Protofibrils Research Articles

Analysis of Interaction Characteristics Between Amyloid β and Lecanemab by HDX‐MS

AbstractBackgroundSoluble amyloid‐β (Aβ) aggregates including protofibrils (PFs) have been reported to be more toxic than insoluble fibrils. Recently, pharmaceutical antibodies targeting these soluble Aβ aggregates have been developed. The structural dynamics and heterogeneity of soluble Aβ aggregates have made it difficult to analyze the structural characteristics and tertiary structure. To investigate conformational recognition of lecanemab to Aβ aggregates, the structural characteristics of Aβ PFs and their change upon binding to lecanemab, an antibody with preferential binding to Aβ protofibrils, were studied using hydrogen‐deuterium exchange mass spectrometry (HDX‐MS).MethodAβ (1‐42) PFs were prepared in vitro. The hydrogen/deuterium (H/D) exchange status for Aβ (1‐42) PFs and monomeric Aβ (1‐40) were studied by HDX‐MS in phosphate buffered saline (PBS) to explore characteristics of their regional structural dynamics. The effects of the anti–Aβ PF antibody lecanemab on H/D status of Aβ (1‐42) PFs and monomeric Aβ (1‐40) were studied to explore the structural basis for the characteristics of antibody‐ Aβ interaction and specificity against Aβ PFs.ResultAβ (1‐42) PFs were more protected from H/D exchange throughout the molecule than monomeric Aβ (1‐40), though the N‐terminal and the mid region of Aβ still showed relatively higher H/D exchange in Aβ (1‐42) PFs. Upon interaction with lecanemab, the mid region of Aβ (1‐42) PFs was also protected further in addition to the N‐terminal region, and its protection level of Aβ (1‐42) PFs was higher than monomeric Aβ (1‐40). The higher protection from H/D exchange both on N‐terminal and mid regions of Aβ PFs is considered to be characteristics of interaction between Aβ (1‐42) PFs and lecanemab.ConclusionThe results indicate that the mid region of Aβ has relatively high flexibility in PFs in addition to N‐terminal region. The mid region is suggested to be involved in the interaction with lecanemab as well as its N‐terminal region, and the conformational epitope formation was expected, though there are still possibility of structural change in the mid region upon lecanemab binding. The results indicated that the combinational action on the N‐terminal and mid regions of Aβ are involved in the modulation of Aβ PFs by lecanemab.

Structural Dynamics of Amyloid-β Protofibrils and Actions of Anti-Amyloid-β Antibodies as Observed by High-Speed Atomic Force Microscopy.

Amyloid-β (Aβ) aggregation intermediates, including oligomers and protofibrils (PFs), have attracted attention as neurotoxic aggregates in Alzheimer's disease. However, due to the complexity of the aggregation pathway, the structural dynamics of aggregation intermediates and how drugs act on them have not been clarified. Here we used high-speed atomic force microscopy to observe the structural dynamics of Aβ42 PF at the single-molecule level and the effect of lecanemab, an anti-Aβ PF antibody with the positive results from Phase 3 Clarity AD. PF was found to be a curved nodal structure with stable binding angle between individual nodes. PF was also a dynamic structure that associates with other PF molecules and undergoes intramolecular cleavage. Lecanemab remained stable in binding to PFs and to globular oligomers, inhibiting the formation of large aggregates. These results provide direct evidence for a mechanism by which antibody drugs interfere with the Aβ aggregation process.

Video imaging of structural dynamics of amyloid β protofibrils.

