α-Synucleinのプリオン様伝播に関する研究

Intercellular abnormal protein aggregates are a common feature of many neurodegenerative diseases. Misfolded proteins are accumulated in neurons and/or glial cells as a fibriller, phosphorylated and partially ubiquitinated forms. The distribution and spreading of these abnormal proteins in brains of patients are shown to correlate with clinical presentation and disease progression. Recent experimental findings support the concept that structurally changed abnormal proteins convert normal proteins into abnormal forms and spread into neighboring cells in a prion-like manner. This prion-like conversion may account for not only the onset but also the progression of neurodegenerative diseases. α-Synuclein (α-syn) is a 140 amino acid protein that is normally located in presynaptic nerve terminals. In 1997, missense mutation in the α-syn gene was discovered in families of Parkinson’s disease (PD), and subsequent immunohistochemical work with anti-α-syn antibodies revealed that α-syn is the major component of Lewy bodies in PD and dementia with Lewy bodies (DLB), and also glial cytoplasmic inclusions (GCIs) in multiple system atrophy. Purified recombinant α-syn assembles into amyloid-like fibrils that share common properties with the fibrils present in pathological human brains in vitro. Preformed α-syn fibrils and brain extracts from α-synucleinopathies’ patients can work as seeds to induce seed-dependent aggregation in cultured cells. Furthermore, injection of preformed α-syn fibrils into brains of wild-type or transgenic mice overexpressing mutant human α-syn led to development of phosphorylated α-syn pathology several months later. However, it has not been fully elucidated what structures of α-syn molecules (fibrils or oligomers, soluble or insoluble) are the most pathogenic, what sizes of these molecules are the most effective, and what mechanisms underlie the cell-to-cell spreading. In this study, I prepared various kinds of α-syn aggregates or intermediates under various conditions and tested their prion-like properties in vitro, in cells, and in mouse experimental models. I found that only fibril form of α-syn induced seed-dependent aggregation of α-syn in cultured cells and in wild-type mouse brains. I also found that sonication of α-syn fibrils accelerate accumulation of phosphorylated α-syn, indicating that fragmented β-sheet rich fibrous structures of α-syn efficiently induce seed-dependent aggregation and prion-like propagation of pathological α-syn in culture cells and wild-type mouse brains. I also characterized these α-syn species by transmission electron microscopy observations and thioflavin fluorescence assays and found that fibrous structures similar to those observed in α-synucleinopathy brains are formed in α-syn fibril-injected mouse brain and that fibrils containing sarkosyl-insoluble fractions extracted from injected mice functioned as seeds for seed-dependent aggregation of α-syn. These results indicate that fragmented amyloid-like α-syn fibrils less than 50 nm in size are the most effective seeds that triggered prion-like conversion. These results may contribute to understand the molecular mechanisms of neurodegenerative diseases. These cellular and mouse models of α-syn propagation should be useful for screening and evaluation of disease-modifying drugs of α-synucleinopathies.首都大学東京, 2017-03-25, 博士（理学）首都大学東

Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

The concept that abnormal protein aggregates show prion-like propagation between cells has been considered to explain the onset and progression of many neurodegenerative diseases. Indeed, both synthetic amyloid-like fibrils and pathogenic proteins extracted from patients’ brains induce self-templated amplification and cell-to-cell transmission in vitro and in vivo. However, it is unclear whether exposure to exogenous prion-like proteins can potentially cause these diseases in humans. Here, we investigated in detail the prion-like seeding activities of several kinds of pathogenic α-synuclein (α-syn), including synthetic fibrils and detergent-insoluble fractions extracted from brains of patients with α-synucleinopathies. Exposure to synthetic α-syn fibrils at concentrations above 100 pg/mL caused seeded aggregation of α-syn in SH-SY5Y cells, and seeded aggregation was also observed in C57BL/6 J mice after intracerebral inoculation of at least 0.1 μg/animal. α-Syn aggregates extracted from brains of multiple system atrophy (MSA) patients showed higher seeding activity than those extracted from patients with dementia with Lewy bodies (DLB), and their potency was similar to that of synthetic α-syn fibrils. We also examined the effects of various methods that have been reported to inactivate abnormal prion proteins (PrPSc), including autoclaving at various temperatures, exposure to sodium dodecyl sulfate (SDS), and combined treatments. The combination of autoclaving and 1% SDS substantially reduced the seeding activities of synthetic α-syn fibrils and α-syn aggregates extracted from MSA brains. However, single treatment with 1% SDS or generally used sterilization conditions proved insufficient to prevent accumulation of pathological α-syn. In conclusion, α-syn aggregates derived from MSA patients showed a potent prion-like seeding activity, which could be efficiently reduced by combined use of SDS and autoclaving

Biochemical classification of tauopathies by immunoblot, protein sequence and mass spectrometric analyses of sarkosyl-insoluble and trypsin-resistant tau

