The Cryo-EM Effect: Structural Biology of Neurodegenerative Disease Aggregates

Abstract

Neurogenerative diseases are characterized by diverse protein aggregates with a variety of microscopic morphologic features. Although ultrastructural studies of human neurodegenerative disease tissues have been conducted since the 1960s, only recently have near-atomic resolution structures of neurodegenerative disease aggregates been described. Solid-state nuclear magnetic resonance spectroscopy and X-ray crystallography have provided near-atomic resolution information about in vitro aggregates but pose logistical challenges to resolving the structure of aggregates derived from human tissues. Recent advances in cryo-electron microscopy (cryo-EM) have provided the means for near-atomic resolution structures of tau, amyloid-β (Aβ), α-synuclein (α-syn), and transactive response element DNA-binding protein of 43 kDa (TDP-43) aggregates from a variety of diseases. Importantly, in vitro aggregate structures do not recapitulate ex vivo aggregate structures. Ex vivo tau aggregate structures indicate individual tauopathies have a consistent aggregate structure unique from other tauopathies. α-syn structures show that even within a disease, aggregate heterogeneity may correlate to disease course. Ex vivo structures have also provided insight into how posttranslational modifications may relate to aggregate structure. Though there is less cryo-EM data for human tissue-derived TDP-43 and Aβ, initial structural studies provide a basis for future endeavors. This review highlights structural variations across neurodegenerative diseases and reveals fundamental differences between experimental systems and human tissue derived protein inclusions.

Keywords: Alzheimer disease; Amyotrophic lateral sclerosis; Frontotemporal degeneration; Lewy body; Multiple system atrophy; Parkinson disease; Tauopathy.

FIGURE 1.

Tau fibril structures, common fragments used of in vitro tau aggregation, and the cores that compose fibrils. (A) The domains of full-length tau showing the N-terminal extension (red) that can have an N1 (orange) or N2 (yellow) insert, proline rich region (green), microtubule binding domain (pink/purple) that can have 3 or 4 repeats, and the C-terminal extension (blue). The common fragments K18 and K11 are shown with the PHF6 peptide, thought to be a nucleus of aggregation, labeled. The amino acids that make up the core of ex vivo fibrils are shown with the different cores that compose in vitro tau fibril structures that have been confirmed by cryo-EM. (B) The paired helical filament (PHF) tau fibril core structure that is found in brains of Alzheimer disease (AD) patients (PDB: 5O3L, EMDB: 3741). (C) The straight filament (SF) tau fibril core structure that is found in the brain of AD patients. It is less prevalent and less twisted than the PHF (PDB: 5O3T, EMDB: 3743). (D) The core folds of different tau protofilaments that have been solved by cryo-EM (PDB: 5O3L, 6GX5, 6NWP, 6TJX, 6QJH, 6QJM, 6QJP, 6QJQ) with the PHF6 (black amino acids) peptide that is thought be a nucleus of aggregation shown in the core.

FIGURE 2.

Ex vivo and in vitro Aβ fibril structures that have been solved by cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance spectroscopy (ssNMR). (A) The 3-fold symmetry structure from “patient 1” ex vivo-seeded fibril. These fibrils were formed by seeded aggregation with recombinant amyloid-β (Aβ) and ex vivo fibrils (PDB: 2M4J). (B) The atomic structure of Morphology I that is currently the only Aβ structure solved of ex vivo fibrils. The density of Morphology II and III are shown with the atomic structure of Morphology I fit into the density, as Morphology II and III are thought to be multiples of Morphology I (PDB: 6SHS, EMDB: 4864, 4866). (C) The right-handedness of the only ex vivo fibril and an example of left-handedness of an in vitro fibril. The ex vivo fibril is the only structure that has been shown to be right-handed, as all of the in vitro structures are left-handed (PDB: 6SHS, 5OQV). (D) The variety of fibril structures of Aβ(1–40) and Aβ(1–42) in vitro fibrils and how their structures are different than that of Morphology I (PDB: 2LMN, 2LMP, 2MVX, 2MPZ, 5AEF, 5KK3, 2NAO, 5OQV).

FIGURE 3.

Ex vivo and in vitro α-synuclein (α-syn) fibril structures and representative α-syn histology. (A) The domains of full-length α-syn, showing the N-terminal domain (red), non-Aβ component (NAC) (blue), and C-terminal tail (pink). Posttranslational modifications, specifically ubiquitination and phosphorylation, that may play a role in α-syn aggregation and disaggregation are labeled (above the domains diagram). Disease-related familial mutations of α-syn are also indicated (below the domains diagram). (B) The image shows a pigmented substantia nigra neuron with a Lewy body (left) adjacent to a normal neuron. Extracellular pigment is also present (right) indicative of neurodegeneration. Lewy bodies are found in a variety of neurodegenerative diseases including dementia with Lewy bodies and Parkinson disease (PD). The right image shows glial cytoplasmic inclusions that are found in multiple system atrophy (MSA). Both Lewy bodies and glial cytoplasmic inclusions contain α-syn. (C) Type I MSA fibril that is made of protofilament IA (light blue) and protofilament IB (dark blue) with its electron density map overlaid. The positively charged cavity made-up of K43, K45, and H50 from both protofilaments (yellow) likely contains a negatively charged nonproteinaceous material. K80 and K60 are both sites of ubiquitination, with K60 only being solvent exposed in IA, not IB. T72 is a site of phosphorylation that is buried in both IA and IB (PDB: 6XYO, EMD: 10650). (D) Type II MSA fibrils with electron density map from Type II₁ fibrils (fibrils with protofilament IIB₁ as opposed to protofilament IIB₂). Type II fibrils are each made of an IIA (light purple) and IIB (dark purple) protofilament. Protofilament IIB₁ and IIB₂ are overlaid and labeled as protofilament IIB. The fibrils have a positively charged cavity made by K43, K45, and H50 of both protofilaments that likely contains a negatively charged nonproteinaceous material. K80 and K60 are both sites of ubiquitnations, with K60 only being solvent exposed in protofilament IIA, not IIB. T72 is a site of phosphorylation that is found in a cavity in protofilament IIA, as opposed to being buried in protofilament IA (PDB: 6XYP, 6XYQ, EMD: 10651). (E) The K80-A91 region of Type IIB₁ and Type IIB₂ protofilaments that has a slightly different conformation. T81 phosphorylation may be what drives protofilaments to take either the IIB₁ or IIB₂ conformation as it is only solvent exposed in IIB₂. (F) The variety of in vitro α-syn fibril structures that mostly differ from the ex vivo MSA fibrils.

FIGURE 4.

In vitro transactive response element DNA-binding protein of 43 kDa (TDP-43) structures of fragments of the low complexity domain (LCD) and the full TDP-43 protein. (A) The domains of full-length TDP-43 including the N-terminal domain (NTD) (red), nuclear localization signal (NLS) (green), RNA recognition motif 1 (RRM1) (purple), RRM2 (pink), and LCD (blue) with SegA and SegB from the LCD indicated. (B) The core structures of in vitro fibrils that have been solved for SegA and SegB. SegA had 3 distinct fibrils, while SegB had one fibril conformation (PDB: 6N37, 6N3B, 6N3A, 6N3C).