Soluble aggregates including oligomers and protofibrils (PFs) of amyloid β (Aβ) have been reported to be more toxic than mature fibrils (MFs). Recently, clinical trials of antibodies targeting these aggregates are also in progress. Their structure is dynamic, which makes it difficult to extract and analyze them in a stable state. Therefore, the molecular process among the soluble Aβ aggregates has been unclear. Development of high-speed atomic force microscopy (HS-AFM) allows video-imaging of structural dynamics of individual amyloid aggregates in liquid. Here, we investigated structural dynamics and mechanical property of Aβ protofibrils and anti-Aβ antibody binding to PFs, using HS-AFM. Aβ1-42 PF and MF were prepared in vitro, which was deposited on the HS-AFM stage. Structural dynamics of the aggregates were observed by HS-AFM in phosphate buffered saline (PBS). Young's moduli of the individual aggregates were measured by force spectrometry mode of HS-AFM in PBS. The anti-Aβ antibody was added in the chamber, followed by HS-AFM observation of the antibody binding to PFs. Aβ1-42 PF showed diffusive 2D motion on bare mica and its typical structure was composed of several globular parts in a polygonal line. The angle between each segment of a PF was maintained during the diffusive motion. The length of PFs showed a single exponential distribution. Its mean length depended on PF concentration. The Young's modulus of PFs was higher than that of MFs in liquid. Lecanemab (BAN2401) bounded to PFs both most rapidly and abundantly, while 4G8 showed the weakest and least binding to PFs. Assembly and disassembly of the globular parts in PFs are dynamic, while their segment structure is stiffer than MFs and the bonds between the globular parts are rigid. These characteristics indicated the difference in internal structure between PFs and MFs, which supports that PFs may not be a direct precursor of MFs. Lecanemab binds to all various parts and length of PFs.

Detection of Amyloid β Oligomers with RNA Aptamers in AppNL-G-F/NL-G-F Mice: A Model of Arctic Alzheimer's Disease.

RNA aptamers have garnered attention for diagnostic applications due to their ability to recognize diverse targets. Oligomers of 42-mer amyloid β-protein (Aβ42), whose accumulation is relevant to the pathology of Alzheimer’s disease (AD), are among the most difficult molecules for aptamer recognition because they are prone to aggregate in heterogeneous forms. In addition to designing haptens for in vitro selection of aptamers, the difficulties involved in determining their effect on Aβ42 oligomerization impede aptamer research. We previously developed three RNA aptamers (E22P-AbD4, -AbD31, and -AbD43) with high affinity for protofibrils (PFs) derived from a toxic Aβ42 dimer. Notably, these aptamers recognized diffuse staining, which likely originated from PFs or higher-order oligomers with curvilinear structures in a knock-in AppNL-G-F/NL-G-F mouse, carrying the Arctic mutation that preferentially induced the formation of PFs, in addition to a PS2Tg2576 mouse. To determine which oligomeric sizes were mainly altered by the aptamer, ion mobility–mass spectrometry (IM–MS) was carried out. One aptamer, E22P-AbD43, formed adducts with the Aβ42 monomer and dimer, leading to suppression of further oligomerization. These findings support the utility of these aptamers as diagnostics for AD.

An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer's disease

Oligomers of β-amyloid 42 (Aβ42), rather than fibrils, drive the pathogenesis of Alzheimer's disease (AD). In particular, toxic oligomeric species called protofibrils (PFs) have attracted significant attention. Herein, we report RNA aptamers with higher affinity toward PFs derived from a toxic Aβ42 dimer than toward fibrils produced from WT Aβ42 or from a toxic, conformationally constrained Aβ42 variant, E22P-Aβ42. We obtained these RNA aptamers by using the preincubated dimer model of E22P-Aβ42, which dimerized via a linker located at Val-40, as the target of in vitro selection. This dimer formed PFs during incubation. Several physicochemical characteristics of an identified aptamer, E22P-AbD43, suggested that preferential affinity of this aptamer toward PFs is due to its higher affinity for the toxic dimer unit (KD = 20 ± 6.0 nm) of Aβ42 than for less-toxic Aβ40 aggregates. Comparison of CD data from the full-length and random regions of E22P-AbD43 suggested that the preferential binding of E22P-AbD43 toward the dimer might be related to the formation of a G-quadruplex structure. E22P-AbD43 significantly inhibited the nucleation phase of the dimer and its associated neurotoxicity in SH-SY5Y human neuroblastoma cells. Of note, E22P-AbD43 also significantly protected against the neurotoxicity of WT Aβ42 and E22P-Aβ42. Furthermore, in an AD mouse model, E22P-AbD43 preferentially recognized diffuse aggregates, which likely originated from PFs or higher-order oligomers with curvilinear structures, compared with senile plaques formed from fibrils. We conclude that the E22P-AbD43 aptamer is a promising research and diagnostic tool for further studies of AD etiology.

