhibited in the presence of serum, which suggested the role of serum factors in the regulation
0006-8993r00r$ - see front matter q 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 1 5 - X
I. Ray et al.r Brain Research 853 (2000) 344–351
of Ab fibrillogenesis w3,27x. Several circulatory proteins such as apolipoprotein Žapo. E, apo J, apo A1, transthyretin and albumin have been shown to bind Ab w1,7,14,19,24x. Recently, we have reported that plasma gelsolin also binds to Ab 1–40rAb 1–42, and forms a sodium dodecyl sulfate ŽSDS.-stable complex w5x. Gelsolin is present intracellularly w25x, and in the plasma and CSF as secreted protein w18x. The cytoplasmic architecture of all cells is maintained by the highly controlled regulation of actin filament assembly. Intracellular gelsolin regulates actin polymerization by binding to actin, and subsequently, capping and severing the actin filaments w11,22x. However, the function of extracellular gelsolin in the plasma and CSF is not known. Here, we report that plasma gelsolin inhibits the fibrillization of soluble Ab 1–40rAb 1–42, and it also defibrillizes the preformed Ab fibrils in a time-dependent manner.
2. Materials and methods 2.1. Materials Purified synthetic Ab 1–40 and Ab 1–42 were purchased from US Peptides Inc., CA. Bioadhesive hydropho-
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bic diagnostic printed slides Ž10 wells. were purchased from Fisher Scientific. The 4G8 monoclonal antibody ŽmAb. that recognizes 17–24 amino acid sequence of Ab was kindly provided by Dr. K.S. Kim of this Institute. Bovine plasma Žfrozen. was from Pel-Freeze Biologicals, AR. Congo red and all other reagents were from Sigma. 2.2. Purification of gelsolin Bovine plasma gelsolin was either purchased from Sigma or purified in the laboratory from the frozen bovine plasma according to the method of Tanaka and Sobue w25x. In brief, Tris, leupeptin, and mercaptoethanol were added to the bovine plasma to obtain a final concentration of 25 mM Tris, leupeptin Ž2 mgrml., 0.1% mercaptoethanol, and pH was adjusted to 8.0. This step was followed by ammonium sulfate precipitation. The fraction between 35% and 50% of ammonium sulfate was collected, and subjected to DE 52 and affinity-gel blue chromatographies. The fractions containing gelsolin were pooled, concentrated and dialyzed against 25 mM Tris–HCl and 1 mM EGTA, pH 7.5. Silver staining of the final preparation of gelsolin showed a single band of 90 kDa molecular weight. The protein content was measured by BioRad assay.
Fig. 1. Congo red staining of Ab 1–40 allowed to age in the absence and presence of purified gelsolin. Samples of soluble Ab 1–40 alone ŽA., gelsolin control ŽG., and Ab mixed at the molar ratio of 40:1 with gelsolin ŽAG. were incubated at 378C. After 5 days Ž5d. and 7 days Ž7d. of incubation, the Congo red stained samples were viewed under polarized light. The green birefringence was observed only in aged Ab samples in the absence of gelsolin ŽA.. Magnification 150 = .
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2.3. Effect of gelsolin on the fibrillization of A b Synthetic Ab 1–40 and Ab 1–42 were solubilized in double distilled water at the concentration of 4 mgrml, and the pH adjusted to 7.5. Gelsolin purified in the laboratory was dissolved in buffer A, i.e., 25 mM Tris–HCl and 1 mM EGTA, pH 7.5, and gelsolin purchased from Sigma was dissolved in 25 mM Tris–HCl, pH 7.5. Ab Ž60 mg. mixed with 35 mg of gelsolin at the molar ratio of 40:1, Ab alone, and gelsolin alone Žcontrol. were allowed to incubate at 378C. The degree of fibrillization in the samples were examined by Congo red staining, and electron microscopy ŽEM. as described below. 2.3.1. Congo red staining This method was used to probe for the presence of cross b-pleated sheet structure in Ab, a defining characteristic of amyloid fibrils. Congo red does not bind to dimers or trimers, but only binds to higher order aggregates so called protofibrils, and amyloid like fibrils. The complex of
Congo red and Ab fibrils gives a green birefringence when viewed under polarized light. We used modified method of Jarrett and Lansbury w12x. At 5th and 7th days of incubation of Ab in the presence and absence of gelsolin, 2 ml from each sample ŽAb, gelsolin, and Ab q gelsolin. was put on the bioadhesive hydrophobic printed slide, and it was allowed to dry for 2 h. Using 1 ml pipette, the dried samples were gently covered with one drop of 1 mM Congo red Žin 100 mM NaCl, 10 mM phosphate buffer, pH 7.4.. After 1 min, the slide was tilted at ; 308 angle, dye was carefully aspirated by slow suction, and sample was rinsed with a drop of water. The slides were dried at room temperature, viewed under polarized light using a Axrophot ŽC. Zeiss. microscope Žequipped with polarizing filter.. 2.3.2. NegatiÕe staining for electron microscopic study The morphological characteristic of structure formed by Ab in the absence and presence of gelsolin was visualized in negative staining in electron microscope. On the 7th day
Fig. 2. Electron micrographs showing inhibition of Ab 1–40 and Ab 1–42 fibrillization in the presence of gelsolin. The samples of Ab 1–40 ŽA40.rAb 1–42 ŽA42., gelsolin control ŽG., Ab and gelsolin at 40:1 molar ratio were incubated for 7 days, and examined by EM in negative staining with uranyl acetate. Incubation of both Ab 1–40 and Ab 1–42 with plasma gelsolin purified in our laboratory ŽA40G, A42G., or gelsolin purchased from Sigma ŽA40GU . caused inhibition of fibril formation. Magnification 120,000 = .
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of incubation of Ab, gelsolin and Ab-gelsolin samples, one drop of each sample was applied to a 400-mesh formvar-coated grid. After 1 min, the sample was washed with 10 consecutive drops of distilled water, followed by
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four drops of 2% uranyl acetate. The last drop of uranyl acetate remained on the grid for 60 s. It was washed with two drops of water, drained, and air-dried. Samples were examined in a Hitachi 7000 electron microscope.
Fig. 3. Effect of gelsolin on the preformed Ab fibrils assessed by Congo red staining. Ab 1–40 Ž2 mgrml. was allowed to incubate in buffer A Ž25 mM Tris–HCl, 1 mM EGTA, pH 7.5. for 6 days to form fibrils ŽAf.. A total of 25 ml of these preformed Ab fibrils was then incubated in the presence of gelsolin Žin buffer A. at a molar ratio of 20:1 ŽAfG. or in buffer A Žcontrol, Af. for 5 days. After 1 h, 2 days Ž2d. and 5 days Ž5d. of incubation, the samples were stained with Congo red, and viewed under polarized light. Magnification 150 = .
