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 different 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.

Tau in Health and Neurodegenerative Diseases

Tau, one of the major microtubule-associated proteins, modulates the dynamic properties of microtubules in the mammalian nervous system. Tau is abundantly expressed in the brain, particularly in the hippocampus. Insoluble and filamentous inclusions of tau in neurons or glia are discovered in neurodegenerative diseases termed ‘tauopathies’, including Alzheimer’s disease (AD), argyrophilic grain disease (AGD), corticobasal degeneration (CBD), frontotemporal dementia (FTD), Pick’s disease (PiD) and progressive supranuclear palsy (PSP). Accumulation of intracellular neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated tau, is directly correlated with the degree of Alzheimer\'s dementia. This chapter reviews the role of tau protein in physiological conditions and the pathological changes of tau related to neurodegenerative diseases. The applications of tau as a therapeutic target are also discussed.

Age-dependent formation of TMEM106B amyloid filaments in human brain

Many age-dependent neurodegenerative diseases, like Alzheimer's and Parkinson's, are characterised by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-β (Aβ), α-synuclein and TDP-43 are the most common. Here, we used electron cryo-microscopy (cryo-EM) structure determination to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in the human brain. We solved cryo-EM structures of TMEM106B filaments from the brains of 22 individuals with neurodegenerative conditions, including sporadic and inherited tauopathies, Aβ-amyloidoses, synucleinopathies and TDP-43opathies, as well as from the brains of two neurologically normal individuals. We observed three different TMEM106B folds, with no clear relationship between folds and diseases. The presence of TMEM106B filaments correlated with that of a 29 kDa sarkosyl-insoluble fragment of the protein on Western blots. The presence of TMEM106B filaments in the brains of older, but not younger, neurologically normal individuals indicates that they form in an age-dependent manner.

Saturated Proteostasis and Nuclear Injuries Defeat Homeostatic Potentials of α-Synuclein Filaments

Biogenesis of inclusion bodies (IBs) contributes to protein quality control (PQC). Perinuclear IBs like aggresomes/JUNQs serve as sites for ubiquitin-proteasome mediated protein degradation. The other canonical IB, IPOD, does not degrade but sequesters non-ubiquitinated terminally aggregated proteins to prevent their promiscuous interactions and interferences with other cellular functions. Here, we show that as a deviation from this convention, misfolding-prone α-Synuclein is simultaneously deposited into two distinct IBs - Syn-aggresomes and seeding based filamentous inclusions (Syn-filaments), both acting as sites for ubiquitin-proteasome mediated protein degradation. Syn-aggresomes buffer the spontaneous turnover of α-Synuclein. Syn-filaments serve the dual purpose of self-sequestration and opportunistic degradation. Counterintuitively, overgrowth of perinuclear Syn-filaments titrates out cellular PQC-pool and challenges the turnover and solubility of other misfolding-prone proteins. Moreover, large Syn-filaments associate with LaminB1, mount mechanical stress on nuclear envelope via dynein, disrupt nuclear integrity, and deregulate stress-triggered transcription of chaperones failing their homeostatic potential.

Structures of α-synuclein filaments from multiple system atrophy

Synucleinopathies are human neurodegenerative diseases that include multiple system atrophy (MSA), Parkinson’s disease, Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB) (1). Existing treatments are at best symptomatic. These diseases are characterised by the presence in brain cells of filamentous inclusions of α-synuclein, the formation of which is believed to cause disease (2, 3). However, the structures of α-synuclein filaments from human brain are not known. Here we show, using electron cryo-microscopy, that α-synuclein inclusions from MSA are made of two types of filaments, each of which consists of two different protofilaments. Non-proteinaceous molecules are present at the protofilament interfaces. By two-dimensional class averaging, we show that α-synuclein filaments from the brains of patients with MSA and DLB are different, suggesting that distinct conformers (or strains) characterise synucleinopathies. As was the case of tau assemblies (4–9), the structures of α-synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, with implications for understanding the mechanisms of aggregate propagation and neurodegeneration in human brain. These findings have diagnostic and potential therapeutic relevance, especially in view of the unmet clinical need to be able to image filamentous α-synuclein inclusions in human brain.

Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer’s and Pick’s diseases

Assembly of microtubule-associated protein tau into filamentous inclusions underlies a range of neurodegenerative diseases. Tau filaments adopt different conformations in Alzheimer’s and Pick’s diseases. Here, we used cryo- and immuno- electron microscopy to characterise filaments that were assembled from recombinant full-length human tau with four (2N4R) or three (2N3R) microtubule-binding repeats in the presence of heparin. 2N4R tau assembles into multiple types of filaments, and the structures of three types reveal similar ‘kinked hairpin’ folds, in which the second and third repeats pack against each other. 2N3R tau filaments are structurally homogeneous, and adopt a dimeric core, where the third repeats of two tau molecules pack in a parallel manner. The heparin-induced tau filaments differ from those of Alzheimer’s or Pick’s disease, which have larger cores with different repeat compositions. Our results illustrate the structural versatility of amyloid filaments, and raise questions about the relevance of in vitro assembly.

Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer’s and Pick’s diseases

The assembly of microtubule-associated protein tau into abundant filamentous inclusions underlies a range of neurodegenerative diseases. The finding that tau filaments adopt different conformations in Alzheimer’s and Pick’s diseases raises the question of what kinds of structures of tau filaments form in vitro. Here, we used electron cryo-microscopy (cryo-EM) and negative-stain immuno-gold electron microscopy (immuno-EM) to characterise filaments that were assembled from recombinant full-length human tau with four (2N4R) or three (2N3R) microtubule-binding repeats in the presence of heparin. 4R tau assembles into at least four different types of filaments. Cryo-EM structures of three types of 4R filaments reveal similar “kinked hairpin” folds, in which the second and third repeats pack against each other. 3R tau filaments are structurally homogeneous, and adopt a dimeric core, where the third repeats of two tau molecules pack against each other in a parallel, yet asymmetric, manner. None of the heparin-induced tau filaments resemble those of Alzheimer’s or Pick’s disease, which have larger cores with different repeat compositions. Our results indicate that tau filaments are structurally versatile, and raise questions about the relevance of in vitro assembled amyloids.