Annular Protofibrils Research Articles

The amyloid concentric β-barrel hypothesis: Models of amyloid beta 42 oligomers and annular protofibrils.

Amyloid beta (Aβ) peptides are a major contributor to Alzheimer's disease. They occur in differing lengths, each of which forms a multitude of assembly types. The most toxic soluble oligomers are formed by Aβ42; some of which have antiparallel β-sheets. Previously, our group proposed molecular models of Aβ42 hexamers in which the C-terminus third of the peptide (S3) forms an antiparallel 6-stranded β-barrel that is surrounded by an antiparallel barrel formed by the more polar N-terminus (S1) and middle (S2) portions. These hexamers were proposed to act as seeds from which dodecamers, octadecamers, both smooth annular protofibrils (sAPFs) and beaded annular protofibrils (bAPFs), and transmembrane channels form. Since then, numerous aspects of our models have been supported by experimental findings. Recently, NMR-based structures have been proposed for Aβ42 tetramers and octamers, and NMR studies have been reported for oligomers composed of ~32 monomers. Here we propose a range of concentric β-barrel models and compare their dimensions to image-averaged electron micrographs of both bAPFs and sAPFs of Aβ42. The smaller oligomers have 6, 8, 12, 16, and 18 monomers. These beads string together to form necklace-like bAPFs. These bAPRs gradually morph into sAPFs in which a S3 β-barrel is shielded on one or both sides by β-barrels formed from S1 and S2 segments.

The amyloid concentric β-barrel hypothesis: Models of synuclein oligomers, annular protofibrils, lipoproteins, and transmembrane channels.

Amyloid beta (Aβ of Alzheimer's disease) and α-synuclein (α-Syn of Parkinson's disease) form large fibrils. Evidence is increasing however that much smaller oligomers are more toxic and that these oligomers can form transmembrane ion channels. We have proposed previously that Aβ42 oligomers, annular protofibrils, and ion channels adopt concentric β-barrel molecular structures. Here we extend that hypothesis to the superfamily of α, β, and γ-synucleins. Our models of numerous synuclein oligomers, annular protofibrils, tubular protofibrils, lipoproteins, and ion channels were developed to be consistent with sizes, shapes, molecular weights, and secondary structures of assemblies as determined by electron microscopy and other studies. The models have the following features: (1) all subunits have identical structures and interactions; (2) they are consistent with conventional β-barrel theory; (3) the distance between walls of adjacent β-barrels is between 0.6 and 1.2 nm; (4) hydrogen bonds, salt bridges, interactions among aromatic side-chains, burial and tight packing of hydrophobic side-chains, and aqueous solvent exposure of hydrophilic side-chains are relatively optimal; and (5) residues that are identical among distantly related homologous proteins cluster in the interior of most oligomers whereas residues that are hypervariable are exposed on protein surfaces. Atomic scale models of some assemblies were developed.

Interplay of isoform 1N4R tau protein and amyloid-β peptide fragment 25-35 in reducing and non-reducing conditions.

Amyloid-β (Aβ) peptide and tau protein are two hallmark proteins in Alzheimer's disease (AD); however, the parameters, which mediate the abnormal aggregation of Aβ and tau, have not been fully discovered. Here, we have provided an optimum method to purify tau protein isoform 1N4R by using nickel-nitrilotriacetic acid agarose chromatography under denaturing condition. The biochemical and biophysical properties of the purified protein were further characterized using in vitro tau filament assembly, tubulin polymerization assay, circular dichroism (CD) spectroscopy and atomic force microscopy. Afterwards, we investigated the effect of tau protein on aggregation of Aβ (25-35) peptide using microscopic imaging and cell viability assay. Incubation of tau at physiologic and supra-physiologic concentrations with Aβ25-35 for 40 days under reducing and non-reducing conditions revealed formation of two types of aggregates with distinct morphologies and dimensions. In non-reducing condition, the co-incubated sample showed granular aggregates, while in reducing condition, they formed annular protofibrils. Results from cell viability assay revealed the increased cell viability for the co-incubated sample. Therefore, the disassembling action shown by tau protein on Aβ25-35 suggests the possibility that tau may have a protective role in preventing Aβ peptide from acquiring the cytotoxic, aggregated form against oxidative stress damages.

The C291R Tau Variant Forms Different Types of Protofibrils.