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2.4. Effect of gelsolin on the preformed A b fibrils The preformed Ab fibrils were obtained by incubating soluble Ab 1–40 Ž2 mgrml. in buffer A for 6 days. The
preformed Ab fibrils Ž52 mg. was then incubated with 60 mg gelsolin in buffer A at the Ab:gelsolin molar ratio of 20:1. In the control sample, buffer A was only added to the preformed Ab fibrils. After 1 h, 5 and 7 days, these
Fig. 4. Electron microscopic studies showing defibrillization of preformed Ab 1–40 fibrils by gelsolin. Ab 1–40 was solubilized Ž2 mgrml. and aged for fibril formation ŽAf.. Gelsolin ŽG. in buffer A Ž25 mM Tris–HCl, 1 mM EGTA, pH 7.5. was added to Af at a molar ratio of 1:20 in the AfG sample. In the control sample ŽAf., buffer A was added. The samples were incubated for 5 days, and examined by EM in negative staining after 1 h, 2 days Ž2d. and 5 days Ž5d. of incubation. Magnification 30,000 = .
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samples were analyzed by Congo red staining and EM as described in 2.3.1 and 2.3.2.
3. Results 3.1. Effect of gelsolin on A b fibrillization Since gelsolin is present in the plasma and CSF, and it binds to Ab w5x, we studied if gelsolin has any effect on the fibrillogenesis of soluble synthetic Ab. The effect of gelsolin on Ab fibrillization was studied by Ža. Congo red staining ŽFig. 1., and Žb. EM in negative staining ŽFig. 2.. When aged Ab samples at 5th and 7th days of incubation were stained with Congo red and viewed under polarized light, they produced a specific green birefringence indicating that Ab was in the fibrillar form. No green birefringence was observed in the Ab sample incubated with gelsolin, indicating that the fibrillization of Ab was completely inhibited in the presence of gelsolin. The control sample, i.e., gelsolin alone, was negative for Congo red staining ŽFig. 1.. On the other hand, green birefringence was observed in Congo red stained samples of Ab 1–40 incubated in the presence of 40:1 Žmolermole. bovine serum albumin ŽBSA. for 5 and 7 days Ždata not shown. indicating that BSA does not affect Ab fibrillization under similar experimental conditions where gelsolin completely inhibits it. The samples containing aged Ab 1–40 or Ab 1–42 alone, and corresponding Ab samples incubated with gelsolin after seven days of aging were also examined by EM in negative staining ŽFig. 2.. Ab 1–40 and Ab 1–42 formed dense network of fibrils with the morphology similar to as reported previously w2–4x. Gelsolin inhibited the fibril formation of both Ab 1–40 and Ab 1–42 by more than 90% of that observed for Abs in the absence of gelsolin. Gelsolin alone Žcontrol. did not make the fibrils by itself ŽFig. 2.. The experiments were first performed using plasma gelsolin ŽSigma., which contained high concentration of salt ŽNaCl, Tris buffer salt.. In order to rule out the possibility of contaminating salt in gelsolin sample having an effect on Ab fibrillization, we repeated the experiment using gelsolin purified in our laboratory from bovine plasma, which was free of salt. Similar results were obtained with gelsolin purchased from Sigma and that purified in the laboratory. 3.2. Defibrillization of preformed A b fibrils by gelsolin Since gelsolin inhibits the fibrillization of Ab, we studied if it could also defibrillize the preformed fibrils of Ab. The preformed fibrils were obtained by incubating soluble Ab for 6 days. After mixing gelsolin to the preformed fibrils, the status of the fibrils was analyzed by Congo red staining and EM at different time periods. The results of Congo red staining of the preformed Ab fibrils
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incubated in buffer Žcontrol., or in the presence of gelsolin for 1 h, 2 and 5 days are shown in Fig. 3. The green birefringent material was observed in the samples of fibrillar Ab stained with Congo red when viewed under polarized light after 1 h, 2 and 5 days of incubation without gelsolin, and after 1 h of incubation with gelsolin. After 2 days, there was remarkable reduction in the amount of green birefringent material in Ab sample containing gelsolin. In fact, only traces of green birefringent material were present. Interestingly, green birefringent material was completely absent after 5 days of incubation with gelsolin ŽFig. 3.. This data suggests that gelsolin was able to defibrillize the preformed fibrils of Ab. The electron microscopic analysis in negative staining of preformed Ab fibrils after incubation for 1 h, 2 and 5 days without gelsolin, and in the presence of gelsolin is shown in Fig. 4. Ab samples incubated without gelsolin after 1 h, 2 and 5 days, showed a dense network of long fibrils of Ab well immunolabeled with mAb 4G8 detecting Ab peptide. After 1 h of incubation with gelsolin, the network of fibrils was degraded to large clusters of long and short fibrils with little or no amorphous material. After 2 days of incubation of Ab q gelsolin sample, long fibrils were absent, only short fibrils or filaments like structures were present in small numbers. The fibrils or filaments were completely absent in Ab samples incubated with gelsolin for 5 days. These results confirmed the results of Congo red staining, and suggest that gelsolin solubilizes the preformed fibrils in a time-dependent manner.