Intracellular filamentous tau pathology is the defining feature of tauopathies, which form a subset of neurodegenerative diseases. We have analyzed pathological tau in Alzheimer’s disease, and in frontotemporal lobar degeneration associated with tauopathy to include cases with Pick bodies, corticobasal degeneration, progressive supranuclear palsy, and ones due to intronic mutations in MAPT. We found that the C-terminal band pattern of the pathological tau species is distinct for each disease. Immunoblot analysis of trypsin-resistant tau indicated that the different band patterns of the 7–18 kDa fragments in these diseases likely reflect different conformations of tau molecular species. Protein sequence and mass spectrometric analyses revealed the carboxyl-terminal region (residues 243–406) of tau comprises the protease-resistant core units of the tau aggregates, and the sequence lengths and precise regions involved are different among the diseases. These unique assembled tau cores may be used to classify and diagnose disease strains. Based on these results, we propose a new clinicopathological classification of tauopathies based on the biochemical properties of tau

Novel tau filament fold in corticobasal degeneration

Corticobasal degeneration (CBD) is a neurodegenerative tauopathy that is characterised by motor and cognitive disturbances (1–3). A higher frequency of the H1 haplotype of MAPT, the tau gene, is present in cases of CBD than in controls (4,5) and genome-wide association studies have identified additional risk factors (6). By histology, astrocytic plaques are diagnostic of CBD (7,8), as are detergent-insoluble tau fragments of 37 kDa by SDS-PAGE (9). Like progressive supranuclear palsy (PSP), globular glial tauopathy (GGT) and argyrophilic grain disease (AGD) (10), CBD is characterised by abundant filamentous tau inclusions that are made of isoforms with four microtubule-binding repeats (4R) (11–15). This distinguishes 4R tauopathies from Pick’s disease, filaments of which are made of three-repeat (3R) tau isoforms, and from Alzheimer’s disease and chronic traumatic encephalopathy (CTE), where both 3R and 4R tau isoforms are found in the filaments (16). Here we report the structures of tau filaments extracted from the brains of three individuals with CBD using electron cryo-microscopy (cryo-EM). They were identical between cases, but distinct from those of Alzheimer’s disease, Pick’s disease and CTE (17–19). The core of CBD filaments comprises residues K274-E380 of tau, spanning the last residue of R1, the whole of R2, R3 and R4, as well as 12 amino acids after R4. It adopts a novel four-layered fold, which encloses a large non-proteinaceous density. The latter is surrounded by the side chains of lysine residues 290 and 294 from R2 and 370 from the sequence after R4. CBD is the first 4R tauopathy with filaments of known structure

Structure-based classification of tauopathies

Ordered assembly of tau protein into filaments characterizes multiple neurodegenerative diseases, which are called tauopathies. We previously reported that by electron cryo-microscopy (cryo-EM), tau filament structures from Alzheimer’s disease (1,2), Pick’s disease (3), chronic traumatic encephalopathy (CTE) (4) and corticobasal degeneration (CBD) (5) are distinct. Here we show that the structures of tau filaments from progressive supranuclear palsy (PSP) define a novel three-layered fold. Moreover, the tau filament structures from globular glial tauopathy (GGT) are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs from the above and resembles the four-layered CBD fold. The AGD fold is also observed in aging-related tau astrogliopathy (ARTAG). Tau protofilament structures from inherited cases with mutations +3 or +16 in intron 10 of MAPT, the microtubule-associated protein tau gene, are also identical to those from AGD, suggesting that relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, tau filament structures from cases of familial British dementia (FBD) and familial Danish dementia (FDD) are the same as those from Alzheimer’s disease and primary age-related tauopathy (PART). These findings suggest a hierarchical classification of tauopathies based on their filament folds, which complements clinical diagnosis and neuropathology, and allows identification of new entities, as we show for a case diagnosed as PSP, but with filament structures that are intermediate between those of GGT and PSP

N-Methyl-D-Aspartate Receptor Link to the MAP Kinase Pathway in Cortical and Hippocampal Neurons and Microglia Is Dependent on Calcium Sensors and Is Blocked by α-Synuclein, Tau, and Phospho-Tau in Non-transgenic and Transgenic APPSw,Ind Mice

N-methyl-D-aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK- and Ca2+-mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APPSw,Ind transgenic Alzheimer’s disease model. Interestingly, a very marked increase in NMDAR–NCS1 complexes was identified in neurons and a marked increase of both NMDAR–NCS1 and NMDAR–CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the signaling of NMDAR to the MAPK pathway and that calcium sensors are important for NMDAR function both in neurons and microglia. Finally, it should be noted that the expression of receptor–calcium sensor complexes, specially those involving NCS1, is altered in neural cells from APPSw,Ind mouse embryos/pups

Additional file 1: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S1. Determination of protein concentration of phosphorylated α-syn in patients’ brains. A, Sarkosyl-insoluble fractions prepared from patients’ brains used in this study were analyzed by immunoblotting with anti-phosphorylated α-syn PSer129 antibody (upper) and anti-tau T46 antibody (lower). B, Standard curve of phosphorylated α-syn, generated by immunoblotting of phosphorylated monomer α-syn. Concentrations of phosphorylated α-syn were determined using this standard curve. Protein concentrations of sarkosyl-insoluble fractions extracted from patients’ brains are shown in Table S2. (PDF 139 kb

Additional file 5: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Table S2. Numbers of mice used in experiments. (PDF 52 kb