Disease-specific tau filaments assemble via polymorphic intermediates.

Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, ß-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.

Age-dependent formation of TMEM106B amyloid filaments in human brains.

Many age-dependent neurodegenerative diseases, such as Alzheimer's and Parkinson's, are characterized by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-ß, α-synuclein and transactive response DNA-binding protein (TARDBP; also known as TDP-43) are the most common1,2. Here we used structure determination by cryogenic electron microscopy to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in human brains. We determined the structures of TMEM106B filaments from a number of brain regions of 22 individuals with abundant amyloid deposits, including those resulting from sporadic and inherited tauopathies, amyloid-ß amyloidoses, synucleinopathies and TDP-43 proteinopathies, as well as from the frontal cortex of 3 individuals with normal neurology and no or only a few amyloid deposits. We observed three TMEM106B folds, with no clear relationships between folds and diseases. TMEM106B filaments correlated with the presence of a 29-kDa sarkosyl-insoluble fragment and globular cytoplasmic inclusions, as detected by an antibody specific to the carboxy-terminal region of TMEM106B. The identification of TMEM106B filaments in the brains of older, but not younger, individuals with normal neurology indicates that they form in an age-dependent manner.

Data-driven regularization lowers the size barrier of cryo-EM structure determination.

Macromolecular structure determination by electron cryo-microscopy (cryo-EM) is limited by the alignment of noisy images of individual particles. Because smaller particles have weaker signals, alignment errors impose size limitations on its applicability. Here, we explore how image alignment is improved by the application of deep learning to exploit prior knowledge about biological macromolecular structures that would otherwise be difficult to express mathematically. We train a denoising convolutional neural network on pairs of half-set reconstructions from the electron microscopy data bank (EMDB) and use this denoiser as an alternative to a commonly used smoothness prior. We demonstrate that this approach, which we call Blush regularization, yields better reconstructions than do existing algorithms, in particular for data with low signal-to-noise ratios. The reconstruction of a protein-nucleic acid complex with a molecular weight of 40 kDa, which was previously intractable, illustrates that denoising neural networks will expand the applicability of cryo-EM structure determination for a wide range of biological macromolecules.

New SNCA mutation and structures of α-synuclein filaments from juvenile-onset synucleinopathy.

A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36-100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.

Cryo-EM structures of amyloid-ß filaments with the Arctic mutation (E22G) from human and mouse brains.

The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-ß (Aß)], causes dominantly inherited Alzheimer's disease. Here, we report the high-resolution cryo-EM structures of Aß filaments from the frontal cortex of a previously described case (AßPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12-V40 (human Arctic fold A) and E11-G37 (human Arctic fold B). They have a substructure (residues F20-G37) in common with the folds of type I and type II Aß42. When compared to the structures of wild-type Aß42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aß molecules and promote filament formation. A minority of Aß42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aß filaments with the Arctic mutation from mouse knock-in line AppNL-G-F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL-G-F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL-G-F murine Arctic fold differs from the human Arctic folds, but shares some substructure.

High-throughput cryo-EM structure determination of amyloids.

The formation of amyloid filaments is characteristic of various degenerative diseases. Recent breakthroughs in electron cryo-microscopy (cryo-EM) have led to atomic structure determination of multiple amyloid filaments, both of filaments assembled in vitro from recombinant proteins, and of filaments extracted from diseased tissue. These observations revealed that a single protein may adopt multiple different amyloid folds, and that in vitro assembly does not necessarily lead to the same filaments as those observed in disease. In order to develop relevant model systems for disease, and ultimately to better understand the molecular mechanisms of disease, it will be important to determine which factors determine the formation of distinct amyloid folds. High-throughput cryo-EM, in which structure determination becomes a tool rather than a project in itself, will facilitate the screening of large numbers of in vitro assembly conditions. To this end, we describe a new filament picking algorithm based on the Topaz approach, and we outline image processing strategies in Relion that enable atomic structure determination of amyloids within days.

Cleaved TMEM106B forms amyloid aggregates in central and peripheral nervous systems.