4. Discussion The fibrillization of Ab is considered a main event in AD pathology that leads to neuronal cell death, and results in cognitive and behavioral decline. Several plasma and CSF constituents have been shown to bind Ab w1,7,14,19,24x. We have reported earlier that serum inhibits the fibrillization of Ab w3,27x, plasma gelsolin binds to Ab w5x, and Ab-gelsolin complex exists under physiological conditions w5x. We now report that gelsolin inhibits the fibrillization of Ab, and it can also defibrillize the preformed Ab fibrils. Gelsolin is known to be present both intracellularly w25x and as secreted protein in the plasma and CSF w18x. Gelsolin acts as a major actin-binding protein in the cytoplasm, where it regulates the assembly of actin filaments in a highly specialized manner. It caps the actin filament’s growing end, stimulates its nucleation, and severs the actin filament w11,22x. Recent studies show the role of gelsolin in several other activities besides control of actin polymerization, namely, modulation of calcium channel and NMDA receptor w6x, apoptosis w13x, and in tumor suppression w15x. However, the function of gelsolin in the plasma and CSF is not yet elucidated. An observed lack of green birefringence upon Congo red staining of aged Ab sam-
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ples in the presence of gelsolin, and inhibition of Ab fibril formation by gelsolin in EM analysis indicate that gelsolin acts as an inhibitor of b-pleated sheet structure characteristic of amyloid fibrils, and the fibril formation of soluble Ab 1–40rAb 1–42. Therefore, gelsolin may act as an antiamyloidogenic agent in the plasma and CSF. Interestingly, gelsolin was also able to solubilize the preformed Ab fibrils in a time-dependent manner, and thus, it may induce the conformational change from b-pleated sheet structure of amyloid fibrils to a-helical structure of soluble amorphous Ab. It is possible that defibrillization of the preformed fibrils of Ab by gelsolin may be the defense mechanism against other pro-amyloidogenic agents. Ab fibrillization is a nucleation-dependent phenomenon. It is not clear at present whether gelsolin inhibits the fibrillization of Ab by blocking the nucleus formation or it inhibits the elongation of Ab fibrils. Recently, we reported that there are two sites of Ab binding on gelsolin with apparent dissociation constants Ž K d . of 1.38 and 2.55 mM w5x. Gelsolin is also known to bind to two actin molecules w11x. Since gelsolin also inhibits the polymerization of actin, the gelsolin-mediated regulation of actin fibrillization and Ab fibrillization may have an analogous mechanism. Further studies are required to address the question if binding of actin and Ab takes place on the same or different region on gelsolin. Several proteins, such as apo E, transthyretin and apo J, that bind to Ab, have been found in the amyloid plaques along with fibrillar Ab. Among these proteins, apo E has been extensively studied for its role in Ab fibrillogenesis. Apo E has been reported to promote the fibrillization of synthetic Ab w17,28x. In fact, apo E4 allele is increased in the genetically linked cases of AD patients w23x. These amyloid associated proteins have been suggested to function as pathological chaperons, and to promote both b-sheet structure and fibril formation w29x. Gelsolin, like other Ab binding proteins, is also present in the plasma and CSF. However, unlike other amyloid associated proteins, gelsolin was not detected in the amyloid plaques of AD w5x. We propose that gelsolin is a major Ab-sequestering protein in the plasma and CSF where it prevents Ab from fibrillization and maintains it in the soluble form. The binding of secretory proteins Žincluding gelsolin. to Ab suggests the presence of a highly complex regulatory pathway for keeping Ab in the soluble form in the plasma and CSF. The imbalance of this regulatory pathway may result in Ab fibrillization in AD. The binding of gelsolin to Ab, and the inhibition of Ab fibril formation in the presence of gelsolin suggest that gelsolin plays a vital role in the regulation of Ab fibrillogenesis. Amyloid formation in AD may result in part from failure of this sequestration mechanism andror imbalance in the levels of gelsolin in the brain, CSF, or plasma. Since fibrillization of Ab is an essential event in the AD pathology, its inhibition or defibrillization by gelsolin suggests that the physiological role of gelsolin in the plasma and
CSF is to prevent Ab from undergoing the conformational change from soluble a-helical to b-pleated sheet structure.
Acknowledgements We would like to dedicate this publication to Dr. H.M. Wisniewski, the late Director of N.Y.S. Institute for Basic Research in Developmental Disabilities. We thank Dr. K.C. Wang and Ms. Deepti Chauhan for technical assistance. This work was supported in part by the funds from the New York State Office of Mental Retardation and Developmental Disabilities, and by NIH grant AG 14199.
References w1x A.L. Biere, B. Ostaszewski, E.R. Stimson, B.T. Hyman, J.E. Maggie, D.J. Selkoe, Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma, J. Biol. Chem. 271 Ž1996. 32916– 32922. w2x A. Chauhan, V.P.S. Chauhan, R. Rubenstein, J. Wegiel, H.M. Wisniewski, Media from rhabdomyosarcoma and neuroblastoma cell cultures stimulate in vitro aggregation and fibrillization of amyloid beta-protein, Neurochem. Res. 22 Ž1997. 227–232. w3x A. Chauhan, V.P.S. Chauhan, J. Wegiel, H.M. Wisniewski, Impact of normal and heat-inactivated serum on in vitro aggregation and fibrillization of synthetic amyloid beta-protein, Alzheimer’s Res. 2 Ž1996. 243–248. w4x V.P.S. Chauhan, I. Ray, A. Chauhan, J. Wegiel, H.M. Wisniewski, Metal cations defibrillize the amyloid beta-protein fibrils, Neurochem. Res. 22 Ž1997. 805–809. w5x V.P.S. Chauhan, I. Ray, A. Chauhan, H.M. Wisniewski, Binding of gelsolin, a secretory protein, to amyloid beta-protein, Biochem. Biophys. Res. Commun. 258 Ž1999. 241–246. w6x K. Furukawa, W. Fu, Y. Li, W. Witke, D.J. Kwiatkowski, M.P. Mattson, The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons, J. Neurosci. 17 Ž1997. 8178–8186. w7x J. Ghiso, E. Matsubara, A. Koudinov, T. Wisniewski, B. Frangione, The cerebrospinal fluid soluble form of Alzheimer’s amyloid b is complexed to SP-40, 40 ŽAPO J., an inhibitor of the complement membrane-attack complex, Biochem. J. 293 Ž1993. 27–30. w8x C. Haas, M.G. Schlossmacher, A.Y. Hung, C. Vigo-Pelfrey, A. Mellon, B.L. Ostaszewski, I. Lieberburg, E.H. Koo, D. Schenk, D.B. Teplow, D. Selkoe, Amyloid beta-peptide is produced by cultured cells during normal metabolism, Nature 359 Ž1992. 325–327. w9x J. Hardy, Amyloid, the presenilins and Alzheimer’s disease, Trends Neurosci. 20 Ž1997. 403–407. w10x C. Hilbich, B. Kisters-Woike, J. Reed, C.L. Masters, K. Beyreuther, Aggregation and secondary structure of synthetic amyloid beta A4 peptides of Alzheimer’s disease, J. Mol. Biol. 218 Ž1991. 149–163. w11x P.A. Janney, C. Chapannier, S.E. Lind, K.S. Zaner, T.P. Stossel, H.L. Yin, Interactions of gelsolin and gelsolin–actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking, Biochemistry 24 Ž1985. 3714–3723. w12x J.T. Jarrett, P.T. Lansbury Jr., Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB, Biochemistry 31 Ž1992. 12345–12352. w13x S. Kothakota, T. Azuma, C. Reinhard, A. Klippel, J. Tang, K. Chu, T.J. McGarry, M.W. Kirschner, K. Koths, D.J. Kwiatkowski, L.T. Williams, Caspase-3-generated fragment of gelsolin: effector of morphological changes in apoptosis, Science 278 Ž1997. 294–298.
