Ultrastructural and biochemical classification of pathogenic tau, α-synuclein and TDP-43

Abstract

Intracellular accumulation of abnormal proteins with conformational changes is the defining neuropathological feature of neurodegenerative diseases. The pathogenic proteins that accumulate in patients' brains adopt an amyloid-like fibrous structure and exhibit various ultrastructural features. The biochemical analysis of pathogenic proteins in sarkosyl-insoluble fractions extracted from patients' brains also shows disease-specific features. Intriguingly, these ultrastructural and biochemical features are common within the same disease group. These differences among the pathogenic proteins extracted from patients' brains have important implications for definitive diagnosis of the disease, and also suggest the existence of pathogenic protein strains that contribute to the heterogeneity of pathogenesis in neurodegenerative diseases. Recent experimental evidence has shown that prion-like propagation of these pathogenic proteins from host cells to recipient cells underlies the onset and progression of neurodegenerative diseases. The reproduction of the pathological features that characterize each disease in cellular and animal models of prion-like propagation also implies that the structural differences in the pathogenic proteins are inherited in a prion-like manner. In this review, we summarize the ultrastructural and biochemical features of pathogenic proteins extracted from the brains of patients with neurodegenerative diseases that accumulate abnormal forms of tau, α-synuclein, and TDP-43, and we discuss how these disease-specific properties are maintained in the brain, based on recent experimental insights.

Keywords: Prion-like propagation; Strains; Synucleinopathy; TDP-43; TDP-43 proteinopathy; Tau; Tauopathy; α-Synuclein.

Fig. 1

Ultrastructural and biochemical characterization of pathogenic tau extracted from human tauopathies. Immunohistochemistry of brain section from patients with tauopathies, stained with AT8 antibody. a Neurofibrillary tangles and neuropil threads in AD. b Pick bodies in PiD. c Tufted astrocyte in PSP. d Astrocytic plaque in CBD. e Neuronal inclusions and glial inclusions in FTDP-17T (+ 16). f Argyrophilic grains in AGD. Scale bar, 50 μm. g Immunoelectron microscopy of sarkosyl-insoluble fractions extracted from brains of tauopathy patients. Electron micrographs show fibrous structures positive for anti-tauC, after labeling with secondary antibody conjugated to 5 nm gold particles. Paired helical filaments (PHF, 10–20 nm in diameter) and straight filaments (SF, 15 nm in diameter) in AD, straight filaments (13–17 nm in diameter) in PiD, twisted ribbon-like filaments (15 nm in diameter) in PSP, twisted filaments (10–30 nm in diameter) in CBD, twisted filaments (7–25 nm in diameter) in FTDP-17T (+ 14, + 16), twisted ribbon-like filaments in GGT, and twisted filaments in AGD were observed. Scale bar, 50 nm. h Immunoblot analyses of sarkosyl-insoluble fractions prepared from brains of tauopathy patients. Sarkosyl-insoluble full-length tau (60, 64 and 68 kDa) and C-terminal fragments were detected with T46 antibody (residues 404–441). Disease-specific C-terminal fragments were also detected: 19, 22, 25, 30, 36 and 40 kDa bands in AD, 21, 34 and 39 kDa bands in PiD, 22 and 33 kDa bands in PSP and GGT, 22, 37 doublet and 43 kDa bands in CBD, FTDP-17T (+ 14, + 16) and AGD. All data are original for this review

Fig. 2

Ultrastructural and biochemical characterization of pathogenic α-syn extracted from human synucleinopathies. Immunohistochemistry of brain section from patients with synucleinopathies, stained with pS129 antibody. a Lewy bodies and Lewy neurites in DLB. b Glial cytoplasmic inclusions in MSA. Scale bar, 50 μm. c Immunoelectron microscopy of sarkosyl-insoluble fractions extracted from brains of synucleinopathy patients. Electron micrographs show fibrous structures with 5–10 nm in diameter positive for pS129, after labeling with secondary antibody conjugated to 5 nm gold particles. Straight filaments in LBD (PDD and DLB), and twisted filaments with 80–100 nm periodicity in MSA were observed. Scale bar, 50 nm. d Immunoblot analyses of sarkosyl-insoluble fractions prepared from brains of synucleinopathy patients. Sarkosyl-insoluble phosphorylated α-syn (17 kDa) was detected with pS129 antibody. Disease-specific high-molecular-weight α-syn species were also observed: 22, 29, and 37 kDa bands in LBD (PDD and DLB), and 22 and 32 kDa bands in MSA. All data are original for this review

