Amyloid Pore Section
In amyloid diseases such as Alzheimer's disease (AD) and Parkinson's (PD), polypeptide aggregation leads to formation of transient (thermodynamically unstable) intermediates which then undergo further aggregation to form more stable oligomers or protofibrils and, eventually, stable mature amyloid fibrils. The presence of such fibrils is a characteristic pathological feature of certain cell populations in AD and PD brains (e.g., Lewy bodies in the case of PD).
Increasing evidence shows that protofibrils (not fibrils) are toxic to cells. Recent studies from ours and other labs support the hypothesis that a-synuclein protofibrils rather than fibrils are toxic to dopaminergic neurons in PD. Growing evidence indicates that during the fibrillization process, smaller oligomers or protofibrils lead to formation of toxic species capable of forming amyloid pores in cellular membranes.
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A number of publications from our lab providing experimental support for the pathogenic role of "amyloid pores" in PD and other amyloid diseases can be found below under the "Selected Publications from Our Lab" section; furthermore, you can access selected recent publications from our laboratory with links to PubMed and access to the Cell, Nature, Science, and Biochemistry Full Texts (HTML and PDF formats) via the "Recent Publications" link.
Please follow the various links below to access additional references relevant to the amyloid pore topic; please note that the links below lead to published references selected by the keyword(s) associated with a given link; e.g., the "amyloid pore" link below contains 27 references found using the search keywords "amyloid" and "pore"; likewise, the "protofibril(s)" link below contains 175 references found using the search keywords "protofibril" and "protofibrils".
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To view the page containing links to external news relevant to this section, please click on the "Amyloid Pore News" link below:
Please use the "Search Form" to search for any keywords or phrases in the information provided in the above hyperlinks or any other part of this web site.
Please note: In addition to the list of
"Selected Publications from Our Lab" (see below), providing
experimental support for the pathogenic role of "amyloid pores" in PD
and other amyloid diseases, a more comprehensive list of recent publications from this laboratory is available through the "Recent Publications"
hyperlink below; for an extended list of publications from our lab
(1990-2003), please follow the "Full Publication List" hyperlink below:
Recent Publications Full Publication List
Selected Publications from Our Lab
(Abstracts included where available.)
Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Cuervo, A.M., Stefanis, L., Fredenburg, R., Lansbury, P.T., and Sulzer, D. Science 2004 Aug 27;305(5688):1292-5. Link to PubMed Abstract.
Back to the future: the 'old-fashioned' way to new medications for neurodegeneration. Peter T. Lansbury, Jr. Nature Reviews Neuroscience 5, S51–S57 (2004).
Link to: Nature
Reviews Neuroscience PDF
Please Note: "Single copy of the article can be downloaded and printed for the reader's personal research and study."
"Reprinted from Nature Reviews Neuroscience 5, S51–S57 (2004), Peter T. Lansbury, Jr. Back to the future: the 'old-fashioned' way to new medications for neurodegeneration. Copyright 2004 Nature Publishing Group, with permission from Nature Publishing Group .
Source: Nature Publishing Group Page: http://www.nature.com/focus/neurodegen/index.html
Please note: Related articles can be found on the above web site.
Rochet J.C., Outeiro T.F., Conway K.A., Ding T.T., Volles M.J., Lashuel H.A., Bieganski R.M., Lindquist S.L., and Lansbury P.T. "Interactions Among alpha-Synuclein, Dopamine, and Biomembranes: Some Clues for Understanding Neurodegeneration in Parkinson's Disease." J Mol Neurosci. 2004; 23(1-2):23-34.
Soumya S. Ray and Peter T. Lansbury, Jr. Proc Natl Acad Sci U S A 2004 Apr. "A possible therapeutic target for Lou Gehrig's disease."
Soumya S. Ray, Richard J. Nowak, Konstantin
Strokovich, Robert H. Brown, Jr., Thomas Walz, and Peter T. Lansbury, Jr.
Web Release Date: 06-Apr-2004; (Accelerated Publication). "An Intersubunit
Disulfide Bond Prevents in Vitro Aggregation of a Superoxide Dismutase-1 Mutant
Linked to Familial Amytrophic Lateral Sclerosis."