I. Ray et al.r Brain Research 853 (2000) 344–351 w14x A. Koudinov, E. Matsubara, B. Frangione, J. Ghiso, The soluble form of Alzheimer’s amyloid beta-protein is complexed to high-density lipoprotein 3 and very high density lipoprotein in normal human plasma, Biochem. Biophys. Res. Commun. 205 Ž1994. 1164–1171. w15x N. Kuzumaki, M. Tanaka, N. Sakai, H. Fujita, Tumor suppressive function of gelsolin, Gan To Kagaku Royoho 24 Ž1997. 1436–1441. w16x A. Lorenzo, B.A. Yankner, b-Amyloid neurotoxicity requires fibril formation, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 12243–12247. w17x J. Ma, A. Yee, H.B. Brewer Jr., S. Das, H. Potter, Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments, Nature 372 Ž1994. 92–94. w18x T. Paunio, S. Kiuru, S.-L. Karonen, J. Palo, L. Peltonen, Quantification of serum and cerebrospinal fluid gelsolin in familial amyloidosis, Finnish type ŽAGel., Amyloid, Int. J. Exp. Clin. Invest. 1 Ž1994. 80–89. w19x A.L. Schwarzman, L. Gregori, M.P. Vitek, S. Lyubski, W.J. Strittmatter, J.J. Enghilde, R. Bhasin, J. Silverman, K.H. Weisgraber, P.K. Coyle, M.G. Zagorski, J. Talafous, M. Eisenberg, A.M. Saunders, A.D. Roses, D. Goldgaber, Transthyretin sequesters amyloid b-protein and prevents amyloid formation, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 8368–8371. w20x P. Seubert, C. Vigo-Pelfrey, F. Esch, M. Lee, H. Dovey, D. Davis, S. Sinha, M. Schlossmacher, J. Whaley, C. Swindlehyurst, R. McCormack, R. Wolfert, D. Selkoe, I. Lieberburg, D. Schenk, Isolation and quantitation of soluble Alzheimer’s beta-peptide from biological fluids, Nature 359 Ž1992. 325–327. w21x L.K. Simmons, P.C. May, K.J. Tomasseli, R.E. Rydel, K.S. Fuson, E.F. Brigham, S. Wright, I. Lieberburg, G.W. Becker, D.N. Brems, W.Y. Li, Secondary structure of amyloid b-peptide correlates with neurotoxic activity in vitro, Mol. Pharmacol. 45 Ž1994. 373–379. w22x T.P. Stossel, Actin-membrane interactions in eukaryotic mammalian cells, in: J.F. Hoffman, G. Giebisch ŽEds.., Current Topics in
w23x
w24x
w25x
w26x
w27x
w28x
w29x w30x w31x
351
Membranes and Transport, Vol. 36, Academic Press, NY, 1990, pp. 97–107. W.J. Strittmatter, A.M. Saunders, D. Schmechel, M. Pericak-Vance, J. Enghild, G.S. Salvesen, A.D. Roses, Increased frequency of type 4 allele in late-onset familial Alzheimer disease. Apolipoprotein E: High-avidity binding to beta-amyloid, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 1977–1981. W.J. Strittmatter, K.H. Weisgraber, D.Y. Huang, L.-M. Dong, G.S. Salvesen, M. Pericak-Vance, D. Schmechel, A.M. Saunders, D. Goldgaber, A.D. Roses, Binding of human apolipoprotein E to synthetic amyloid b-peptide: isoform-specific effects and implications for late-onset Alzheimer’s disease, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 8098–8102. J. Tanaka, K. Sobue, Localization and characterization of gelsolin in nervous tissues: gelsolin is specifically enriched in myelin-forming cells, J. Neurosci. 14 Ž1994. 1038–1052. C. Vigo-Pelfrey, D. Lee, P. Keim, I. Lieberburg, D.B. Schenk, Characterization of b-amyloid peptide from human cerebrospinal fluid, J. Neurochem. 61 Ž1993. 1965–1968. J. Wegiel, A. Chauhan, H.M. Wisniewski, J. Nowakowski, K.C. Wang, H. Levine, Promotion of synthetic amyloid b-peptide fibrillization by cell culture media and abolishment of fibrillization by serum, Neurosci. Lett. 211 Ž1996. 151–154. T. Wisniewski, E.M. Castano, A. Golabek, T. Vogel, B. Frangione, Acceleration of Alzheimer’s fibril formation by apolipoprotein E in vitro, Am. J. Pathol. 145 Ž1994. 1030–1035. T. Wisniewski, J. Ghiso, B. Frangione, Alzheimer’s disease and soluble Ab, Neurobiol. Aging 15 Ž1994. 143–152. T. Wisniewski, J. Ghiso, B. Frangione, Biology of Ab amyloid in Alzheimer’s disease, Neurobiol. Dis. 4 Ž1997. 313–328. S.G. Younkin, Evidence that Ab 42 is the real culprit in Alzheimer’s disease, Ann. Neurol. 37 Ž1995. 287–288.
Research report
Gelsolin inhibits the fibrillization of amyloid beta-protein, and also defibrillizes its preformed fibrils Indrani Ray, Abha Chauhan, Jerzy Wegiel, Ved P.S. Chauhan
)
New York State Institute for Basic Research in DeÕelopmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314-6399, USA Accepted 2 November 1999
Abstract Amyloid beta-protein ŽAb . is present in soluble form in the plasma and cerebrospinal fluid ŽCSF. of normal people and patients with Alzheimer’s disease ŽAD.. However, in AD patients, Ab gets fibrillized as the main constituent of amyloid plaques in the brain. Soluble synthetic Ab also forms amyloid-like fibrils when it is allowed to age. The mechanism that prevents soluble Ab from fibrillization in biological fluids is not clear. We recently reported that gelsolin, a secretory protein, binds to Ab, and that gelsolinrAb complex is present in the plasma wV.P.S. Chauhan, I. Ray, A. Chauhan, H.M. Wisniewski, Biochem. Biophys. Res. Commun. 258 Ž1999. 241–246.x. We now studied the effect of gelsolin on Ab fibrillization. Congo red staining and electron microscopic examination in negative staining of aged samples of Ab alone and Ab incubated with gelsolin showed that gelsolin inhibits the fibrillization of synthetic Ab 1–40 and Ab 1–42 at gelsolin to Ab molar ratio of 1:40. In addition, gelsolin also defibrillized the preformed fibrils of Ab 1–40 and Ab 1–42 in a time-dependent manner. These results suggest that gelsolin functions as an anti-amyloidogenic protein in the plasma and CSF, where it prevents Ab from fibrillization, and helps to maintain it in the soluble form. q 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Amyloid beta-protein; Alzheimer’s disease; Gelsolin; Cerebrospinal fluid; Plasma; Fibrillization
1. Introduction Alzheimer’s disease ŽAD., a neurodegenerative disorder among the elderly, is characterized by two pathological hallmarks: fibrillization and deposition of amyloid betaprotein ŽAb . of 39–43 amino acids as amyloid plaques, and the formation of neurofibrillary tangles composed of paired helical filaments wreviewed in Ref. w30xx. AD can be divided into an early-onset form Žonset - 60 years. and the more common late-onset form Žonset ) 60 years.. In both cases, Ab fibrillization has been suggested to be a major event in the pathology of AD. Ab is produced by the proteolytic cleavage of b-amyloid precursor protein Žb-APP., and is present as soluble protein in cerebrospinal fluid ŽCSF. and sera of normal individuals as well as AD patients, and in the conditioned media of many types of cultured cells w8,20,26x.