Fig. 3

Ultrastructural and biochemical characterization of pathogenic TDP-43 extracted from human TDP-43 proteinopathies. Immunohistochemistry of brain section from patients with TDP-43 proteinopathies, stained with pS409/pS410 antibody. a Neuronal cytoplasmic inclusions (NCIs) and short degenerative neurites (DNs) in FTLD-TDP Type A. b NCIs in FTLD-TDP Type B. c Long DNs in FTLD-TDP Type C. Scale bar, 50 μm. d Immunoelectron microscopy of sarkosyl-insoluble fractions extracted from FTLD-TDP (type A, type B, type C) patients. Electron micrographs show fibrous structures with 10–15 nm in diameter positive for pS409/pS410, after labeling with secondary antibody conjugated to 5 nm gold particles. Scale bar, 50 nm. e Immunoblot analyses of sarkosyl-insoluble fractions prepared from brains of patients withTDP-43 proteinopathies. Sarkosyl-insoluble phosphorylated TDP-43 (45 kDa) was detected with pS409/410 antibody. Subtype-specific C-terminal fragments were also observed: 18, 19, 23, 24 and 26 kDa bands in type A, 18, 19, 23, 24 and 26 kDa bands in type B, 18, 19, 23 and 24 kDa bands in type C. All data are original for this review

Fig. 4

Full matching of amino acid sequence between template and substrate is essential for template-dependent amplification of tau filaments. Fibrous core regions of tau filaments extracted from brains of AD, PiD, PSP and CBD patients identified by cryogenic electron microscopic studies explain the templated tau amplification and tau filament formation observed in our cellular model. The core region of AD-tau (G273-E380 in 3R tau and G304-E380 in 4R tau), which recruits both 3R tau and 4R tau for seeded aggregation, consists of amino acid sequences common to 3R tau and 4R tau. On the other hand, PiD-tau, PSP-tau and CBD-tau consist of 3R tau or 4R tau specific amino acid sequences: K254-F378 in 3R tau, G272-N381 in 4R tau and K274-E380 in 4R tau. Therefore, PiD-tau, PSP-tau and CBD-tau recruit only tau substrates that match the template for seeded aggregation, and do not induce aggregation when the template and substrate are mismatched. The amplification and intracerebral expansion of tau filaments with the same structure by this mechanism leads to the pathological diversity of tauopathies

Fig. 5

Strain formation and cell-to-cell transmission in neurodegenerative diseases. a Prion-like proteins, including tau, α-syn and TDP-43, can adopt various misfolded forms. The variety of misfolding is caused by genetic and/or environmental factors, resulting in the formation of strains with distinct conformations. Disease-associated mutations alter the core structure of filaments and the interaction between two protofilaments. Differences in the cellular environment between neuronal and glial cells may also contribute to the various types of misfolding. The interaction of prion-like proteins with cell type-specific co-factors during misfolding would lead to the formation of disease-specific filaments. Post-translational modifications, interactions with other proteins, lipids and nucleic acids, as well as differences in salt concentration, pH and metals, may also be involved in the formation of distinct strains. b Pathogenic proteins amplified and accumulated in cells have proposed to transmit as seeds from cell to cell and then spread throughout the brain. Possible mechanisms of cell-to-cell transmission include extracellular release of pathogenic seeds via exocytosis or in synaptic vesicles or exosomes, followed by incorporation into neighboring cells either directly or via macropinocytosis or receptor-mediated endocytosis. Alternatively pathogenic seeds may be taken up into cells by cell membrane fusion of exosomes containing seeds. Cell-to-cell transmission of pathogenic seeds via tunneling nanotubes has also been suggested