Mutations in the MAPT gene can lead to disease-associated variants of tau. However, the pathological mechanisms behind these genetic tauopathies are poorly understood. Here, we characterized the aggregation stages and conformational changes of tau C291R, a recently described MAPT mutation with potential pathogenic functions. The C291R variant of the tau four-repeat domain (tau-K18; a functional fragment with increased aggregation propensity compared with the full-length protein), aggregated into a mix of granular oligomers, amorphous and annular pore-like aggregates, in native-state and heparin-treated reactions as observed using atomic force microscopy (AFM) and negative-stained electron microscopy. On extended incubation in the native-state, tau-K18 C291R oligomers, unlike wild type (WT) tau-K18, aggregated to form protofibrils of four different phenotypes: (1) spherical annular; (2) spherical annular encapsulating granular oligomers; (3) ring-like annular but non-spherical; and (4) linear protofibrils. The ring-like tau-K18 C291R aggregates shared key properties of annular protofibrils previously described for other amyloidogenic proteins, in addition to two unique features: irregular/non-spherical-shaped annular protofibrils, and spherical protofibrils encapsulating granular oligomers. Tau-K18 C291R monomers had a circular dichroism (CD) peak at ~210 nm compared with ~199 nm for tau-K18 WT. These data suggest mutation-enhanced β-sheet propensity. Together, we describe the characterization of tau-K18 C291R, the first genetic mutation substituting a cysteine residue. The aggregation mechanism of tau-K18 C291R appears to involve β-sheet-rich granular oligomers which rearrange to form unique protofibrillar structures.

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers.

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ, in the absence of molecular handles and using persistence length-fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head-to-head orientation with a nonnative interface along their β-strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.

Cu(II) promotes amyloid pore formation

The aggregation of α-synuclein is associated with dopamine neuron death in Parkinson's disease. There is controversy in the field over the question of which species of the aggregates, fibrils or protofibrils, are toxic. Moreover, compelling evidence suggested the exposure to heavy metals to be a risk of PD. Nevertheless, the mechanism of metal ions in promoting PD remains unclear. In this research, we investigated the structural basis of Cu(II) induced aggregation of α-synuclein. Using transmission electron microscopy experiments, Cu(II) was found to promote in vitro aggregation of α-synuclein by facilitating annular protofibril formation rather than fibril formation. Furthermore, neuroprotective baicalein disaggregated annular protofibrils accompanied by considerable decrease of β-sheet content. These results strongly support the hypothesis that annular protofibrils are the toxic species, rather than fibrils, thereby inspiring us to search novel therapeutic strategies for the suppression of the toxic annular protofibril formation.

Quaternary Structure Defines a Large Class of Amyloid-β Oligomers Neutralized by Sequestration

The accumulation of amyloid-β (Aβ) as amyloid fibrils and toxic oligomers is an important step in the development of Alzheimer's disease (AD). However, there are numerous potentially toxic oligomers and little is known about their neurological effects when generated in the living brain. Here we show that Aβ oligomers can be assigned to one of at least two classes (type 1 and type 2) based on their temporal, spatial, and structural relationships to amyloid fibrils. The type 2 oligomers are related to amyloid fibrils and represent the majority of oligomers generated in vivo, but they remain confined to the vicinity of amyloid plaques and do not impair cognition at levels relevant to AD. Type 1 oligomers are unrelated to amyloid fibrils and may have greater potential to cause global neural dysfunction in AD because they are dispersed. These results refine our understanding of the pathogenicity of Aβ oligomers in vivo.

The formation of tau pore-like structures is prevalent and cell specific: possible implications for the disease phenotypes.

Pathological aggregation of the microtubule-associated protein tau and subsequent accumulation of neurofibrillary tangles (NFTs) or other tau-containing inclusions are defining histopathological features of many neurodegenerative diseases, which are collectively known as tauopathies. Due to conflicting results regarding a correlation between the presence of NFTs and disease progression, the mechanism linking pathological tau aggregation with cell death is poorly understood. An emerging view is that NFTs are not the toxic entity in tauopathies; rather, tau intermediates between monomers and NFTs are pathogenic. Several proteins associated with neurodegenerative diseases, such as β-amyloid (Aβ) and α-synuclein, have the tendency to form pore-like amyloid structures (annular protofibrils, APFs) that mimic the membrane-disrupting properties of pore-forming protein toxins. The present study examined the similarities of tau APFs with other tau amyloid species and showed for the first time the presence of tau APFs in brain tissue from patients with progressive supranuclear palsy (PSP) and dementia with Lewy bodies (DLB), as well as in the P301L mouse model, which overexpresses mutated tau. Furthermore, we found that APFs are preceded by tau oligomers and do not go on to form NFTs, evading fibrillar fate. Collectively, our results demonstrate that in vivo APF formation depends on mutations in tau, phosphorylation levels, and cell type. These findings establish the pathological significance of tau APFs in vivo and highlight their suitability as therapeutic targets for several neurodegenerative tauopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/2051-5960-2-56) contains supplementary material, which is available to authorized users.