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Ab exists in solution in a monomeric a-helical conformation. Ab fibril formation is a multi step process that is preceded by oligomerization and aggregation of Ab, and involves conformational change of the peptide from ahelical to cross b-pleated sheet secondary conformation w10x. Several investigators have reported that neurotoxicity of Ab is directly correlated to its degree of fibrillization w16x which in turn is related to the b-sheet structure w21x and the size of Ab peptides w10,31x. Recently, it has been reported that amyloid formation by Ab 1–42 plays a key role in promoting AD, specifically genetically linked early onset AD w9x. The genes responsible for the genetic cases have mutations in the b-APP, presenilin ŽPS. 1 and PS 2. Mutations in PS 1 and PS 2 result in the selective processing of b-APP to produce preferentially Ab 1–42 rather than Ab 1–40. Ab 1–42 has been reported to be more fibrillogenic and cytotoxic than shorter Abs w31x. The molecular mechanisms by which the normally produced soluble Ab forms amyloid fibrils are not understood well. We have reported earlier that the fibrillization of Ab 1–40rAb 1–42 is inhibited in the presence of serum, which suggested the role of serum factors in the regulation
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of Ab fibrillogenesis w3,27x. Several circulatory proteins such as apolipoprotein Žapo. E, apo J, apo A1, transthyretin and albumin have been shown to bind Ab w1,7,14,19,24x. Recently, we have reported that plasma gelsolin also binds to Ab 1–40rAb 1–42, and forms a sodium dodecyl sulfate ŽSDS.-stable complex w5x. Gelsolin is present intracellularly w25x, and in the plasma and CSF as secreted protein w18x. The cytoplasmic architecture of all cells is maintained by the highly controlled regulation of actin filament assembly. Intracellular gelsolin regulates actin polymerization by binding to actin, and subsequently, capping and severing the actin filaments w11,22x. However, the function of extracellular gelsolin in the plasma and CSF is not known. Here, we report that plasma gelsolin inhibits the fibrillization of soluble Ab 1–40rAb 1–42, and it also defibrillizes the preformed Ab fibrils in a time-dependent manner.
2. Materials and methods 2.1. Materials Purified synthetic Ab 1–40 and Ab 1–42 were purchased from US Peptides Inc., CA. Bioadhesive hydropho-
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bic diagnostic printed slides Ž10 wells. were purchased from Fisher Scientific. The 4G8 monoclonal antibody ŽmAb. that recognizes 17–24 amino acid sequence of Ab was kindly provided by Dr. K.S. Kim of this Institute. Bovine plasma Žfrozen. was from Pel-Freeze Biologicals, AR. Congo red and all other reagents were from Sigma. 2.2. Purification of gelsolin Bovine plasma gelsolin was either purchased from Sigma or purified in the laboratory from the frozen bovine plasma according to the method of Tanaka and Sobue w25x. In brief, Tris, leupeptin, and mercaptoethanol were added to the bovine plasma to obtain a final concentration of 25 mM Tris, leupeptin Ž2 mgrml., 0.1% mercaptoethanol, and pH was adjusted to 8.0. This step was followed by ammonium sulfate precipitation. The fraction between 35% and 50% of ammonium sulfate was collected, and subjected to DE 52 and affinity-gel blue chromatographies. The fractions containing gelsolin were pooled, concentrated and dialyzed against 25 mM Tris–HCl and 1 mM EGTA, pH 7.5. Silver staining of the final preparation of gelsolin showed a single band of 90 kDa molecular weight. The protein content was measured by BioRad assay.
Fig. 1. Congo red staining of Ab 1–40 allowed to age in the absence and presence of purified gelsolin. Samples of soluble Ab 1–40 alone ŽA., gelsolin control ŽG., and Ab mixed at the molar ratio of 40:1 with gelsolin ŽAG. were incubated at 378C. After 5 days Ž5d. and 7 days Ž7d. of incubation, the Congo red stained samples were viewed under polarized light. The green birefringence was observed only in aged Ab samples in the absence of gelsolin ŽA.. Magnification 150 = .
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2.3. Effect of gelsolin on the fibrillization of A b Synthetic Ab 1–40 and Ab 1–42 were solubilized in double distilled water at the concentration of 4 mgrml, and the pH adjusted to 7.5. Gelsolin purified in the laboratory was dissolved in buffer A, i.e., 25 mM Tris–HCl and 1 mM EGTA, pH 7.5, and gelsolin purchased from Sigma was dissolved in 25 mM Tris–HCl, pH 7.5. Ab Ž60 mg. mixed with 35 mg of gelsolin at the molar ratio of 40:1, Ab alone, and gelsolin alone Žcontrol. were allowed to incubate at 378C. The degree of fibrillization in the samples were examined by Congo red staining, and electron microscopy ŽEM. as described below. 2.3.1. Congo red staining This method was used to probe for the presence of cross b-pleated sheet structure in Ab, a defining characteristic of amyloid fibrils. Congo red does not bind to dimers or trimers, but only binds to higher order aggregates so called protofibrils, and amyloid like fibrils. The complex of
Congo red and Ab fibrils gives a green birefringence when viewed under polarized light. We used modified method of Jarrett and Lansbury w12x. At 5th and 7th days of incubation of Ab in the presence and absence of gelsolin, 2 ml from each sample ŽAb, gelsolin, and Ab q gelsolin. was put on the bioadhesive hydrophobic printed slide, and it was allowed to dry for 2 h. Using 1 ml pipette, the dried samples were gently covered with one drop of 1 mM Congo red Žin 100 mM NaCl, 10 mM phosphate buffer, pH 7.4.. After 1 min, the slide was tilted at ; 308 angle, dye was carefully aspirated by slow suction, and sample was rinsed with a drop of water. The slides were dried at room temperature, viewed under polarized light using a Axrophot ŽC. Zeiss. microscope Žequipped with polarizing filter.. 2.3.2. NegatiÕe staining for electron microscopic study The morphological characteristic of structure formed by Ab in the absence and presence of gelsolin was visualized in negative staining in electron microscope. On the 7th day
Fig. 2. Electron micrographs showing inhibition of Ab 1–40 and Ab 1–42 fibrillization in the presence of gelsolin. The samples of Ab 1–40 ŽA40.rAb 1–42 ŽA42., gelsolin control ŽG., Ab and gelsolin at 40:1 molar ratio were incubated for 7 days, and examined by EM in negative staining with uranyl acetate. Incubation of both Ab 1–40 and Ab 1–42 with plasma gelsolin purified in our laboratory ŽA40G, A42G., or gelsolin purchased from Sigma ŽA40GU . caused inhibition of fibril formation. Magnification 120,000 = .