Aβ42 Neurotoxicity Is Mediated by Ongoing Nucleated Polymerization Process Rather than by Discrete Aβ42 Species

The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aβ42 preparations, containing a monomeric and heterogeneous mixture of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.

Suppression of Map Kinases Inhibits Microglial Activation and Attenuates Neuronal Cell Death Induced by α-Synuclein Protofibrils

Alpha-Synuclein (alpha-Syn) accounts, as a major component of Lewy bodies (LB), for the filamentous deposits in many cases of neurodegenerative diseases. Yet, little is known about the molecular mechanisms of neuronal loss in these diseases. The correlation between alpha-Syn oligomerization/aggregation and pathologies raises the key question of which molecular form of alpha-Syn (i.e. monomeric alpha-Syn, protofibrils or mature fibrils) represents the damage-inducing culprit in the scenario of synucleinopathies. We show that human alpha-Syn protofibrils (PFs) are potent activators of parallel proinflammatory signalling pathways (p38 and ERK1/2 MAP kinases and NF-kappaB) in microglial cells in vitro. Furthermore, stereotactic injection of alpha-Syn PFs into the substantia nigra of adult rats leads to a profound activation of microglia and adjacent neuronal cell loss, which can be attenuated by the MAP kinase inhibitor semapimod. We propose that the neurodegenerative process of alpha-synucleinopathies involves microglial activation through alpha-Syn released or extruded from cells with pathogenic alpha-Syn metabolism. Compounds that inhibit the MAPK/NF-kappaB pathways might be a promising pharmacological strategy for the treatment of the inflammatory component of synucleinopathies including PD.

Dopamine Affects the Stability, Hydration, and Packing of Protofibrils and Fibrils of the Wild Type and Variants of α-Synuclein

Parkinson's disease (PD) is characterized by the presence of cytoplasmic inclusions composed of alpha-synuclein (alpha-syn) in dopaminergic neurons. This suggests a pivotal role of dopamine (DA) on PD development. Here, we show that DA modulates differently the stability of protofibrils (PF) and fibrils (F) composed of wild type or variants of alpha-syn (A30P and A53T) as probed by high hydrostatic pressure (HHP). While in the absence of DA, all alpha-syn PF exhibited identical stability, in its presence, the variant-composed PF acquired a greater stability (DAPFwt < DAPFA30P = DAPFA53T), implying that they would last longer, which could shed light onto why these mutations are so aggressive. When alpha-syn was incubated for long times (18 days) in the presence of DA, we observed the formation of F by electronic microscopy, suggesting that the PF trapped in the presence of DA in short times can evolve into F. The stability of F was also altered by DA. DAFwt was more labile than Fwt, indicating that the former would be more susceptible to breakage. PFA30P and DAPFA30P, when added to mesencephalic and cortical neurons in culture, decreased the number and length of neurites and increased the number of apoptotic cells. Surprisingly, these toxic effects of PFA30P and DAPFA30P were practically abolished with HHP treatment, which was able to break the PF into smaller aggregates, as seen by atomic force microscopy. These results suggest that strategies aimed at breaking and/or clearing these aggregates is promising in alleviating the symptoms of PD.

Apomyoglobin reveals a random-nucleation mechanism in amyloid protofibril formation

Protofibrils (PFs) represent the earliest fibrillar species that occur in the course of amyloid fibril formation. Using apomyoglobin, we report here that PFs arise from a multi-step reaction and that they are preceded by an ensemble of non-fibrillar particles (NFPs). These intermediate aggregates encompass nascent elements of amyloid structure and can act as seeds in PF formation. Taken together with the observation that PFs often protrude from NFPs, our data suggest that PFs form by a random nucleation mechanism in which the polypeptide chains sample many different aggregated conformations. Once the appropriate structural characteristics are acquired, PFs are formed by addition of further polypeptide chains.