Kheterpal, I., H. A. Lashuel, et al. (2003). "Abeta Protofibrils Possess a Stable Core Structure Resistant to Hydrogen Exchange." Biochemistry 42(48): 14092-14098.
Protofibrils are transient structures observed during in vitro formation of mature amyloid fibrils and have been implicated as the toxic species responsible for cell dysfunction and neuronal loss in Alzheimer's disease (AD) and other protein aggregation diseases. To better understand the roles of protofibrils in amyloid assembly and Alzheimer's disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. We chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Abeta(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We show that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We find that about 40% of the backbone amide hydrogens of Abeta protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also find a surprising degree of specificity in amyloid assembly, in that wild type Abeta is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides. These and other data are interpreted and discussed in terms of the role of protofibrils in fibril assembly and in disease.
Lashuel, H. A., D. M. Hartley, et al. (2003). "Mixtures of Wild-type and a Pathogenic (E22G) Form of Abeta40 in Vitro Accumulate Protofibrils, Including Amyloid Pores." J Mol Biol 332(4): 795-808.
Although APP mutations associated with inherited forms of Alzheimer's disease (AD) are relatively rare, detailed studies of these mutations may prove critical for gaining important insights into the mechanism(s) and etiology of AD. Here, we present a detailed biophysical characterization of the structural properties of protofibrils formed by the Arctic variant (E22G) of amyloid-beta protein (Abeta40(ARC)) as well as the effect of Abeta40(WT) on the distribution of the protofibrillar species formed by Abeta40(ARC) by characterizing biologically relevant mixtures of both proteins that may mimic the situation in the heterozygous patients. These studies revealed that the Arctic mutation accelerates both Abeta oligomerization and fibrillogenesis in vitro. In addition, Abeta40(ARC) was observed to affect both the morphology and the size distribution of Abeta protofibrils. Electron microscopy examination of the protofibrils formed by Abeta40(ARC) revealed several morphologies, including: (1) relatively compact spherical particles roughly 4-5 nm in diameter; (2) annular pore-like protofibrils; (3) large spherical particles 18-25 nm in diameter; and (4) short filaments with chain-like morphology. Conversion of Abeta40(ARC) protofibrils to fibrils occurred more rapidly than protofibrils formed in mixed solutions of Abeta40(WT)/Abeta40(ARC), suggesting that co-incubation of Abeta40(ARC) with Abeta40(WT) leads to kinetic stabilization of Abeta40(ARC) protofibrils. An increase in the ratio of Abeta(WT)/Abeta(MUT(Arctic)), therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimer's disease mutation carriers.
Volles, M. J. and P. T. Lansbury, Jr. (2003). "Zeroing in on the Pathogenic Form of alpha-Synuclein and Its Mechanism of Neurotoxicity in Parkinson's Disease." Biochemistry 42(26): 7871-8.
Parkinson's disease (PD) is linked to mutations in the protein alpha-synuclein, which can exist in vitro in several aggregation states, including a natively unfolded monomer, a beta-sheet rich oligomer, or protofibril, and a stable amyloid fibril. This work reviews the current literature that is relevant to two linked questions: which of these species is pathogenic, and what is the mechanism of neurotoxicity? The amyloid fibril, fibrillar aggregates, Lewy bodies, and the alpha-synuclein monomer, which is normally expressed at high levels, are all unlikely to be pathogenic, for reasons discussed here. We therefore favor a toxic protofibril scenario, and propose that the pathogenic species is transiently populated during the process of fibrillization. Toxicity may arise from pore-like protofibrils that cause membrane permeabilization. An approach to testing this hypothesis is discussed.
Caughey, B. and P. T. Lansbury, Jr. (2003). "Protofibrils, Pores, Fibrils, and Neurodegeneration: Separating the Responsible Protein Aggregates from the Innocent Bystanders." Annu Rev Neurosci.
Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates. Expected online publication date for the Annual Review of Neuroscience Volume 26 is June 16, 2003. Please see http://www.annualreviews.org/catalog/pub_dates.asp for revised estimates.
Park, J. Y. and P. T. Lansbury, Jr. (2003). "beta-Synuclein Inhibits Formation of alpha-Synuclein Protofibrils: A Possible Therapeutic Strategy against Parkinson's Disease." Biochemistry 42(13): 3696-700.