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of incubation of Ab, gelsolin and Ab-gelsolin samples, one drop of each sample was applied to a 400-mesh formvar-coated grid. After 1 min, the sample was washed with 10 consecutive drops of distilled water, followed by
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four drops of 2% uranyl acetate. The last drop of uranyl acetate remained on the grid for 60 s. It was washed with two drops of water, drained, and air-dried. Samples were examined in a Hitachi 7000 electron microscope.
Fig. 3. Effect of gelsolin on the preformed Ab fibrils assessed by Congo red staining. Ab 1–40 Ž2 mgrml. was allowed to incubate in buffer A Ž25 mM Tris–HCl, 1 mM EGTA, pH 7.5. for 6 days to form fibrils ŽAf.. A total of 25 ml of these preformed Ab fibrils was then incubated in the presence of gelsolin Žin buffer A. at a molar ratio of 20:1 ŽAfG. or in buffer A Žcontrol, Af. for 5 days. After 1 h, 2 days Ž2d. and 5 days Ž5d. of incubation, the samples were stained with Congo red, and viewed under polarized light. Magnification 150 = .
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2.4. Effect of gelsolin on the preformed A b fibrils The preformed Ab fibrils were obtained by incubating soluble Ab 1–40 Ž2 mgrml. in buffer A for 6 days. The
preformed Ab fibrils Ž52 mg. was then incubated with 60 mg gelsolin in buffer A at the Ab:gelsolin molar ratio of 20:1. In the control sample, buffer A was only added to the preformed Ab fibrils. After 1 h, 5 and 7 days, these
Fig. 4. Electron microscopic studies showing defibrillization of preformed Ab 1–40 fibrils by gelsolin. Ab 1–40 was solubilized Ž2 mgrml. and aged for fibril formation ŽAf.. Gelsolin ŽG. in buffer A Ž25 mM Tris–HCl, 1 mM EGTA, pH 7.5. was added to Af at a molar ratio of 1:20 in the AfG sample. In the control sample ŽAf., buffer A was added. The samples were incubated for 5 days, and examined by EM in negative staining after 1 h, 2 days Ž2d. and 5 days Ž5d. of incubation. Magnification 30,000 = .
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samples were analyzed by Congo red staining and EM as described in 2.3.1 and 2.3.2.
3. Results 3.1. Effect of gelsolin on A b fibrillization Since gelsolin is present in the plasma and CSF, and it binds to Ab w5x, we studied if gelsolin has any effect on the fibrillogenesis of soluble synthetic Ab. The effect of gelsolin on Ab fibrillization was studied by Ža. Congo red staining ŽFig. 1., and Žb. EM in negative staining ŽFig. 2.. When aged Ab samples at 5th and 7th days of incubation were stained with Congo red and viewed under polarized light, they produced a specific green birefringence indicating that Ab was in the fibrillar form. No green birefringence was observed in the Ab sample incubated with gelsolin, indicating that the fibrillization of Ab was completely inhibited in the presence of gelsolin. The control sample, i.e., gelsolin alone, was negative for Congo red staining ŽFig. 1.. On the other hand, green birefringence was observed in Congo red stained samples of Ab 1–40 incubated in the presence of 40:1 Žmolermole. bovine serum albumin ŽBSA. for 5 and 7 days Ždata not shown. indicating that BSA does not affect Ab fibrillization under similar experimental conditions where gelsolin completely inhibits it. The samples containing aged Ab 1–40 or Ab 1–42 alone, and corresponding Ab samples incubated with gelsolin after seven days of aging were also examined by EM in negative staining ŽFig. 2.. Ab 1–40 and Ab 1–42 formed dense network of fibrils with the morphology similar to as reported previously w2–4x. Gelsolin inhibited the fibril formation of both Ab 1–40 and Ab 1–42 by more than 90% of that observed for Abs in the absence of gelsolin. Gelsolin alone Žcontrol. did not make the fibrils by itself ŽFig. 2.. The experiments were first performed using plasma gelsolin ŽSigma., which contained high concentration of salt ŽNaCl, Tris buffer salt.. In order to rule out the possibility of contaminating salt in gelsolin sample having an effect on Ab fibrillization, we repeated the experiment using gelsolin purified in our laboratory from bovine plasma, which was free of salt. Similar results were obtained with gelsolin purchased from Sigma and that purified in the laboratory. 3.2. Defibrillization of preformed A b fibrils by gelsolin Since gelsolin inhibits the fibrillization of Ab, we studied if it could also defibrillize the preformed fibrils of Ab. The preformed fibrils were obtained by incubating soluble Ab for 6 days. After mixing gelsolin to the preformed fibrils, the status of the fibrils was analyzed by Congo red staining and EM at different time periods. The results of Congo red staining of the preformed Ab fibrils
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incubated in buffer Žcontrol., or in the presence of gelsolin for 1 h, 2 and 5 days are shown in Fig. 3. The green birefringent material was observed in the samples of fibrillar Ab stained with Congo red when viewed under polarized light after 1 h, 2 and 5 days of incubation without gelsolin, and after 1 h of incubation with gelsolin. After 2 days, there was remarkable reduction in the amount of green birefringent material in Ab sample containing gelsolin. In fact, only traces of green birefringent material were present. Interestingly, green birefringent material was completely absent after 5 days of incubation with gelsolin ŽFig. 3.. This data suggests that gelsolin was able to defibrillize the preformed fibrils of Ab. The electron microscopic analysis in negative staining of preformed Ab fibrils after incubation for 1 h, 2 and 5 days without gelsolin, and in the presence of gelsolin is shown in Fig. 4. Ab samples incubated without gelsolin after 1 h, 2 and 5 days, showed a dense network of long fibrils of Ab well immunolabeled with mAb 4G8 detecting Ab peptide. After 1 h of incubation with gelsolin, the network of fibrils was degraded to large clusters of long and short fibrils with little or no amorphous material. After 2 days of incubation of Ab q gelsolin sample, long fibrils were absent, only short fibrils or filaments like structures were present in small numbers. The fibrils or filaments were completely absent in Ab samples incubated with gelsolin for 5 days. These results confirmed the results of Congo red staining, and suggest that gelsolin solubilizes the preformed fibrils in a time-dependent manner.