Amyloid β-protein induced electrophysiological changes are dependent on aggregation state: N-methyl- d-aspartate (NMDA) versus non-NMDA receptor/channel activation

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, however, the underlying mechanism driving this condition is unknown. Unexplored is the possibility that the time-dependent generation of different Aβ assemblies may underlie the pathogenic cascade with biophysically distinct structures interacting with unique biological targets. Thus, the presence of subtle alterations in synaptic function during the earliest clinical phase of AD may be mediated by diffusible assemblies of the amyloid β-protein (Aβ). Using primary neocortical cultures, here we compare the synaptic responses induced by two different Aβ assemblies, protofibrils (PFs) and fibrils (FBs), and demonstrate for the first time that neuronal activation was selectively dependent on the assembly state of Aβ. PF-induced activity was specifically attenuated by the N-methyl- d-aspartate (NMDA) receptor antagonist, d-APV. In contrast, the non-NMDA glutamate receptor antagonist, NBQX, preferentially reduced FB-induced activity. In support, removal of Mg 2+ from the medium, which enhances NMDA channels, increased both PF- or FB-induced activation, but d-APV was more effective in attenuating PF-induced excitatory activity. These findings suggest that PFs may activate neurons differently than fibrils and lend support to the hypothesis that pre-fibrillar assemblies of Aβ may play an important role in the development of AD-type synaptic deficits.

Protofibrils of amyloid β-protein inhibit specific K + currents in neocortical cultures

Protofibrils (PFs) are recently described intermediate assemblies formed during the fibrillogenesis of amyloidogenic proteins and may play an important pathogenic role in Parkinson’s and Alzheimer’s disease (AD). Here we show for the first time that amyloid β-protein (Aβ) attenuation of specific K + currents is dependent on the aggregation state; PFs inhibit K + currents, whereas low-molecular-weight assemblies have no effect. Using patch clamp analysis in whole cell current-clamp mode, we showed that at low nanomolar concentrations Aβ 1–42 PFs induce reversible, Ca 2+-dependent increases in spontaneous action potentials and membrane depolarizations. The low nanomolar PF concentrations used, the instantaneous responses observed, and the reversibility of the effect all suggest that PFs may bind to specific channels or membrane proteins. Switching to voltage-clamp mode, we found that PFs at 1–2 μM can inhibit specifically the 4AP-sensitive K + currents, A-type and D-type, but not other outward or inward rectifying K + channels. Finally, we show that a consequence of PF-induced membrane activity is an increase in intracellular Ca 2+ spikes that are dependent on synaptic connections in the neural network formed in culture. Our data strongly support the concept that PFs can induce subtle synaptic alterations that may underlie early symptoms of AD.

Protofibrillar Intermediates of Amyloid β-Protein Induce Acute Electrophysiological Changes and Progressive Neurotoxicity in Cortical Neurons

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is thought to be caused in part by the age-related accumulation of amyloid beta-protein (Abeta). The presence of neuritic plaques containing abundant Abeta-derived amyloid fibrils in AD brain tissue supports the concept that fibril accumulation per se underlies neuronal dysfunction in AD. Recent observations have begun to challenge this assumption by suggesting that earlier Abeta assemblies formed during the process of fibrillogenesis may also play a role in AD pathogenesis. Here, we present the novel finding that protofibrils (PF), metastable intermediates in amyloid fibril formation, can alter the electrical activity of neurons and cause neuronal loss. Both low molecular weight Abeta (LMW Abeta) and PF reproducibly induced toxicity in mixed brain cultures in a time- and concentration-dependent manner. No increase in fibril formation during the course of the experiments was observed by either Congo red binding or electron microscopy, suggesting that the neurotoxicity of LMW Abeta and PF cannot be explained by conversion to fibrils. Importantly, protofibrils, but not LMW Abeta, produced a rapid increase in EPSPs, action potentials, and membrane depolarizations. These data suggest that PF have inherent biological activity similar to that of mature fibrils. Our results raise the possibility that the preclinical and early clinical progression of AD is driven in part by the accumulation of specific Abeta assembly intermediates formed during the process of fibrillogenesis.