Parkinson's disease (PD) is an age-associated and progressive movement disorder that is characterized by dopaminergic neuronal loss in the substantia nigra and, at autopsy, by fibrillar alpha-synuclein inclusions, or Lewy bodies. Despite the qualitative correlation between alpha-synuclein fibrils and disease, in vitro biophysical studies strongly suggest that prefibrillar alpha-synuclein oligomers, or protofibrils, are pathogenic. Consistent with this proposal, transgenic mice that express human alpha-synuclein develop a Parkinsonian movement disorder concurrent with nonfibrillar alpha-synuclein inclusions and the loss of dopaminergic terminii. Double-transgenic progeny of these mice that also express human beta-synuclein, a homologue of alpha-synuclein, show significant amelioration of all three phenotypes. We demonstrate here that beta- and gamma-synuclein (a third homologue that is expressed primarily in peripheral neurons) are natively unfolded in monomeric form, but structured in protofibrillar form. beta-Synuclein protofibrils do not bind to or permeabilize synthetic vesicles, unlike protofibrils comprising alpha-synuclein or gamma-synuclein. Significantly, beta-synuclein inhibits the generation of A53T alpha-synuclein protofibrils and fibrils. This finding provides a rationale for the phenotype of the double-transgenic mice and suggests a therapeutic strategy for PD.
Kessler, J. C., J. C. Rochet, et al. (2003). "The N-Terminal Repeat Domain of alpha-Synuclein Inhibits beta-Sheet and Amyloid Fibril Formation." Biochemistry 42(3): 672-8.
The conversion of alpha-synuclein into amyloid fibrils in the substantia nigra is linked to Parkinson's disease. alpha-Synuclein is natively unfolded in solution, but can be induced to form either alpha-helical or beta-sheet structure depending on its concentration and the solution conditions. The N-terminus of alpha-synuclein comprises seven 11-amino acid repeats (XKTKEGVXXXX) which can form an amphipathic alpha-helix. Why seven repeats, rather than six or eight, survived the evolutionary process is not clear. To probe this question, two sequence variants of alpha-synuclein, one with two fewer (del2) and one with two additional (plus2) repeats, were studied. As compared to wild-type alpha-synuclein, the plus2 variant disfavors the formation of beta-sheet-rich oligomers, including amyloid fibrils. In contrast, the truncated variant, del2, favors beta-sheet and fibril formation. We propose that the repeat number in WT alpha-synuclein represents an evolutionary balance between the functional conformer of alpha-synuclein (alpha-helix and/or random coil) and its pathogenic beta-sheet conformation. N-Terminal truncation of alpha-synuclein may promote pathogenesis.
Lashuel, H., B. Petre, et al. (2002). "alpha-Synuclein, Especially the Parkinson's Disease-associated Mutants, Forms Pore-like Annular and Tubular Protofibrils." J Mol Biol 322(5): 1089.
Two mutations in the alpha-synuclein gene (A30P and A53T) have been linked to autosomal dominant early-onset Parkinson's disease (PD). Both mutations promote the formation of transient protofibrils (prefibrillar oligomers), suggesting that protofibrils are linked to cytotoxicity. In this work, the effect of these mutations on the structure of alpha-synuclein oligomers was investigated using electron microscopy and digital image processing. The PD-linked mutations (A30P and A53T) were observed to affect both the morphology and the size distribution of alpha-synuclein protofibrils (measured by analytical ultracentrifugation and scanning transmission electron microscopy). The A30P variant was observed to promote the formation of annular, pore-like protofibrils, whereas A53T promotes formation of annular and tubular protofibrillar structures. Wild-type alpha-synuclein also formed annular protofibrils, but only after extended incubation. The formation of pore-like oligomeric structures may explain the membrane permeabilization activity of alpha-synuclein protofibrils. These structures may contribute to the pathogenesis of PD.
Anguiano, M., R. J. Nowak, et al. (2002). "Protofibrillar Islet Amyloid Polypeptide Permeabilizes Synthetic Vesicles by a Pore-like Mechanism that May Be Relevant to Type II Diabetes." Biochemistry 41(38): 11338-43.