4. Discussion The fibrillization of Ab is considered a main event in AD pathology that leads to neuronal cell death, and results in cognitive and behavioral decline. Several plasma and CSF constituents have been shown to bind Ab w1,7,14,19,24x. We have reported earlier that serum inhibits the fibrillization of Ab w3,27x, plasma gelsolin binds to Ab w5x, and Ab-gelsolin complex exists under physiological conditions w5x. We now report that gelsolin inhibits the fibrillization of Ab, and it can also defibrillize the preformed Ab fibrils. Gelsolin is known to be present both intracellularly w25x and as secreted protein in the plasma and CSF w18x. Gelsolin acts as a major actin-binding protein in the cytoplasm, where it regulates the assembly of actin filaments in a highly specialized manner. It caps the actin filament’s growing end, stimulates its nucleation, and severs the actin filament w11,22x. Recent studies show the role of gelsolin in several other activities besides control of actin polymerization, namely, modulation of calcium channel and NMDA receptor w6x, apoptosis w13x, and in tumor suppression w15x. However, the function of gelsolin in the plasma and CSF is not yet elucidated. An observed lack of green birefringence upon Congo red staining of aged Ab sam-
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ples in the presence of gelsolin, and inhibition of Ab fibril formation by gelsolin in EM analysis indicate that gelsolin acts as an inhibitor of b-pleated sheet structure characteristic of amyloid fibrils, and the fibril formation of soluble Ab 1–40rAb 1–42. Therefore, gelsolin may act as an antiamyloidogenic agent in the plasma and CSF. Interestingly, gelsolin was also able to solubilize the preformed Ab fibrils in a time-dependent manner, and thus, it may induce the conformational change from b-pleated sheet structure of amyloid fibrils to a-helical structure of soluble amorphous Ab. It is possible that defibrillization of the preformed fibrils of Ab by gelsolin may be the defense mechanism against other pro-amyloidogenic agents. Ab fibrillization is a nucleation-dependent phenomenon. It is not clear at present whether gelsolin inhibits the fibrillization of Ab by blocking the nucleus formation or it inhibits the elongation of Ab fibrils. Recently, we reported that there are two sites of Ab binding on gelsolin with apparent dissociation constants Ž K d . of 1.38 and 2.55 mM w5x. Gelsolin is also known to bind to two actin molecules w11x. Since gelsolin also inhibits the polymerization of actin, the gelsolin-mediated regulation of actin fibrillization and Ab fibrillization may have an analogous mechanism. Further studies are required to address the question if binding of actin and Ab takes place on the same or different region on gelsolin. Several proteins, such as apo E, transthyretin and apo J, that bind to Ab, have been found in the amyloid plaques along with fibrillar Ab. Among these proteins, apo E has been extensively studied for its role in Ab fibrillogenesis. Apo E has been reported to promote the fibrillization of synthetic Ab w17,28x. In fact, apo E4 allele is increased in the genetically linked cases of AD patients w23x. These amyloid associated proteins have been suggested to function as pathological chaperons, and to promote both b-sheet structure and fibril formation w29x. Gelsolin, like other Ab binding proteins, is also present in the plasma and CSF. However, unlike other amyloid associated proteins, gelsolin was not detected in the amyloid plaques of AD w5x. We propose that gelsolin is a major Ab-sequestering protein in the plasma and CSF where it prevents Ab from fibrillization and maintains it in the soluble form. The binding of secretory proteins Žincluding gelsolin. to Ab suggests the presence of a highly complex regulatory pathway for keeping Ab in the soluble form in the plasma and CSF. The imbalance of this regulatory pathway may result in Ab fibrillization in AD. The binding of gelsolin to Ab, and the inhibition of Ab fibril formation in the presence of gelsolin suggest that gelsolin plays a vital role in the regulation of Ab fibrillogenesis. Amyloid formation in AD may result in part from failure of this sequestration mechanism andror imbalance in the levels of gelsolin in the brain, CSF, or plasma. Since fibrillization of Ab is an essential event in the AD pathology, its inhibition or defibrillization by gelsolin suggests that the physiological role of gelsolin in the plasma and
CSF is to prevent Ab from undergoing the conformational change from soluble a-helical to b-pleated sheet structure.
Acknowledgements We would like to dedicate this publication to Dr. H.M. Wisniewski, the late Director of N.Y.S. Institute for Basic Research in Developmental Disabilities. We thank Dr. K.C. Wang and Ms. Deepti Chauhan for technical assistance. This work was supported in part by the funds from the New York State Office of Mental Retardation and Developmental Disabilities, and by NIH grant AG 14199.
References w1x A.L. Biere, B. Ostaszewski, E.R. Stimson, B.T. Hyman, J.E. Maggie, D.J. Selkoe, Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma, J. Biol. Chem. 271 Ž1996. 32916– 32922. w2x A. Chauhan, V.P.S. Chauhan, R. Rubenstein, J. Wegiel, H.M. Wisniewski, Media from rhabdomyosarcoma and neuroblastoma cell cultures stimulate in vitro aggregation and fibrillization of amyloid beta-protein, Neurochem. Res. 22 Ž1997. 227–232. w3x A. Chauhan, V.P.S. Chauhan, J. Wegiel, H.M. Wisniewski, Impact of normal and heat-inactivated serum on in vitro aggregation and fibrillization of synthetic amyloid beta-protein, Alzheimer’s Res. 2 Ž1996. 243–248. w4x V.P.S. Chauhan, I. Ray, A. Chauhan, J. Wegiel, H.M. Wisniewski, Metal cations defibrillize the amyloid beta-protein fibrils, Neurochem. Res. 22 Ž1997. 805–809. w5x V.P.S. Chauhan, I. Ray, A. Chauhan, H.M. Wisniewski, Binding of gelsolin, a secretory protein, to amyloid beta-protein, Biochem. Biophys. Res. Commun. 258 Ž1999. 241–246. w6x K. Furukawa, W. Fu, Y. Li, W. Witke, D.J. Kwiatkowski, M.P. Mattson, The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons, J. Neurosci. 17 Ž1997. 8178–8186. w7x J. Ghiso, E. Matsubara, A. Koudinov, T. Wisniewski, B. Frangione, The cerebrospinal fluid soluble form of Alzheimer’s amyloid b is complexed to SP-40, 40 ŽAPO J., an inhibitor of the complement membrane-attack complex, Biochem. J. 293 Ž1993. 27–30. w8x C. Haas, M.G. Schlossmacher, A.Y. Hung, C. Vigo-Pelfrey, A. Mellon, B.L. Ostaszewski, I. Lieberburg, E.H. Koo, D. Schenk, D.B. Teplow, D. Selkoe, Amyloid beta-peptide is produced by cultured cells during normal metabolism, Nature 359 Ž1992. 325–327. w9x J. Hardy, Amyloid, the presenilins and Alzheimer’s disease, Trends Neurosci. 20 Ž1997. 403–407. w10x C. Hilbich, B. Kisters-Woike, J. Reed, C.L. Masters, K. Beyreuther, Aggregation and secondary structure of synthetic amyloid beta A4 peptides of Alzheimer’s disease, J. Mol. Biol. 218 Ž1991. 149–163. w11x P.A. Janney, C. Chapannier, S.E. Lind, K.S. Zaner, T.P. Stossel, H.L. Yin, Interactions of gelsolin and gelsolin–actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking, Biochemistry 24 Ž1985. 3714–3723. w12x J.T. Jarrett, P.T. Lansbury Jr., Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB, Biochemistry 31 Ž1992. 12345–12352. w13x S. Kothakota, T. Azuma, C. Reinhard, A. Klippel, J. Tang, K. Chu, T.J. McGarry, M.W. Kirschner, K. Koths, D.J. Kwiatkowski, L.T. Williams, Caspase-3-generated fragment of gelsolin: effector of morphological changes in apoptosis, Science 278 Ž1997. 294–298.
