Is there a cause-and-effect relationship between alpha-synuclein fibrillization and Parkinson's disease?

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.

Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease.

Two mutations in the gene encoding alpha-synuclein have been linked to early-onset Parkinson's disease (PD). alpha-Synuclein is a component of Lewy bodies, the fibrous cytoplasmic inclusions characteristic of nigral dopaminergic neurons in the PD brain. This connection between genetics and pathology suggests that the alpha-synuclein mutations may promote PD pathogenesis by accelerating Lewy body formation. To test this, we studied alpha-synuclein folding and aggregation in vitro, in the absence of other Lewy body-associated molecules. We demonstrate here that both mutant forms of alpha-synuclein (A53T and A30P) are, like wild-type alpha-synuclein (WT), disordered in dilute solution. However, at higher concentrations, Lewy body-like fibrils and discrete spherical assemblies are formed; most rapidly by A53T. Thus, mutation-induced acceleration of alpha-synuclein fibril formation may contribute to the early onset of familial PD.

A detergent-insoluble membrane compartment contains A beta in vivo.

Ordered assembly of the amyloid-beta protein (A beta) into amyloid fibrils is a critical step in Alzheimer's disease (AD). To release the amyloidogenic peptide A beta from the Alzheimer amyloid precursor protein (APP), two secretases act sequentially: first, beta-secretase cleaves close to the membrane within the ectodomain and then gamma-secretase cuts within the transmembrane domain. The sites of gamma-secretase cleavage are after residues 40 or 42 of A beta. Except in those rare cases of AD caused by a mutation, levels of secreted A beta are not elevated; thus, the secretory pathway may be unaffected, and factors other than the extracellular concentration of A beta may contribute to the aggregation properties of the peptide. A beta is also present in intracellular compartments. The two gamma-secretase cleavage products, A beta42 and A beta40, were found in different compartments: A beta42 in the endoplasmic reticulum (ER)/intermediate compartment, and A beta40 in the trans-Golgi network (TGN). The cellular compartments that harbor A beta are target sites for therapeutic intervention. Here we report that in the brain, the principal compartment in which A beta resides is a detergent-insoluble glycolipid-enriched membrane domain (DIG). Also present in the DIG fractions are the endoproteolytic fragments of presenilin-1 (PS1) and APP. The presence of these proteins, which all contribute to the generation of A beta, indicates that the DIG fraction is probably where the intramembranous cleavage of APP occurs.

Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct.

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.

Yeast prions: bungee cord domains' balancing act.

The yeast prion-like protein Sup35 has repeats responsible for the reversible induction of an altered, but advantageous phenotype. The expansion of similar repeat domains in several mammalian proteins is associated with neurodegenerative disease - so why are these 'bungee cord' domains conserved?

Amyloid fibrillogenesis: themes and variations.

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.

Inhibition of amyloid formation: a strategy to delay the onset of Alzheimer's disease.

Converging evidence suggests that the formation of amyloid plaques may play a central role in the pathogenesis of Alzheimer's disease and that blocking amyloid formation may be an effective therapeutic strategy.

Neurodegeneration: new clues on inclusions.

The rare neurological disorders frontotemporal dementia and British dementia have been linked to two mutant genes whose products constitute the fibrils that define the two disease pathologies. Two recent studies add to the mounting circumstantial case that protein fibrillization, inside (neurofibrillary tangles) or outside (amyloid plaques) of the neuron, may be pathogenic and suggest that either or both of these mechanisms could initiate Alzheimer's disease.

The chemistry of scrapie infection: implications of the 'ice 9' metaphor.

The transmissible spongiform encephalopathies pose an increasing problem for animal, and perhaps human, health. The infectious agent seems to lack a nucleic acid component, posing the question of how it can reproduce. A model of reproduction by nucleated polymerization suggests a number of novel approaches to the problem.

The core Alzheimer's peptide NAC forms amyloid fibrils which seed and are seeded by beta-amyloid: is NAC a common trigger or target in neurodegenerative disease?