Islet amyloid polypeptide (IAPP) and insulin are copackaged and cosecreted by pancreatic islet beta-cells. Non-insulin-dependent (type II) diabetes mellitus (NIDDM) is characterized by dysfunction and depletion of these beta-cells and also, in more than 90% of patients, amyloid plaques containing fibrillar IAPP. An aggregated but not necessarily fibrillar form of IAPP is toxic in cell culture, suggesting that prefibrillar oligomeric (protofibrillar) IAPP may be pathogenic. We report here that IAPP generates oligomeric species in vitro that are consumed as beta-sheet-rich fibrils grow. Protofibrillar IAPP, like protofibrillar alpha-synuclein, which is implicated in Parkinson's disease pathogenesis, permeabilizes synthetic vesicles by a pore-like mechanism. The formation of the IAPP amyloid pore is temporally correlated to the formation of early IAPP oligomers and its disappearance to the appearance of amyloid fibrils. Neither pores nor oligomers were formed by the nonfibrillogenic rat IAPP variant. The IAPP amyloid pore may be critical to the pathogenic mechanism of NIDDM, as other amyloid pores may be to Alzheimer's disease and Parkinson's disease.
Ding, T. T., S. J. Lee, et al. (2002). "Annular alpha-Synuclein Protofibrils Are Produced When Spherical Protofibrils Are Incubated in Solution or Bound to Brain-Derived Membranes." Biochemistry 41(32): 10209-17.
The Parkinson's disease substantia nigra is characterized by the loss of dopaminergic neurons and the presence of cytoplasmic fibrillar Lewy bodies in surviving neurons. The major fibrillar protein of Lewy bodies is alpha-synuclein. Two point mutations in the alpha-synuclein gene are associated with autosomal-dominant Parkinson's disease (FPD). Studies of the in vitro fibrillization behavior of the mutant proteins suggest that fibril precursors, or alpha-synuclein protofibrils, rather than the fibrils, may be pathogenic. Atomic force microscopy (AFM) revealed two distinct forms of protofibrillar alpha-synuclein: rapidly formed spherical protofibrils and annular protofibrils, which were produced on prolonged incubation of spheres. The spherical protofibrils bound to brain-derived membrane fractions much more tightly than did monomeric or fibrillar alpha-synuclein, and membrane-associated annular protofibrils were observed. The structural features of alpha-synuclein annular protofibrils are reminiscent of bacterial pore-forming toxins and are consistent with their porelike activity in vitro. Thus, abnormal membrane permeabilization may be a pathogenic mechanism in PD.
Lashuel, H. A., D. Hartley, et al. (2002). "Neurodegenerative disease: Amyloid pores from pathogenic mutations." Nature 418(6895): 291.
Alzheimer's and Parkinson's diseases are associated with the formation in the brain of amyloid fibrils from beta-amyloid and alpha-synuclein proteins, respectively. It is likely that oligomeric fibrillization intermediates (protofibrils), rather than the fibrils themselves, are pathogenic, but the mechanism by which they cause neuronal death remains a mystery. We show here that mutant amyloid proteins associated with familial Alzheimer's and Parkinson's diseases form morphologically indistinguishable annular protofibrils that resemble a class of pore-forming bacterial toxins, suggesting that inappropriate membrane permeabilization might be the cause of cell dysfunction and even cell death in amyloid diseases.
Volles, M. J. and P. T. Lansbury, Jr. (2002). "Vesicle permeabilization by protofibrillar alpha-synuclein is sensitive to Parkinson's disease-linked mutations and occurs by a pore-like mechanism." Biochemistry 41(14): 4595-602.
Two mutations in the protein alpha-synuclein (A30P and A53T) are linked to an autosomal dominant form of Parkinson's disease. Both mutations accelerate the formation of prefibrillar oligomers (protofibrils) in vitro, but the mechanism by which they promote toxicity is unknown. Protofibrils of wild-type alpha-synuclein bind and permeabilize acidic phospholipid vesicles. This study examines the relative membrane permeabilizing activities of the wild type, mutant, and mouse variants of protofibrillar alpha-synuclein and the mechanism of membrane permeabilization. Protofibrillar A30P, A53T, and mouse variants were each found to have greater permeabilizing activities per mole than the wild-type protein. The leakage of vesicular contents induced by protofibrillar alpha-synuclein exhibits a strong preference for low-molecular mass molecules, suggesting a pore-like mechanism for permeabilization. Under conditions in which the vesicular membrane is less stable (lack of calcium as a phospholipid counterion), protofibril permeabilization is less size-selective and monomeric alpha-synuclein can permeabilize via a detergent-like mechanism. We conclude that the pathogenesis of Parkinson's disease may involve membrane permeabilization by protofibrillar alpha-synuclein, the extent of which will be strongly dependent on the in vivo conditions.