In Vitro Study of α-Synuclein Protofibrils by Cryo-EM Suggests a Cu2+-Dependent Aggregation Pathway

The aggregation of α-synuclein is thought to play a role in the death of dopamine neurons in Parkinson’s disease (PD). Alpha-synuclein transitions itself through an aggregation pathway consisting of pathogenic species referred to as protofibrils (or oligomer), which ultimately convert to mature fibrils. The structural heterogeneity and instability of protofibrils has significantly impeded advance related to the understanding of their structural characteristics and the amyloid aggregation mystery. Here, we report, to our knowledge for the first time, on α-synuclein protofibril structural characteristics with cryo-electron microscopy. Statistical analysis of annular protofibrils revealed a constant wall thickness as a common feature. The visualization of the assembly steps enabled us to propose a novel, to our knowledge, mechanisms for α-synuclein aggregation involving ring-opening and protofibril-protofibril interaction events. The ion channel-like protofibrils and their membrane permeability have also been found in other amyloid diseases, suggesting a common molecular mechanism of pathological aggregation. Our direct visualization of the aggregation pathway of α-synuclein opens up fresh opportunities to advance the understanding of protein aggregation mechanisms relevant to many amyloid diseases. In turn, this information would enable the development of additional therapeutic strategies aimed at suppressing toxic protofibrils of amyloid proteins involved in neurological disorders.

Specific Soluble Oligomers of Amyloid-β Peptide Undergo Replication and Form Non-fibrillar Aggregates in Interfacial Environments

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

Astrocytes contain amyloid-β annular protofibrils in Alzheimer’s disease brains

Annular protofibrils (APFs) represent a newly described and distinct class of amyloid structures formed by disease-associated proteins. In vitro, these pore-like structures have been implicated in membrane permeabilization and ion homeostasis via pore formation. Still, their formation and relevance in vivo are poorly understood. Herein, we report that APFs are in human Alzheimer’s disease brain samples and that amyloid-β APFs were associated with activated astrocytes. Moreover, we show that amyloid-β APFs in astrocytes adopt a conformation in which the N-terminal region is buried inside the wall of the pore. Our results together with previous studies suggest that the formation of amyloid-β APFs in astrocytes could be a relevant event in the pathogenesis of Alzheimer’s disease and validate this amyloidogenic structure as a target for the prevention of the disease.

Amyloid-β Annular Protofibrils Evade Fibrillar Fate in Alzheimer Disease Brain

Annular protofibrils (APFs) represent a new and distinct class of amyloid structures formed by disease-associated proteins. In vitro, these pore-like structures have been implicated in membrane permeabilization and ion homeostasis via pore formation. Still, evidence for their formation and relevance in vivo is lacking. Herein, we report that APFs are in a distinct pathway from fibril formation in vitro and in vivo. In human Alzheimer disease brain samples, amyloid-β APFs were associated with diffuse plaques, but not compact plaques; moreover, we show the formation of intracellular APFs. Our results together with previous studies suggest that the prevention of amyloid-β annular protofibril formation could be a relevant target for the prevention of amyloid-β toxicity in Alzheimer disease.

Aluminum, copper, iron and zinc differentially alter amyloid-Aβ1–42 aggregation and toxicity

Amyloid-β1–42 (Aβ) is believed to play a crucial role in the ethiopathogenesis of Alzheimer's Disease (AD). In particular, its interactions with biologically relevant metal ions may lead to the formation of highly neurotoxic complexes. Here we describe the species that are formed upon reacting Aβ with several biometals, namely copper, zinc, iron, and with non-physiological aluminum to assess whether different metal ions are able to differently drive Aβ aggregation. The nature of the resulting Aβ–metal complexes and of the respective aggregates was ascertained through a number of biophysical techniques, including electrospray ionization mass spectrometry, dynamic light scattering, fluorescence, transmission electron microscopy and by the use of conformation-sensitive antibodies (OC, αAPF). Metal binding to Aβ is shown to confer highly different chemical properties to the resulting complexes; accordingly, their overall aggregation behaviour was deeply modified. Both aluminum(III) and iron(III) ions were found to induce peculiar aggregation properties, ultimately leading to the formation of annular protofibrils and of fibrillar oligomers. Notably, only Aβ-aluminum was characterized by the presence of a relevant percentage of aggregates with a mean radius slightly smaller than 30nm. In contrast, both zinc(II) and copper(II) ions completely prevented the formation of soluble fibrillary aggregates. The biological effects of the various Aβ–metal complexes were studied in neuroblastoma cell cultures: Aβ–aluminum turned out to be the only species capable of triggering amyloid precursor and tau181 protein overproduction. Our results point out that Al can effectively interact with Aβ, forming “structured” aggregates with peculiar biophysical properties which are associated with a high neurotoxicity.