BACKGROUND: NAC is a 35-amino-acid peptide which has been isolated from the insoluble core of Alzheimer's disease (AD) amyloid plaque. It is a fragment of alpha-synuclein (or NACP), a neuronal protein of unknown function. We noted a striking sequence similarity between NAC, the carboxyl terminus of the beta-amyloid protein, and a region of the scrapie prion protein (PrP) which has been implicated in amyloid formation. RESULTS: NAC was prepared by chemical synthesis and was found to form amyloid fibrils via a nucleation-dependent polymerization mechanism. NAC amyloid fibrils effectively seed beta 1-40 amyloid formation. Amyloid fibrils comprising peptide models of the homologous beta and PrP sequences were also found to seed amyloid formation by NAC. CONCLUSIONS: The in vitro model studies presented here suggest that seeding of NAC amyloid formation by the beta-amyloid protein, or seeding of amyloid fibrils of the beta-amyloid protein by NAC, may occur in vivo. Accumulation of ordered NAC aggregates in the synapse may be responsible for the neurodegeneration observed in AD and the prion disorders. Alternatively, neurodegeneration may be caused by the loss of alpha-synuclein (NACP) function.

Aggregates of scrapie-associated prion protein induce the cell-free conversion of protease-sensitive prion protein to the protease-resistant state.

INTRODUCTION: Scrapie infection instigates the in vivo conversion of normal, protease-sensitive prion protein (PrPC) into a protease-resistant form (PrPSc) by an unknown mechanism. In vitro studies have indicated that PrPSc can induce this conversion, consistent with proposals that PrPSc itself might be the infectious scrapie agent. Using this cell-free model of the PrPC to PrPSc conversion, we have studied the dependence of conversion on reactant concentration, and the properties of the PrPSc-derived species that has converting activity. RESULTS: The cell-free conversion of 35S PrPC to the proteinase K-resistant form was dependent on the reaction time and initial concentrations of PrPSc (above an apparent minimum threshold concentration) and 35S PrPC. Analysis of the physical size of the converting activity indicated that detectable converting activity was associated only with aggregates. Under mildly chaotropic conditions, which partially disaggregated PrPSc and enhanced the converting activity, the active species were heterogeneous in size, but larger than either effectively solubilized PrP or molecular weight standards of approximately 2000 kDa. CONCLUSIONS: The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrPSc. These results are consistent with a nucleated polymerization mechanism of PrPSc formation and inconsistent with a heterodimer mechanism.

Amyloid probes based on Congo Red distinguish between fibrils comprising different peptides.

BACKGROUND: Amyloid plaques, which characterize degenerating tissue in Alzheimer's disease (brain) and type II diabetes (pancreas), were first visualized by staining with the dye Congo Red (CR). The ability of CR to recognize amyloid fibrils comprising diverse proteins suggests that the binding site includes an unidentified structural feature common to all amyloid fibrils. We set out to design and synthesize analogs of CR that could distinguish between fibrils comprising different peptides. RESULTS: Relative affinities of several CR analogs for two model amyloid fibrils were measured and compared to that of CR. Amyloid fibrils comprising peptides based on the critical carboxyl terminus of the Alzheimer's disease amyloid protein beta1-42 (beta34-42) and the critical region of the type II diabetes pancreatic amyloid protein, IAPP (IAPP20-29) were tested. The ratio of affinities of each individual CR analog for the two amyloid fibrils varied considerably. Complexation of certain metal ions (Cu(II), Zn(II), Ni(II), Cd(II)) by a CR analog did not abolish its affinity for amyloid but changed the affinity ratio significantly. CONCLUSIONS: This study demonstrates that small organic and organometallic molecules are capable of detecting differences in amyloid fibril structure and/or amyloid protein sequence. Molecules of this type could have utility as neuropathological probes or imaging agents, since they are much easier to prepare and functionalize than antibodies and are specific for the fibrillar form of the amyloid proteins.

Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-beta protein.

BACKGROUND: Amyloid plaques composed of the fibrillar form of the amyloid-beta protein (Abeta) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Abeta to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Abeta amyloid protofibril, has been identified. Here, we report studies of Abeta amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy. RESULTS: Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Abeta influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Abeta. Finally, branched and partially wound fibrils were observed. CONCLUSIONS: The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.

Observation of metastable Abeta amyloid protofibrils by atomic force microscopy.