Shtilerman, M. D., T. T. Ding, et al. (2002). "Molecular crowding accelerates fibrillization of alpha-synuclein: could an increase in the cytoplasmic protein concentration induce Parkinson's disease?" Biochemistry 41(12): 3855-60.
Parkinson's disease (PD) is one of many neurodegenerative diseases that are characterized by amyloid fibril formation. Alpha-synuclein is a primary component of the fibrillar neuronal inclusions, known as Lewy bodies, that are diagnostic of PD. In addition, the alpha-synuclein gene is linked to familial PD. Fibril formation by alpha-synuclein proceeds via discrete beta-sheet-rich oligomers, or protofibrils, that are consumed as fibrils grow. Both FPD mutations accelerate formation of protofibrils, suggesting that these intermediates, rather than the fibril product, trigger neuronal loss. In idiopathic PD, other factors may be responsible for accelerating protofibril formation by wild-type alpha-synuclein. One possible factor could be molecular crowding in the neuronal cytoplasm. We demonstrate here that crowding using inert polymers significantly reduced the lag time for protofibril formation and the conversion of the protofibril to the fibril, but did not affect the morphology of either species. Physiologically realistic changes in the degree of in vitro crowding have significant kinetic consequences. Thus, nonspecific changes in the total cytoplasmic protein concentration, induced by cell volume changes and/or altered protein degradation, could promote formation of and stabilize the alpha-synuclein protofibril.
Volles, M. J., S. J. Lee, et al. (2001). "Vesicle permeabilization by protofibrillar alpha-synuclein: implications for the pathogenesis and treatment of Parkinson's disease." Biochemistry 40(26): 7812-9.
Fibrillar alpha-synuclein is a component of the Lewy body, the characteristic neuronal inclusion of the Parkinson's disease (PD) brain. Both alpha-synuclein mutations linked to autosomal dominant early-onset forms of PD promote the in vitro conversion of the natively unfolded protein into ordered prefibrillar oligomers, suggesting that these protofibrils, rather than the fibril itself, may induce cell death. We report here that protofibrils differ markedly from fibrils with respect to their interactions with synthetic membranes. Protofibrillar alpha-synuclein, in contrast to the monomeric and the fibrillar forms, binds synthetic vesicles very tightly via a beta-sheet-rich structure and transiently permeabilizes these vesicles. The destruction of vesicular membranes by protofibrillar alpha-synuclein was directly observed by atomic force microscopy. The possibility that the toxicity of alpha-synuclein fibrillization may derive from an oligomeric intermediate, rather than the fibril, has implications regarding the design of therapeutics for PD.
Lansbury, P. T., Jr. (2001). "Following nature's anti-amyloid strategy." Nat Biotechnol 19(2): 112-3.
Conway, K. A., J. C. Rochet, et al. (2001). "Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct." Science 294(5545): 1346-9.
The substantia nigra in Parkinson's disease (PD) is depleted of dopaminergic neurons and contains fibrillar Lewy bodies comprising primarily alpha-synuclein. We screened a library to identify drug-like molecules to probe the relation between neurodegeneration and alpha-synuclein fibrilization. All but one of 15 fibril inhibitors were catecholamines related to dopamine. The inhibitory activity of dopamine depended on its oxidative ligation to alpha-synuclein and was selective for the protofibril-to-fibril conversion, causing accumulation of the alpha-synuclein protofibril. Adduct formation provides an explanation for the dopaminergic selectivity of alpha-synuclein-associated neurotoxicity in PD and has implications for current and future PD therapeutic and diagnostic strategies.
Rochet, J. C. and P. T. Lansbury, Jr. (2000). "Amyloid fibrillogenesis: themes and variations." Curr Opin Struct Biol 10(1): 60-8.
