A new era for understanding amyloid structures and disease

Abstract

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-β structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention.

Fig. 1. Progression of amyloid structure research over close to 400 years that has culminated in the first atomic structures of amyloid fibrils.

The timeline displays the history of key discoveries in the amyloid field from the initial identification of amyloid to discoveries that led to the first structures of amyloid fibrils associated with disease in all-atom detail^–. Aβ, amyloid-β; β₂m, β₂-microglobulin; cryo-EM, cryo-electron microscopy; cryo-ET, cryo-electron tomography; EM, electron microscopy; micro-ED, micro-electron diffraction; polyA, poly-alanine; polyQ, poly-glutamine; ssNMR, solid-state NMR spectroscopy. Images reproduced with permission from REF., Cold Spring Harbor Laboratory Press; REF.^,, Elsevier; REF., American Society for Clinical Investigation; reFs^,, John Wiley and Sons; REF., American Chemical Society; REFS^,, Springer Nature Limited.

Fig. 2. Schematic of amyloid formation.

Native proteins are in dynamic equilibrium with their less-structured, partially folded and/or unfolded states. One (or possibly several) of these states initiates amyloid fibril formation by assembling into oligomeric species. The precursor of aggregation (native, partially folded or unfolded) may differ for different protein sequences. Oligomeric species can then assemble further to form higher-order oligomers, one or more of which can form a fibril nucleus, which, by rapidly recruiting other monomers, can nucleate assembly into amyloid fibrils. This process occurs in the lag time (nucleation phase) of assembly. As fibrils grow, they can fragment, yielding more fibril ends that are capable of elongation by the addition of new aggregation-prone species^,,. This elongation results in an exponential growth of fibrillar material (blue line) until nearly all free monomer is converted into a fibrillar form. Fibrils are dynamic and can release oligomers that may or may not be toxic. Fibrils can also associate further with each other, with other proteins and with non-proteinaceous factors (not shown here) to form the amyloid plaques and intracellular inclusions characteristic of amyloid disease. Note that any and/or all of these steps are potential points for drug intervention.

Fig. 3. Amyloid aggregates can cause cell disruption by a variety of mechanisms.

Amyloid aggregates can deposit extracellularly or intracellularly, and both can give rise to cellular dysfunction and disease. The aggregates that form from different protein precursors may have different cellular effects, but deconvoluting the toxic mechanism of an individual protein and its ensemble of misfolded or aggregated states (misfolded monomers, oligomers, fibrils or plaques and intracellular inclusions) remains a challenge. Plaques and inclusions sequester a range of other molecules that include glycosaminoglycans^,, lipids^, and metal ions, which stabilize their assembly. Plaques are physically large and can disrupt organ function by their sheer size. Small fibrils can also be taken up into a cell via endocytosis, but this can be perturbed by preventing binding to certain cell surface receptors such as lymphocyte activation gene 3 protein. Within endosomes and lysosomes, fibrils can release toxic oligomers and can disrupt the endosomal and lysosomal function and dynamics because fibrils are highly resilient to degradation^,. Fibrils can also access the intracellular space following release from cells, thus spreading disease by uptake into adjacent cells. Other effects of aggregates within cells include disruption of endoplasmic reticulum (ER) dynamics, release of reactive oxygen species (ROS) from mitochondria and the induction of stress responses (not shown here).

Fig. 4. Structural motifs that stabilize amyloid fibrils.

a | A ten-residue peptide from transthyretin (TTR), showing β-sheet stacking in which each β-strand ‘rung’ is stabilized by hydrogen bonds (denoted by fine black dotted lines) between the polypeptide backbones of precursors, which are separated by the canonical 4.7–4.8 Å repeat of the cross-β amyloid fold (PDB accession number 2nm5 (REF.)). Further stabilization is provided by a steric zipper between the β-sheets, which stabilizes the fibril core. b | The β-helix of HET-S illustrating its steric zippers (PDB accession number 2rnm (REF.)). c | A structure of amyloid-β (Aβ)42 fibrils (PDB accession number 5oqv (REF.)) illustrating the variety of interactions that stabilize the fibril, including β-strand stacking (top left), formation of inter-protofilament salt bridges (top right), intra-protofilament steric zippers (bottom left) and inter-protofilament steric zippers (bottom right).

Fig. 5. Subunit packing in amyloid fibrils.

Space-filling representations of near-atomic-resolution models of different amyloid fibrils, each filtered to 4 Å. Individual subunits are coloured in red to highlight different inter-protofilament packing in different fibril types. a | The β-helix of HET-S that forms a single filament (PDB accession number 2lbu (REF.)). b | Two polymorphs of amyloid-β (Aβ)42 fibrils formed under different growth conditions (PDB accession number 5oqv (left) and PDB accession number 5kk3 (REF.) (right)). c | Two polymorphs of Aβ40. Fibrils formed under the same solution conditions but propagated from seeds with different morphologies (2A, PDB accession number 2lmn (REF.) (left) and 3Q, PDB accession number 2lmp (REF.) (right)). d | Two polymorphs of tau fibrils: paired helical (PHF) (left) (PDB accession number 5o3l (REF.) and straight (SF) (right) (PDB accession number 5o3t (REF.)). e | The single filament of α-synuclein fibrils (PDB accession number 2n0a). The main chain of the top layer of polypeptide chain in each fibril is shown in red. ‘2A’ indicates fibrils with two-fold symmetry; ‘3Q’ indicates fibrils with three-fold symmetry.

Fig. 6. How changes in primary sequence affect amyloid disease.

Top left panel: various diseases are caused by poly-glutamine (polyQ) expansion disorders. Depending on the specific disease (shown in the figure), polyQ repeat lengths exceeding a critical threshold can cause disease, whereas fewer repeats are innocuous. Data were taken from reFs^,. Lower left panel: the age of onset of patients with Parkinson disease (PD) is influenced by the copy number of the α-synuclein gene (duplication (2SNCA), triplication (3SNCA) or quadruplication (4SNCA)), with increased expression correlating with earlier onset. Age of onset and disease duration are also influenced by single point mutations, which may result in different aggregation pathways and/or kinetics or different fibril architectures resulting in different disease phenotypes. Data were taken from reFs^,. Top right panel: the pathology of Alzheimer disease (AD) can be influenced by fibril morphology. In particular, typical-AD (t-AD) and a rapidly progressive form of AD (r-AD) show similar fibril architecture monitored by solid-state NMR spectroscopy (ssNMR) but have varied ages of onset and disease duration. However, in posterior cortical atrophy AD (PCA-AD), fibrils with a different structure are formed. The age of onset and disease duration for PCA-AD are similar to t-AD and r-AD, but the disease primarily affects the cerebellum rather than the temporal lobe. Centre panel: a diagram of the brain highlighting the regions primarily affected by each of the diseases shown. CACNA1A, voltage-dependent P/Q-type calcium channel subunit α1A; HD, Huntington disease; TBP, TATA-box-binding protein; WT, wild type. The top right panel was adapted from REF..