I. Ray et al.r Brain Research 853 (2000) 344–351 w14x A. Koudinov, E. Matsubara, B. Frangione, J. Ghiso, The soluble form of Alzheimer’s amyloid beta-protein is complexed to high-density lipoprotein 3 and very high density lipoprotein in normal human plasma, Biochem. Biophys. Res. Commun. 205 Ž1994. 1164–1171. w15x N. Kuzumaki, M. Tanaka, N. Sakai, H. Fujita, Tumor suppressive function of gelsolin, Gan To Kagaku Royoho 24 Ž1997. 1436–1441. w16x A. Lorenzo, B.A. Yankner, b-Amyloid neurotoxicity requires fibril formation, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 12243–12247. w17x J. Ma, A. Yee, H.B. Brewer Jr., S. Das, H. Potter, Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments, Nature 372 Ž1994. 92–94. w18x T. Paunio, S. Kiuru, S.-L. Karonen, J. Palo, L. Peltonen, Quantification of serum and cerebrospinal fluid gelsolin in familial amyloidosis, Finnish type ŽAGel., Amyloid, Int. J. Exp. Clin. Invest. 1 Ž1994. 80–89. w19x A.L. Schwarzman, L. Gregori, M.P. Vitek, S. Lyubski, W.J. Strittmatter, J.J. Enghilde, R. Bhasin, J. Silverman, K.H. Weisgraber, P.K. Coyle, M.G. Zagorski, J. Talafous, M. Eisenberg, A.M. Saunders, A.D. Roses, D. Goldgaber, Transthyretin sequesters amyloid b-protein and prevents amyloid formation, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 8368–8371. w20x P. Seubert, C. Vigo-Pelfrey, F. Esch, M. Lee, H. Dovey, D. Davis, S. Sinha, M. Schlossmacher, J. Whaley, C. Swindlehyurst, R. McCormack, R. Wolfert, D. Selkoe, I. Lieberburg, D. Schenk, Isolation and quantitation of soluble Alzheimer’s beta-peptide from biological fluids, Nature 359 Ž1992. 325–327. w21x L.K. Simmons, P.C. May, K.J. Tomasseli, R.E. Rydel, K.S. Fuson, E.F. Brigham, S. Wright, I. Lieberburg, G.W. Becker, D.N. Brems, W.Y. Li, Secondary structure of amyloid b-peptide correlates with neurotoxic activity in vitro, Mol. Pharmacol. 45 Ž1994. 373–379. w22x T.P. Stossel, Actin-membrane interactions in eukaryotic mammalian cells, in: J.F. Hoffman, G. Giebisch ŽEds.., Current Topics in
w23x
w24x
w25x
w26x
w27x
w28x
w29x w30x w31x
351
Membranes and Transport, Vol. 36, Academic Press, NY, 1990, pp. 97–107. W.J. Strittmatter, A.M. Saunders, D. Schmechel, M. Pericak-Vance, J. Enghild, G.S. Salvesen, A.D. Roses, Increased frequency of type 4 allele in late-onset familial Alzheimer disease. Apolipoprotein E: High-avidity binding to beta-amyloid, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 1977–1981. W.J. Strittmatter, K.H. Weisgraber, D.Y. Huang, L.-M. Dong, G.S. Salvesen, M. Pericak-Vance, D. Schmechel, A.M. Saunders, D. Goldgaber, A.D. Roses, Binding of human apolipoprotein E to synthetic amyloid b-peptide: isoform-specific effects and implications for late-onset Alzheimer’s disease, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 8098–8102. J. Tanaka, K. Sobue, Localization and characterization of gelsolin in nervous tissues: gelsolin is specifically enriched in myelin-forming cells, J. Neurosci. 14 Ž1994. 1038–1052. C. Vigo-Pelfrey, D. Lee, P. Keim, I. Lieberburg, D.B. Schenk, Characterization of b-amyloid peptide from human cerebrospinal fluid, J. Neurochem. 61 Ž1993. 1965–1968. J. Wegiel, A. Chauhan, H.M. Wisniewski, J. Nowakowski, K.C. Wang, H. Levine, Promotion of synthetic amyloid b-peptide fibrillization by cell culture media and abolishment of fibrillization by serum, Neurosci. Lett. 211 Ž1996. 151–154. T. Wisniewski, E.M. Castano, A. Golabek, T. Vogel, B. Frangione, Acceleration of Alzheimer’s fibril formation by apolipoprotein E in vitro, Am. J. Pathol. 145 Ž1994. 1030–1035. T. Wisniewski, J. Ghiso, B. Frangione, Alzheimer’s disease and soluble Ab, Neurobiol. Aging 15 Ž1994. 143–152. T. Wisniewski, J. Ghiso, B. Frangione, Biology of Ab amyloid in Alzheimer’s disease, Neurobiol. Dis. 4 Ž1997. 313–328. S.G. Younkin, Evidence that Ab 42 is the real culprit in Alzheimer’s disease, Ann. Neurol. 37 Ž1995. 287–288.