Recent progress has improved our knowledge of how proteins form amyloid fibrils. Both 'natively unfolded' and globular proteins have been shown to initiate fibrillization by adopting a partially structured conformation. Oligomeric prefibrillar intermediates have been extensively characterized with respect to their morphology and temporal evolution. Three-dimensional models obtained using biophysical and computational methods have provided information about fibril structure. All of these advances suggest common features of self-assembly pathways, with subtle variations accounting for differences among distinct amyloid fibrils.
Rochet, J. C., K. A. Conway, et al. (2000). "Inhibition of fibrillization and accumulation of prefibrillar oligomers in mixtures of human and mouse alpha-synuclein." Biochemistry 39(35): 10619-26.
Parkinson's disease (PD) is a neurodegenerative disorder attributed to the loss of dopaminergic neurons from the substantia nigra. Some surviving neurons are characterized by cytoplasmic Lewy bodies, which contain fibrillar alpha-synuclein. Two mutants of human alpha-synuclein (A53T and A30P) have been linked to early-onset, familial PD. Oligomeric forms of these mutants accumulate more rapidly and/or persist for longer periods of time than oligomeric, human wild-type alpha-synuclein (WT), suggesting a link between oligomerization and cell death. The amino acid sequences of the mouse protein and WT differ at seven positions. Mouse alpha-synuclein, like A53T, contains a threonine residue at position 53. We have assessed the conformational properties and fibrillogenicity of the murine protein. Like WT and the two PD mutants, mouse alpha-synuclein adopts a "natively unfolded" or disordered structure. However, at elevated concentrations, the mouse protein forms amyloid fibrils more rapidly than WT, A53T, or A30P. The fibrillization of mouse alpha-synuclein is slowed by WT and A53T. Inhibition of fibrillization leads to the accumulation of nonfibrillar, potentially toxic oligomers. The results are relevant to the interpretation of the phenotypes of transgenic animal models of PD and suggest a novel approach for testing the cause and effect relationship between fibrillization and neurodegeneration.
Goldberg, M. S. and P. T. Lansbury, Jr. (2000). "Is there a cause-and-effect relationship between alpha-synuclein fibrillization and Parkinson's disease?" Nat Cell Biol 2(7): E115-9.
The first gene to be linked to Parkinson's disease encodes the neuronal protein alpha-synuclein. Recent mouse and Drosophila models of Parkinson's disease support a central role for the process of alpha-synuclein fibrillization in pathogenesis. However, some evidence indicates that the fibril itself may not be the pathogenic species. Our own biophysical studies suggest that a structured fibrillization intermediate or an alternatively assembled oligomer may be responsible for neuronal death. This speculation can now be experimentally tested in the animal models. Such experiments will have implications for the development of new therapies for Parkinson's disease and related neurodegenerative diseases.
Conway, K. A., S. J. Lee, et al. (2000). "Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy." Proc Natl Acad Sci U S A 97(2): 571-6.
The Parkinson's disease (PD) substantia nigra is characterized by the presence of Lewy bodies containing fibrillar alpha-synuclein. Early-onset PD has been linked to two point mutations in the gene that encodes alpha-synuclein, suggesting that disease may arise from accelerated fibrillization. However, the identity of the pathogenic species and its relationship to the alpha-synuclein fibril has not been elucidated. In this in vitro study, the rates of disappearance of monomeric alpha-synuclein and appearance of fibrillar alpha-synuclein were compared for the wild-type (WT) and two mutant proteins, as well as equimolar mixtures that may model the heterozygous PD patients. Whereas one of the mutant proteins (A53T) and an equimolar mixture of A53T and WT fibrillized more rapidly than WT alpha-synuclein, the other (A30P) and the corresponding equimolar mixture with WT fibrillized more slowly. However, under conditions that ultimately produced fibrils, the A30P monomer was consumed at a comparable rate or slightly more rapidly than the WT monomer, whereas A53T was consumed even more rapidly. The difference between these trends suggested the existence of nonfibrillar alpha-synuclein oligomers, some of which were separated from fibrillar and monomeric alpha-synuclein by sedimentation followed by gel-filtration chromatography. Spheres (range of heights: 2-6 nm), chains of spheres (protofibrils), and rings resembling circularized protofibrils (height: ca. 4 nm) were distinguished from fibrils (height: ca. 8 nm) by atomic force microscopy. Importantly, drug candidates that inhibit alpha-synuclein fibrillization but do not block its oligomerization could mimic the A30P mutation and thus may accelerate disease progression.
