Aberrant alpha-synuclein degradation is implicated in Parkinson's disease pathogenesis because the protein accumulates in the Lewy inclusion bodies associated with the disease. Little is known, however, about the pathways by which wild-type alpha-synuclein is normally degraded. We found that wild-type alpha-synuclein was selectively translocated into lysosomes for degradation by the chaperone-mediated autophagy pathway. The pathogenic A53T and A30P alpha-synuclein mutants bound to the receptor for this pathway on the lysosomal membrane, but appeared to act as uptake blockers, inhibiting both their own degradation and that of other substrates. These findings may underlie the toxic gain-of-function by the mutants.

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Several variants of the beta amyloid protein, differing only at their carboxy terminus (beta 1-39, beta 1-40, beta 1-42, and beta 1-43), have been identified as the major components of the cerebral amyloid deposits which are characteristic of Alzheimer's disease. Kinetic studies of aggregation by three naturally occurring beta protein variants (beta 1-39, beta 1-40, beta 1-42) and four model peptides (beta 26-39, beta 26-40, beta 26-42, beta 26-43) demonstrate that amyloid formation, like crystallization, is a nucleation-dependent phenomenon. This discovery has practical consequences for studies of the beta amyloid protein. The length of the C-terminus is a critical determinant of the rate of amyloid formation ("kinetic solubility") but has only a minor effect on the thermodynamic solubility. Amyloid formation by the kinetically soluble peptides (e.g., beta 1-39, beta 1-40, beta 26-39, beta 26-40) can be nucleated, or "seeded", by peptides which include the critical C-terminal residues (beta 1-42, beta 26-42, beta 26-43, beta 34-42). These results suggest that nucleation may be the rate-determining step of in vivo amyloidogenesis and that beta 1-42 and/or beta 1-43, rather than beta 1-40, may be the pathogenic protein(s) in AD.

Ordered protein aggregation in the brain is a hallmark of Alzheimer's disease and scrapie. The disease-specific amyloid fibrils comprise primarily a single protein, amyloid beta, in Alzheimer's disease, and the prion protein in scrapie. These proteins can be induced to form aggregates in vitro that are indistinguishable from brain-derived fibrils. Consequently, much effort has been invested in the development of in vitro model systems to study the details of the aggregation processes and the effects of endogenous molecules that have been implicated in disease. Selected studies of this type are reviewed herein. A simple mechanistic model has emerged for both processes that involves a nucleation-dependent polymerization. This mechanism dictates that aggregation is dependent on protein concentration and time. Furthermore, amyloid formation can be seeded by a preformed fibril. The physiological consequences of this mechanism are discussed.

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.

The Parkinson's disease (PD) substantia nigra is characterized by the presence of Lewy bodies containing fibrillar ␣-synuclein. Earlyonset PD has been linked to two point mutations in the gene that encodes ␣-synuclein, suggesting that disease may arise from accelerated fibrillization. However, the identity of the pathogenic species and its relationship to the ␣-synuclein fibril has not been elucidated. In this in vitro study, the rates of disappearance of monomeric ␣-synuclein and appearance of fibrillar ␣-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 ␣-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 ␣-synuclein oligomers, some of which were separated from fibrillar and monomeric ␣-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 ␣-synuclein fibrillization but do not block its oligomerization could mimic the A30P mutation and thus may accelerate disease progression.amyloid ͉ aggregation ͉ protofibril ͉ atomic force microscopy (AFM) P arkinson's disease (PD) is an age-related neurodegenerative disorder characterized by difficulty in initiating movements, rigidity, and resting tremor (1). In PD, neuronal death is localized to dopaminergic neurons in the substantia nigra region of the brain stem and precedes appearance of symptoms; ca. 70% of neurons may have died by the time symptoms become apparent (1). The postmortem PD substantia nigra is characterized by sporadic intraneuronal cytoplasmic inclusions known as Lewy bodies (LB), the fibrous portion of which contains the protein ␣-synuclein (2, 3). Lewy bodies themselves could be neurotoxic, analogous to the proposed toxicity of amyloid plaques in Alzheimer's disease (AD) (4); the frequency of cortical Lewy bodies correlates with the severity of the AD-like dementia diffuse Lewy body disease (5). Alternatively, Lewy bodies may be an epiphenomenon, induced by neuronal death. Finally, it is possible that Lewy bodies are an inert end point of a process that, early on, produces a neurotoxic species. This scenario would predict that Lewy body formation may protect against neuronal death by sequestering the toxic species. Two neuropathological observations may be relevant to this issue.First, inclusion-bearing neurons appear to be more healthy than neighboring ...

The "non-A beta component of Alzheimer's disease amyloid plaque" (NAC) is a minor peptide component of the insoluble fibrillar core of the Alzheimer's disease (AD) neuritic plaque. NAC amyloid fibrils seed the polymerization of A beta 1-40, the major AD amyloid protein. NAC is derived from a 14 kDa precursor protein, designated NACP, a member of a highly conserved family of heat-stable brain-specific acidic proteins which have been suggested to be involved in synapse formation and/or stabilization. NACP has also been suggested to play a role in AD. We present herein a conformational analysis of human NACP. NACP has a much larger Stokes radius (34 A) but sedimented more slowly (s20,w = 1.7S) than globular proteins of similar molecular weight, indicating that the native protein is elongated. Circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR) indicate the absence of significant amounts of secondary structure in NACP, while CD and ultraviolet spectroscopy suggest the lack of a hydrophobic core. The conformational properties of NACP were unchanged by boiling and were independent of concentration, pH, salt, and chemical denaturants. These features indicate that NACP exists as a mixture of rapidly equilibrating extended conformers and is representative of a class of "natively unfolded" proteins, many of which potentiate protein-protein interactions.

ARTICLES Fig. 1 Circular dichroism spectra. a, WT α-synuclein and the two mutant proteins, A30P and A53T, were mainly random coil in PBS. b, Helicity could be induced by the addition of 0.5%SDS or 10% HFIP (data not shown). The two mutant proteins, A30P and A53T, were indistinguishable from WT under these conditions.