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Parkinson's disease (PD) is pathologically characterized by intraneuronal α-synuclein (α-Syn) aggregates called Lewy bodies (LBs) as well as the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). On the basis of autopsy studies, Braak et al. hypothesized that Lewy pathology initially occurs in the enteric nervous system, subsequently spreading to the dorsal motor nucleus of the vagus nerve (dmX) and then ascending in the brainstem to the SNpc. However, this hypothetical progression lacks adequate experimental evidence. We previously reported that inoculation of α-Syn preformed fibrils (PFFs) into the gastric wall of wild-type (WT) mice induced LB-like α-Syn aggregates in the dmX via the vagus nerve. However, α-Syn pathology did not spread beyond the dmX up to 12 months postinoculation. In the present study, we inoculated α-Syn PFFs into the gastric wall of bacterial artificial chromosome (BAC) transgenic mice harboring the human α-Syn gene with an A53 T mutation and analyzed the pathology. The transgenic mice had ∼1.5-fold overexpression of α-Syn in the brains and ∼6-fold overexpression of α-Syn in the stomach compared with WT mice. After inoculation of α-Syn PFFs, the transgenic mice developed a higher number of phosphorylated α-Syn (p-α-Syn)-positive neurons in the dmX compared with similarly inoculated WT mice. However, the number of p-α-Syn-positive neurons in the dmX decreased over time, and α-Syn pathology was not observed in other brain regions except in the ambiguous nucleus up to 8 months postinoculation. Taken together, BAC transgenic expression of α-Syn facilitated induction of α-Syn pathology in the brainstem, but not subsequent caudo-rostral spread in accordance with Braak's hypothesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Intraneuronal α-synuclein (α-Syn) aggregates known as Lewy bodies (LBs) and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are the pathological hallmarks of Parkinson's disease (PD). Braak's hypothesis based on autopsy studies suggests that Lewy pathology initially occurs in the enteric nervous system (ENS) and then travels retrogradely to the dorsal motor nucleus of the vagus nerve (dmX), proceeding from there in a caudo-rostral direction. Recent evidence that α-Syn aggregates propagate between interconnected neurons supports this hypothesis. However, there is no direct evidence demonstrating this transmission from the ENS to the dmX and then to the SNpc., Methods: We inoculated α-Syn preformed fibrils (PFFs) or phosphate-buffered saline (PBS) into the mouse gastric wall and analyzed the progression of the pathology., Results: The mice inoculated with α-Syn PFFs, but not with PBS, developed phosphorylated α-Syn (p-α-Syn)-positive LB-like aggregates in the dmX at 45 days postinoculation. This aggregate formation was completely abolished when vagotomy was performed prior to inoculation of α-Syn PFFs, suggesting that the aggregates in the dmX were retrogradely induced via the vagus nerve. Unexpectedly, the number of neurons containing p-α-Syn-positive aggregates in the dmX decreased over time, and no further caudo-rostral propagation beyond the dmX was observed up to 12 months postinoculation. P-α-Syn-positive aggregates were also present in the myenteric plexus at 12 months postinoculation. However, unlike in patients with PD, there was no cell-type specificity in neurons containing those aggregates in this model., Conclusions: These results indicate that α-Syn PFF inoculation into the mouse gastrointestinal tract can induce α-Syn pathology resembling that of very early PD, but other factors are apparently required if further progression of PD pathology is to be replicated in this animal model.

α-synuclein (α-syn) is the main component of Lewy bodies, which are neuropathological hallmarks of patients with Parkinson's disease. As it has been controversial whether human α-syn from erythrocytes exists as a tetramer under physiological conditions, we tried solving this issue by the small-angle X-ray solution scattering method. Under two different conditions (high ionic strength with a Tris buffer and low ionic strength with an ammonium acetate buffer), no evidence was found for the presence of tetramer. When comparing erythrocyte and recombinant α-syn molecules, we found no significant difference of the molecular weight and the secondary structure although the buffer conditions strongly affect the radius of gyration of the protein. The results indicate that, even though a stable tetramer may not be formed, conformation of α-syn depends much on its environment, which may be the reason for its tendency to aggregate in cells.