Conway, K. A., J. D. Harper, et al. (2000). "Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid." Biochemistry 39(10): 2552-63.
Two missense mutations in the gene encoding alpha-synuclein have been linked to rare, early-onset forms of Parkinson's disease (PD). These forms of PD, as well as the common idiopathic form, are characterized by the presence of cytoplasmic neuronal deposits, called Lewy bodies, in the affected region of the brain. Lewy bodies contain alpha-synuclein in a form that resembles fibrillar Abeta derived from Alzheimer's disease (AD) amyloid plaques. One of the mutant forms of alpha-synuclein (A53T) fibrillizes more rapidly in vitro than does the wild-type protein, suggesting that a correlation may exist between the rate of in vitro fibrillization and/or oligomerization and the progression of PD, analogous to the relationship between Abeta fibrillization in vitro and familial AD. In this paper, fibrils generated in vitro from alpha-synuclein, wild-type and both mutant forms, are shown to possess very similar features that are characteristic of amyloid fibrils, including a wound and predominantly unbranched morphology (demonstrated by atomic force and electron microscopies), distinctive dye-binding properties (Congo red and thioflavin T), and antiparallel beta-sheet structure (Fourier transform infrared spectroscopy and circular dichroism spectroscopy). alpha-Synuclein fibrils are relatively resistant to proteolysis, a property shared by fibrillar Abeta and the disease-associated fibrillar form of the prion protein. These data suggest that PD, like AD, is a brain amyloid disease that, unlike AD, is characterized by cytoplasmic amyloid (Lewy bodies). In addition to amyloid fibrils, a small oligomeric form of alpha-synuclein, which may be analogous to the Abeta protofibril, was observed prior to the appearance of fibrils. This species or a related one, rather than the fibril itself, may be responsible for neuronal death.
Conway, K. A., S. J. Lee, et al. (2000). "Accelerated oligomerization by Parkinson's disease linked alpha-synuclein mutants." Ann N Y Acad Sci 920: 42-5.
Lansbury, P. T., Jr. (1999). "Evolution of amyloid: what normal protein folding may tell us about fibrillogenesis and disease." Proc Natl Acad Sci U S A 96(7): 3342-4.
Koo, E. H., P. T. Lansbury, Jr., et al. (1999). "Amyloid diseases: abnormal protein aggregation in neurodegeneration." Proc Natl Acad Sci U S A 96(18): 9989-90.
Harper, J. D., S. S. Wong, et al. (1999). "Assembly of A beta amyloid protofibrils: an in vitro model for a possible early event in Alzheimer's disease." Biochemistry 38(28): 8972-80.
Amyloid fibrils comprising primarily the peptides A beta 40 and A beta 42 are a defining feature of the Alzheimer's disease (AD) brain, and convergent evidence suggests that the process of their formation plays a central role in the AD pathogenic pathway. Elucidation of fibril assembly is critical for the discovery of potential AD diagnostics and therapeutics, since the pathogenic entity is not necessarily the product fibril, but could be a precursor species whose formation is linked to fibrillogenesis in vivo. Atomic force microscopy allowed the identification of an unanticipated intermediate in in vitro fibril formation, the A beta amyloid protofibril. This manuscript describes studies of the structure of the A beta 40 protofibril and its in vitro assembly and disassembly using atomic force microscopy (AFM). The A beta 40 protofibril has a height of ca. 4.3 +/- 0.5 nm and a periodicity of ca. 20 +/- 4.7 nm. The rate of its elongation depends on the total concentration of A beta 40, the temperature, and ionic strength of the medium. A beta 42 and A beta 40 protofibrils elongate at a comparable rate. Statistical analysis of AFM data reveals a decrease in the number of protofibrils with time, indicating that coalescence of smaller protofibrils contributes to protofibril elongation. Similar analysis reveals that protofibrils shorten while the number of protofibrils also decrease following dilution, indicating that protofibril disassembly does not proceed by a reverse of the assembly process. These investigations provide systematic data defining factors affecting A beta fibrillization and, thus, should be valuable in the design of high-throughput assays to identify agents which alter A beta protofibril assembly.
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