BACKGROUND: Brain amyloid plaque, a diagnostic feature of Alzheimer's disease (AD), contains an insoluble fibrillar core that is composed primarily of variants of the beta-amyloid protein (Abeta). As Abeta amyloid fibrils may initiate neurodegeneration, the inhibition of fibril formation is a possible therapeutic strategy. Very little is known about the early steps of the process, however. RESULTS: Atomic force microscopy was used to follow amyloid fibril formation in vitro by the Abeta variants Abeta1-40 and Abeta1-42. Both variants first form small ordered aggregates that grow slowly and then rapidly disappear, while prototypical amyloid fibrils of two discrete morphologies appear. Abeta1-42 aggregates much more rapidly than Abeta1-40, which is consistent with its connection to early-onset AD. We propose that the metastable intermediate species be called Abeta amyloid protofibrils. CONCLUSIONS: Abeta protofibrils are likely to be intermediates in the in vitro assembly of Abeta amyloid fibrils, but their in vivo role has yet to be determined. Numerous reports of a nonfibrillar form of Abeta aggregate in the brains of individuals who are predisposed to AD suggest the existence of a precursor form, possibly the protofibril. Thus, stabilization of Abeta protofibrils may be a useful therapeutic strategy.

Synthesis and amyloid binding properties of rhenium complexes: preliminary progress toward a reagent for SPECT imaging of Alzheimer's disease brain.

The definitive diagnosis of Alzheimer's disease (AD) requires the detection of amyloid plaques in postmortem brain. Although the amount of fibrillar amyloid roughly correlates with the severity of symptoms at the time of death, the temporal relationship between amyloid deposition, neuronal loss, and cognitive decline is unclear. To elucidate this relationship, a noninvasive, practical method for the quantitation of brain amyloid deposition is required. We describe herein the initial stages of a strategy to accomplish this goal by single photon computed tomographic imaging. The amyloid-binding dye Congo Red was modified to allow its conjugation to the monoamine-monoamide bis(thiol) ligand. This ligand complexes technetium(V) in its neutral oxo form. A biphenyl-containing building block was conjugated to the protected ligand, and the product was coupled to the relevant aromatic compounds. Rhenium oxo complexes, which are isosteric, but nonradioactive, analogues of the potential imaging agent technetium oxo complexes, were synthesized. These complexes bound to Abeta amyloid fibrils produced in vitro and stained amyloid plaques and vascular amyloid in AD brain sections.

An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments.

Synthetic amyloid beta-protein (A beta) is used widely to study fibril formation and the physiologic effects of low molecular weight and fibrillar forms of the peptide on cells in culture or in experimental animals. Not infrequently, conflicting results have arisen in these studies, in part due to variation in the starting conformation and assembly state of A beta. To avoid these problems, we sought a simple, reliable means of preparing A beta for experimental use. We found that solvation of synthetic peptide with sodium hydroxide (A beta x NaOH), followed by lyophilization, produced stocks with superior solubility and fibrillogenesis characteristics. Solubilization of the pretreated material with neutral buffers resulted in a pH transition from approximately 10.5 to neutral, avoiding the isoelectric point of A beta (pI approximately 5.5), at which A beta precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (A beta x TFA) or hydrochloric acid (A beta x HCl) salts of A beta, yields of "low molecular weight A beta" (monomers and/or dimers) were improved significantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that A beta x NaOH formed fibrils more readily than did the other A beta preparations and that these fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for the preparation of A beta for biophysical and physiologic studies.

The C-terminus of the beta protein is critical in amyloidogenesis.

The beta amyloid protein found in extracellular deposits in Alzheimer's disease (AD) is heterogeneous at its C-terminus; proteins ending at residues 40, 42, and 43 have been identified in neuritic deposits, while protein in vascular amyloid appears to end at residue 39 or 40. Studies of synthetic beta proteins (beta 1-39, beta 1-40, beta 1-42), and model peptides (beta 26-39, beta 26-40, beta 26-42, beta 26-43) demonstrate that amyloid formation is a nucleation-dependent phenomenon. Peptides ending at residues 39 or 40 were kinetically soluble for hours to days, while peptides ending at residues 42 or 43 aggregated immediately; all eventually reached similar thermodynamic solubility. The kinetically soluble variants could be seeded with the kinetically insoluble variants. The secondary structure of beta 26-39 fibrils was different from that of beta 26-42 fibrils, however, seeding beta 26-39 with beta 26-42 produces mixed fibrils with structure similar to beta 26-42. These results suggest that neuritic plaques may be seeded by their minor component; this may determine the structure and properties of amyloid in AD.

Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems. An update.

Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease-sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.