Fibrillar Oligomers Nucleate the Oligomerization of Monomeric Amyloid β but Do Not Seed Fibril Formation

Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid beta (Abeta) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 x g, rich in beta-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Abeta FOs do not seed the formation of thioflavin T-positive fibrils from Abeta monomers but instead seed the formation of FOs from Abeta monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is kinetically more favorable than the assembly of the oligomers into fibrils via sheet stacking interaction. These studies provide novel structural insight into the relationship between fibrils and FOs and suggest that the increased toxicity of FOs may be due to their ability to replicate and the exposure of hydrophobic sheet surfaces that are otherwise obscured by sheet-sheet interactions between protofilaments in a fibril.

Structural Diversity of Amyloid Oligomers

Recent evidence suggests that soluble amyloid oligomers may represent the primary pathological species of protein aggregates in neurodegenerative diseases. Conformation-dependent, aggregation specific antisera indicate that there are three widely distributed and distinct classes of oligomer structures: prefibrillar oligomers, fibrillar oligomers and pore like annular protofibrils. Fibrillar oligomers are soluble at 100,000 x g, rich in β-sheet structures but yet bind weakly to thioflavin T. EPR spectroscopy indicates that fibrillar oligomers display significantly more spin-spin interaction at multiple labeled sites than prefibrillar oligomers and are more structurally similar to fibrils.

Amyloid Oligomer Structures and Toxicity

Amyloid accumulation is commonly associated with a number of important human degenerative diseases and recent findings indicate that soluble amyloid oligomers may represent the primary pathological species in degenerative diseases. Amyloid oligomers are structurally and morphologically diverse, raising the question on whether this diversity is pathologically significant and whether different types of oligomers may have different toxic activities. Many of the amyloids associated with neurodegenerative diseases form three immunologically distinct types of oligomers. Fibrillar oligomers are structurally related to fibrils and may represent small pieces of fibrils or fibril protofilaments. Prefibrillar oligomers are kinetic intermediates in fibril formation and annular protofibrils that resemble membrane pores. These three classes of oligomers share common structures and toxic activities. Focus on these common mechanisms of toxicity provides a means of simplifying the list of primary disease mechanisms and opens the possibility of developing broad spectrum therapeutics that target several amyloid related degenerative diseases.

Annular Protofibrils Are a Structurally and Functionally Distinct Type of Amyloid Oligomer

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (alphaAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in alphaAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric alpha-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a beta barrel structural motif.

Amyloid Beta Annular Protofibrils in Cell Processes and Synapses Accumulate with Aging and Alzheimer-Associated Genetic Modification

Amyloid β (Aβ) annular protofibrils (APFs) have been described where the structure is related to that of β barrel pore-forming bacterial toxins and exhibits cellular toxicity. To investigate the relationship of Aβ APFs to disease and their ultrastructural localization in brain tissue, we conducted a pre-embedding immunoelectron microscopic study using anti-annular protofibril antiserum. We examined brain tissues of young- and old-aged amyloid precursor protein transgenic mice (APP23), neprilysin knockout APP23 mice, and nontransgenic littermates. αAPF-immunoreactions tended to be found (1) on plasma membranes and vesicles inside of cell processes, but not on amyloid fibrils, (2) with higher density due to aging, APP transgene, and neprilysin deficiency, and (3) with higher positive rate at synaptic compartments in aged APP23, especially in neprilysin knockout APP23 mice. These findings imply that APFs are distinct from amyloid fibrils, interact with biological membranes, and might be related to synaptic dysfunction in Alzheimer model mouse brains.

Thermodynamic and kinetic origins of Alzheimer's and related diseases: A chemical engineer's perspective

Thermodynamic and kinetic origins of Alzheimer's and related diseases: A chemical engineer's perspective

Mapping the Structural Transition in an Amyloidogenic Apolipoprotein A-I

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIOWA) leads to the formation of beta-secondary structure rich amyloid fibrils in vivo. Here we show that full-length apoA-IIOWA has a decreased lipid-binding capability, an increased amino-terminal sensitivity to protease, and a propensity to form annular protofibrils visible by electron microscopy. The molecular basis for the conversion of apolipoprotein A-I to a proamyloidogenic form was examined by electron paramagnetic resonance spectroscopy. Our recent findings [Lagerstedt, J. O., Budamagunta, M. S., Oda, M. N., and Voss, J. C. (2007) J. Biol. Chem. 282, 9143-9149] indicate that Gly26 in the native apoprotein separates a preceding beta-strand structure (residues 20-25) from a downstream largely alpha-helical region. The current study demonstrates that the G26R variant promotes a structural transition of positions 27-56 to a mixture of coil and beta-strand secondary structure. Microscopy and staining by amyloidophilic dyes suggest that this alteration extends throughout the protein within 1 week of incubation in vitro, leading to insoluble aggregates of distinct morphology. The severe consequences of the Iowa mutation likely arise from the combination of losing the contribution of the native Gly residue in terminating beta-strand propagation and the promotion of beta-structure when an Arg is introduced adjacent to the succeeding residue of identical charge and size, Arg27.