Filaments made of residues 120-254 of transmembrane protein 106B (TMEM106B) form in an age-dependent manner and can be extracted from the brains of neurologically normal individuals and those of subjects with a variety of neurodegenerative diseases. TMEM106B filament formation requires cleavage at residue 120 of the 274 amino acid protein; at present, it is not known if residues 255-274 form the fuzzy coat of TMEM106B filaments. Here we show that a second cleavage appears likely, based on staining with an antibody raised against residues 263-274 of TMEM106B. We also show that besides the brain TMEM106B inclusions form in dorsal root ganglia and spinal cord, where they were mostly found in non-neuronal cells. We confirm that in the brain, inclusions were most abundant in astrocytes. No inclusions were detected in heart, liver, spleen or hilar lymph nodes. Based on their staining with luminescent conjugated oligothiophenes, we confirm that TMEM106B inclusions are amyloids. By in situ immunoelectron microscopy, TMEM106B assemblies were often found in structures resembling endosomes and lysosomes.

Tau filaments with the Alzheimer fold in cases with MAPT mutations V337M and R406W.

Frontotemporal dementia (FTD) and Alzheimer's disease are the most common forms of early-onset dementia. Dominantly inherited mutations in MAPT, the microtubule-associated protein tau gene, cause FTD and parkinsonism linked to chromosome 17 (FTDP-17). Individuals with FTDP-17 develop abundant filamentous tau inclusions in brain cells. Here we used electron cryo-microscopy to determine the structures of tau filaments from the brains of individuals with MAPT mutations V337M and R406W. Both mutations gave rise to tau filaments with the Alzheimer fold, which consisted of paired helical filaments in all V337M and R406W cases and of straight filaments in two V337M cases. We also identified a new assembly of the Alzheimer fold into triple tau filaments in a V337M case. Filaments assembled from recombinant tau(297-391) with mutation V337M had the Alzheimer fold and showed an increased rate of assembly.

Super-resolution imaging unveils the self-replication of tau aggregates upon seeding.

Tau is a soluble protein interacting with tubulin to stabilize microtubules. However, under pathological conditions, it becomes hyperphosphorylated and aggregates, a process that can be induced by treating cells with exogenously added tau fibrils. Here, we employ single-molecule localization microscopy to resolve the aggregate species formed in early stages of seeded tau aggregation. We report that entry of sufficient tau assemblies into the cytosol induces the self-replication of small tau aggregates, with a doubling time of 5 h inside HEK cells and 1 day in murine primary neurons, which then grow into fibrils. Seeding occurs in the vicinity of the microtubule cytoskeleton, is accelerated by the proteasome, and results in release of small assemblies into the media. In the absence of seeding, cells still spontaneously form small aggregates at lower levels. Overall, our work provides a quantitative picture of the early stages of templated seeded tau aggregation in cells.

Cryo-EM structures of tau filaments from SH-SY5Y cells seeded with brain extracts from cases of Alzheimer's disease and corticobasal degeneration.

The formation of amyloid filaments through templated seeding is believed to underlie the propagation of pathology in most human neurodegenerative diseases. A widely used model system to study this process is to seed amyloid filament formation in cultured cells using human brain extracts. Here, we report the electron cryo-microscopy structures of tau filaments from  undifferentiated seeded SH-SY5Y cells that transiently expressed N-terminally HA-tagged 1N3R or 1N4R human tau, using brain extracts from individuals with Alzheimer's disease or corticobasal degeneration. Although the resulting filament structures differed from those of the brain seeds, some degrees of structural templating were observed. Studying templated seeding in cultured cells, and determining the structures of the resulting filaments, can thus provide insights into the cellular aspects underlying neurodegenerative diseases.

Assembly of recombinant tau into filaments identical to those of Alzheimer's disease and chronic traumatic encephalopathy.

