Cryo-EM structures of amyloid-β 42 filaments from human brains

Alzheimer’s disease is characterized by a loss of memory and other cognitive functions and the filamentous assembly of Aβ and tau in the brain. The assembly of Aβ peptides into filaments that end at residue 42 is a central event. Yang et al. used electron cryo–electron microscopy to determine the structures of Aβ42 filaments from human brain (see the Perspective by Willem and Fändrich). They identified two types of related S-shaped filaments, each consisting of two identical protofilaments. These structures will inform the development of better in vitro and animal models, inhibitors of Aβ42 assembly, and imaging agents with increased specificity and sensitivity. —SM

Structural studies of the in vitro assembly of tau and α-synuclein amyloids

Neurodegenerative diseases are characterised by the accumulation of filamentous protein aggregates, which are composed of amyloids. Cryo-EM studies of amyloid filaments isolated from human brains have revealed that specific conformers of tau and α-synuclein are associated with different diseases. These findings suggest that specific molecular mechanisms underlie the formation of filaments in the different diseases. However, studying the molecular mechanisms of amyloid assembly in *post mortem* brains is difficult to study. In my PhD research, I studied the molecular mechanisms of amyloid formation for tau and α-synuclein by developing in vitro amyloid assembly reactions that replicate the same structures as observed in diseased brains. My results are divided into three parts. First, I describe the seeded assembly of recombinant α-synuclein filaments with seeds from brains with Multiple System Atrophy (MSA), and show that seeded assembly does not necessarily replicate the structures of the seeds. The results in this section have important implications when interpreting seeded assembly assays. In the future, it will be important to identify the factors that determine which structures are formed in seeded aggregation experiments. Second, I focused on the *in vitro* assembly of tau. I identified truncated tau constructs, lacking the N- and C-termini, and the *in vitro* assembly conditions, which can accurately replicate disease-relevant folds observed in Alzheimer’s Disease (AD) and Chronic Traumatic Encephalopathy (CTE). These findings are the first to describe the formation of disease specific structures of any amyloid, using recombinant protein *in vitro*. The conditions identified in this study can be used for the development of high affinity binders which are specific to the AD and CTE folds. Finally, I studied the time-resolved filament formation of tau into AD and CTE filament folds. I show that tau filament formation is a step-wise and dynamic process, characterised by the formation of initial intermediate filaments, which I call First Intermediate Amyloids (FIAs), that subsequently mature into AD and CTE folds through a variety of later intermediate amyloid structures. This study is the first to provide a tangible structural characterisation of intermediates of amyloid filament formation.MR

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

Disease-specific tau filaments assemble via polymorphic intermediates.

Acknowledgements: We thank C. Charlier and F. Ferrage for providing the Mathematica script for IMPACT analysis; H. Wang and T. P. J. Knowles for helpful discussions; J. Grimmett, T. Darling and I. Clayson for help with high-performance computing; and M. Wilkinson and R. A. Crowther for critical reading of the manuscript. This work was supported by the facilities for biophysics, electron microscopy, NMR and scientific computing of the MRC Laboratory of Molecular Biology, and by the Francis Crick Institute through provision of access to the MRC Biomedical NMR Centre. The Francis Crick Institute receives its core funding from Cancer Research UK (CC1078), the UK MRC (CC1078) and the Wellcome Trust (CC1078). This work was supported by the MRC, as part of United Kingdom Research and Innovation (MC_U105184291 to M.G. and MC_UP_A025-1013 to S.H.W.S.), and a Marshall scholarship to D.L.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, β-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

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

Funder: Mochida Memorial Foundation for Medical and Pharmaceutical Research; doi: http://dx.doi.org/10.13039/501100005865The 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

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

Cleaved TMEM106B forms amyloid aggregates in central and peripheral nervous systems

Acknowledgements: We are grateful to the patients’ families for donating brain tissues. We thank Brad Glazier, Rose Richardson, Max Jacobsen, for help with neuropathology and Kieren Allinson and John Xuereb for help with neuropathological diagnosis. We are grateful to Markus Tolnay (Institute of Pathology, Department of Neuropathology of Basel University, Switzerland) for the neuropathological diagnosis and providing tissues, the Edinburgh Brain and Tissue Bank for providing brain and peripheral tissue samples from neurologically normal individuals; the Cambridge Brain Bank; the Queen Square Brain Bank; the Indiana University Dementia Laboratory Brain Library and the Parkinson’s Brain Bank, for providing tissues. The electron microscopy work on brain sections was supported by the Center for Electron Microscopy at Indiana University School of Medicine (iCEM).AbstractFilaments 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.</jats:p

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.This work was supported by the UK Dementia Research Institute, which receives its funding from DRI Ltd., funded by the UK Medical Research Council, Alzheimer’s Society, and Alzheimer’s Research. T.K. and W.A.M. have received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement 116060 (IMPRiND). This Joint Undertaking receives support from the European Union’s Horizon 2020 Research and Innovation Program and EFPIA. This work is supported by the Swiss State Secretariat for Education, Research, and Innovation (SERI) under contract 17.00038. WAM was funded by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number 206248/Z/17/Z) and by the Lister Institute for Preventative Medicine. During this work E.D. was funded by a Deutsche Forschungsgemeinschaft Research Fellowship (426806622) and an EMBO Fellowship (ALTF 173-2019). J.Y.L.L. is supported by the Croucher Foundation Limited (Hong Kong)

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

Acknowledgements: This work was supported by the Electron Microscopy Facility of the MRC Laboratory of Molecular Biology. We thank Jake Grimmett, Toby Darling and Ivan Clayson for help with high-performance computing, and Rose Richardson and Max Jacobsen for help with neuropathology. We acknowledge Diamond Light Source for access and support of the cryo-EM facilities at the UK’s Electron Bio-Imaging Centre (under proposal bi23268), funded by the Wellcome Trust, the MRC and the Biotechnology and Biological Sciences Research Council (BBSRC). M.G. is an Associate Member of the UK Dementia Research Institute. For the purpose of open access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising.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-β filaments with the Arctic mutation (E22G) from human and mouse brains.

Funder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-β (Aβ)], causes dominantly inherited Alzheimer's disease. Here, we report the high-resolution cryo-EM structures of Aβ filaments from the frontal cortex of a previously described case (AβPParc1) 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 Aβ42. When compared to the structures of wild-type Aβ42 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 Aβ molecules and promote filament formation. A minority of Aβ42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aβ 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