Fibrils Formed in Vitro from α-Synuclein and Two Mutant Forms Linked to Parkinson's Disease are Typical Amyloid

Abstract

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.

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

“…It is apparent that the type 2 aggregates observed resemble α-Syn fibrils grown at high bulk concentrations in solution, which typically show a variable and stochastic lag phase followed by a rapid elongation process. 7,13,14,50,60 Elongated, linear, or dendrite-like fibrils of length up to several micrometers were frequently reported in the context of α-Syn fibrillation. 7,13,14,50 In addition, aggregation mechanisms for amyloidogenic proteins at interfaces in agreement with our observations are comprehensively described in the literature.…”

“…7,13,14,50,60 Elongated, linear, or dendrite-like fibrils of length up to several micrometers were frequently reported in the context of α-Syn fibrillation. 7,13,14,50 In addition, aggregation mechanisms for amyloidogenic proteins at interfaces in agreement with our observations are comprehensively described in the literature. 61,62 For instance, Zhu et al reported two distinct onsurface aggregation mechanisms for the amyloidogenic protein SMA predominantly differing in their growth rates.…”

“…3−5 Aggregates produced from recombinant α-Syn in vitro are indistinguishable from those extracted from Lewy bodies of PD patients. 6,7 While most cases of PD are of the late-onset sporadic type, there are rare cases of inherited early onset PD caused by α-Syn gene triplications 8 or by the presence of point mutations that are reported to increase the oligomerization and fibrillation propensities of α-Syn. 9−11 Several in vitro studies have shown that the process of α-Syn aggregation in solution is concentration dependent and typically requires bulk protein concentrations above 100 μM.…”

Discovery and characterization of a mammalian amyloid disaggregation activity

“…The tight linkage of disaggregation and proteolysis may be due to a physical linkage of proteins or protein complexes that carry out these activities. Additionally, because amyloid is known to be protease resistant, [39][40][41] it is likely that disaggregation is required for proteolysis, and this relationship would necessitate a linear correlation between disaggregation and proteolysis (Fig. 7).…”

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

“…Recombinant, human wild-type α-synuclein was produced in E. coli and purified as described previously [35,36]. The lyophilized protein was dissolved in 10 mM sodium phosphate buffer (pH 7.0) with 100 mM NaCl and filtered through a 0.22 μm nylon spin filter (Costar) to remove high-molecular mass aggregates.…”