Abundant filamentous inclusions of tau are characteristic of more than 20 neurodegenerative diseases that are collectively termed tauopathies. Electron cryo-microscopy (cryo-EM) structures of tau amyloid filaments from human brain revealed that distinct tau folds characterise many different diseases. A lack of laboratory-based model systems to generate these structures has hampered efforts to uncover the molecular mechanisms that underlie tauopathies. Here, we report in vitro assembly conditions with recombinant tau that replicate the structures of filaments from both Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE), as determined by cryo-EM. Our results suggest that post-translational modifications of tau modulate filament assembly, and that previously observed additional densities in AD and CTE filaments may arise from the presence of inorganic salts, like phosphates and sodium chloride. In vitro assembly of tau into disease-relevant filaments will facilitate studies to determine their roles in different diseases, as well as the development of compounds that specifically bind to these structures or prevent their formation.

Many neurodegenerative diseases, including Alzheimer's disease, the most common form of dementia, are characterised by knotted clumps of a protein called tau. In these diseases, tau misfolds, stacks together and forms abnormal filaments, which have a structured core and fuzzy coat. These sticky, misfolded proteins are thought to be toxic to brain cells, the loss of which ultimately causes problems with how people move, think, feel or behave. Reconstructing the shape of tau filaments using an atomic-level imaging technique called electron cryo-microscopy, or cryo-EM, researchers have found distinct types of tau filaments present in certain diseases. In Alzheimer's disease, for example, a mixture of paired helical and straight filaments is found. Different tau filaments are seen again in chronic traumatic encephalopathy (CTE), a condition associated with repetitive brain trauma. It remains unclear, however, how tau folds into these distinct shapes and under what conditions it forms certain types of filaments. The role that distinct tau folds play in different diseases is also poorly understood. This is largely because researchers making tau proteins in the lab have yet to replicate the exact structure of tau filaments found in diseased brain tissue. Lövestam et al. describe the conditions for making tau filaments in the lab identical to those isolated from the brains of people who died from Alzheimer's disease and CTE. Lövestam et al. instructed bacteria to make tau protein, optimised filament assembly conditions, including shaking time and speed, and found that bona fide filaments formed from shortened versions of tau. On cryo-EM imaging, the lab-produced filaments had the same left-handed twist and helical symmetry as filaments characteristic of Alzheimer's disease. Adding salts, however, changed the shape of tau filaments. In the presence of sodium chloride, otherwise known as kitchen salt, tau formed filaments with a filled cavity at the core, identical to tau filaments observed in CTE. Again, this structure was confirmed on cryo-EM imaging. Being able to make tau filaments identical to those found in human tauopathies will allow scientists to study how these filaments form and elucidate what role they play in disease. Ultimately, a better understanding of tau filament formation could lead to improved diagnostics and treatments for neurodegenerative diseases involving tau.

Cryo-EM structures of amyloid-ß 42 filaments from human brains.

Filament assembly of amyloid-ß peptides ending at residue 42 (Aß42) is a central event in Alzheimer's disease. Here, we report the cryo­electron microscopy (cryo-EM) structures of Aß42 filaments from human brains. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains of individuals with sporadic Alzheimer's disease, and type II filaments were found in individuals with familial Alzheimer's disease and other conditions. The structures of Aß42 filaments from the brain differ from those of filaments assembled in vitro. By contrast, in AppNL-F knock-in mice, Aß42 deposits were made of type II filaments. Knowledge of Aß42 filament structures from human brains may lead to the development of inhibitors of assembly and improved imaging agents.

Seeded assembly in vitro does not replicate the structures of α-synuclein filaments from multiple system atrophy.

The propagation of conformational strains by templated seeding is central to the prion concept. Seeded assembly of α-synuclein into filaments is believed to underlie the prion-like spreading of protein inclusions in a number of human neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We previously determined the atomic structures of α-synuclein filaments from the putamen of five individuals with MSA. Here, we used filament preparations from three of these brains for the in vitro seeded assembly of recombinant human α-synuclein. We find that the structures of the seeded assemblies differ from those of the seeds, suggesting that additional, as yet unknown, factors play a role in the propagation of the seeds. Identification of these factors will be essential for understanding the prion-like spreading of α-synuclein proteinopathies.