α-synuclein (αSyn) is a protein consisting of 140 amino acid residues and is abundant in the presynaptic nerve terminals in the brain. Although its precise function is unknown, the filamentous aggregates (amyloid fibrils) of αSyn have been shown to be involved in the pathogenesis of Parkinson's disease, which is a progressive neurodegenerative disorder. To understand the pathogenesis mechanism of this disease, the mechanism of the amyloid fibril formation of αSyn must be elucidated. Purified αSyn from bacterial expression is monomeric but intrinsically disordered in solution and forms amyloid fibrils under various conditions. As a first step toward elucidating the mechanism of the fibril formation of αSyn, we investigated dynamical behavior of the purified αSyn in the monomeric state and the fibril state using quasielastic neutron scattering (QENS). We prepared the solution sample of 9.5 mg/ml purified αSyn, and that of 46 mg/ml αSyn in the fibril state, both at pD 7.4 in D2O. The QENS experiments on these samples were performed using the near-backscattering spectrometer, BL02 (DNA), at the Materials and Life Science Facility at the Japan Accelerator Research Complex, Japan. Analysis of the QENS spectra obtained shows that diffusive global motions are observed in the monomeric state but largely suppressed in the fibril state. However, the amplitude of the side chain motion is shown to be larger in the fibril state than in the monomeric state. This implies that significant solvent space exists within the fibrils, which is attributed to the αSyn molecules within the fibrils having a distribution of conformations. The larger amplitude of the side chain motion in the fibril state than in the monomeric state implies that the fibril state is entropically favorable.

Lewy bodies (LBs), which mainly consist of α-synuclein (α-syn), are neuropathological hallmarks of patients with Parkinson's disease (PD). The fine structure of LBs is unknown, and LBs cannot be made artificially. Nevertheless, many studies have described fibrillisation using recombinant α-syn purified from E. coli. An extremely fundamental problem is whether the structure of LBs is the same as that of recombinant amyloid fibrils. Thus, we used synchrotron Fourier transform infrared micro-spectroscopy (FTIRM) to analyse the fine structure of LBs in the brain of PD patients. Our results showed a shift in the infrared spectrum that indicates abundance of a β-sheet-rich structure in LBs. Also, 2D infrared mapping of LBs revealed that the content of the β-sheet structure is higher in the halo than in the core, and the core contains a large amount of proteins and lipids.

Ultrasonication can be used to break the supersaturation of α-synuclein, a protein associated with Parkinson's disease, at pH7.4 above the critical concentration of fibrillation, thereby inducing the formation of amyloid fibrils. We speculated that ultrasonication could also be used to depolymerize preformed fibrils below the critical concentration. However, extensive ultrasonic irradiation transformed preformed fibrils into amorphous aggregates even above the critical concentration. Exposing preformed fibrils to the hydrophobic air-water interface of cavitation bubbles may have destabilized the fibrils and stabilized amorphous aggregates. Upon extensive ultrasonic irradiation, the accompanying decomposition of chemical structures was suggested when monitored by analytical ultracentrifugation. Amorphous aggregates produced by extensive ultrasonication showed higher cytotoxicity, suggesting that, although ultrasonication might be a useful approach for inactivating amyloid fibrils, potential cytotoxicity of amorphous aggregates should be considered., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α-Synuclein fibrils cold-denatured to monomers at 0-20 °C and heat-denatured at 60-110 °C. Meanwhile, the fibrils of β2-microglobulin, Alzheimer's Aβ1-40/Aβ1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α-synuclein fibrils. We propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation arising from the unfavorable burial of charged side-chains in fibril cores., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

alpha-Synuclein is one of the causative proteins of the neurodegenerative disorder, Parkinson's disease. Deposits of alpha-synuclein called Lewy bodies are a hallmark of this disorder, which is implicated in its progression. In order to understand the mechanism of amyloid fibril formation of alpha-synuclein in more detail, in this study we have isolated a specific, ~20 residue peptide region of the alpha-synuclein fibril core, using a combination of Edman degradation and mass-spectroscopy analyses of protease-resistant samples. Starting from this core peptide sequence, we then synthesized a series of peptides that undergo aggregation and fibril formation under similar conditions. Interestingly, in a derivative peptide where a crucial phenylalanine residue was changed to a glycine, the ability to initiate spontaneous fibril formation was abolished, while the ability to extend from preexisting fibril seeds was conserved. This fibril extension occurred irrespective of the source of the initial fibril seed, as demonstrated in experiments using fibril seeds of insulin, lysozyme, and GroES. This interesting ability suggests that this peptide might form the basis for a possible diagnostic tool useful in detecting the presence of various fibrillogenic factors., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.