Primary Toxic Species Research Articles

Chemical regulation of Tau oligomers in phase separation in Alzheimer’s disease

A huge number of patients suffering from certain neurodegenerative disorder, which are collectively called as tauopathies, may exhibit pathological tau protein aggregates in their brains. This category of diseases includes Alzheimer's disease (AD). In AD, post translational modifications such as phos phorylations, glycosylations, truncations, and subsequent aggregation into oligomers, paired helical filaments (PHFs), and neurofibrillary tangles (NFTs) are closely associated with cognitive decline and neurodegeneration. As a result, tau oligomers have emerged as the primary toxic species in AD and tauo pathies. Tau oligomers are soluble, self-assembled tau proteins that are formed prior to fibrils and have been demonstrated to play a pivotal role in neuronal cell death and the induction of neurodegeneration in animal models. In this succinct review, we collate and summarize literature pertaining to tau oligomer formation and its role in Alzheimer's disease. Secondly, we explore the crucial role of zinc ions (Zn²⁺) in tau aggregation, as studies suggest that zinc induces reversible tau oligomerization and can lead to tau hyper phosphorylation. The concentration of zinc is critical, as excessive levels can promote harmful tau aggregation, while normal levels are essential for physiological functions. We also examine natural and chemical compounds that can modulate tau aggregation, and lastly, we discuss how Tau protein can undergo liquid-liquid phase separation (LLPS) in neurons, forming droplets that can later develop into toxic oligomers, which are the primary hallmark of AD. We mention some molecules, such as proteins, nucleic acids, and metal ions, that influence tau LLPS and aggregation.

Recent advances in cellular biosensor technology to investigate tau oligomerization.

Tau is a microtubule binding protein which plays an important role in physiological functions but it is also involved in the pathogenesis of Alzheimer's disease and related tauopathies. While insoluble and β‐sheet containing tau neurofibrillary tangles have been the histopathological hallmark of these diseases, recent studies suggest that soluble tau oligomers, which are formed prior to fibrils, are the primary toxic species. Substantial efforts have been made to generate tau oligomers using purified recombinant protein strategies to study oligomer conformations as well as their toxicity. However, no specific toxic tau species has been identified to date, potentially due to the lack of cellular environment. Hence, there is a need for cell‐based models for direct monitoring of tau oligomerization and aggregation. This review will summarize the recent advances in the cellular biosensor technology, with a focus on fluorescence resonance energy transfer, bimolecular fluorescence complementation, and split luciferase complementation approaches, to monitor formation of tau oligomers and aggregates in living cells. We will discuss the applications of the cellular biosensors in examining the heterogeneous tau conformational ensembles and factors affecting tau self‐assembly, as well as detecting cell‐to‐cell propagation of tau pathology. We will also compare the advantages and limitations of each type of tau biosensors, and highlight their translational applications in biomarker development and therapeutic discovery.

Single-Molecule Characterization of Heterogeneous Soluble Protein Oligomers

Soluble protein oligomers are the primary toxic species in a number of human diseases including Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, cancer, and even certain viral infections. A complicating factor in establishing structure/dynamics/toxicity relationships for soluble oligomers is divergent experimental conditions, such as oligomer preparation protocols, which heavily influence experimental outcomes. Similarly, the low abundance and the structural and dynamic heterogeneity of oligomers are challenges to traditional (ensemble-averaged) biophysical/biochemical characterization. Single-molecule methods are powerful tools for probing this heterogeneity, since oligomers can be observed one at a time. We used single-molecule Förster resonance energy transfer (smFRET) to characterize the heterogeneous structures and dynamics of soluble amyloid-beta (aβ) oligomers prepared using several disparate protocols. The improved comparison between aβ oligomers generated under disparate experimental conditions will better enable correlations to be made between biophysical observables and possible pathways for mediating disease (e.g., direct membrane damage, redox activity, etc.). These correlations are a prerequisite to identifying molecular determinants of oligomer toxicity and therefore means of interfering therapeutically with their mechanisms.

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies

ObjectiveUnderstanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. BackgroundTauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. New/Updated HypothesisWe hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-of-concept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. Major Challenges for the HypothesisOur approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide high-resolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. Linkage to Other Major TheoriesOur biosensor technology is broadly applicable to other areas of tauopathy therapeutic development. These biosensors can be readily modified for different isoforms of tau, specific post-translational modifications, and familial Alzheimer's disease–associated mutations. We are eager to explore tau interactions with chaperone proteins, monitor cross-reactivity with other intrinsically disordered proteins, and target seeded oligomer pathology.

Large fatty acid-derived Aβ42 oligomers form ring-like assemblies.

As the primary toxic species in the etiology of Alzheimer disease (AD) are low molecular weight oligomers of Aβ, it is crucial to understand the structure of Aβ oligomers for gaining molecular insights into AD pathology. We have earlier demonstrated that in the presence of fatty acids, Aβ42 peptides assemble as 12-24mer oligomers. These Large Fatty Acid-derived Oligomers (LFAOs) exist predominantly as 12mers at low and as 24mers at high concentrations. The 12mers are more neurotoxic than the 24mers and undergo self-replication, while the latter propagate to morphologically distinct fibrils with succinct pathological consequences. In order to glean into their functional differences and similarities, we have determined their structures in greater detail by combining molecular dynamic simulations with biophysical measurements. We conjecture that the LFAO are made of Aβ units in an S-shaped conformation, with the 12mers forming a double-layered hexamer ring (6 × 2) while the structure of 24mers is a double-layered dodecamer ring (12 × 2). A closer inspection of the (6 × 2) and (12 × 2) structures reveals a concentration and pH dependent molecular reorganization in the assembly of 12 to 24mers, which seems to be the underlying mechanism for the observed biophysical and cellular properties of LFAOs.

Computational Study of the Driving Forces and Dynamics of Curcumin Binding to Amyloid-β Protofibrils

Oligomeric aggregates of the amyloid-β (Aβ) peptide are believed to be the primary toxic species that initiate events leading to neurodegeneration and cognitive decline in Alzheimer's disease (AD). Small molecules that interfere with Aβ aggregation and/or neurotoxicity are being investigated as potential therapeutics for AD, including naturally occurring polyphenols. We have recently shown that curcumin exerts a neuroprotective effect against Aβ40-induced toxicity on cultured neuronal cells through two possible concerted pathways, ameliorating Aβ oligomer-induced toxicity and inducing the formation of nontoxic Aβ oligomers, both of which involve curcumin binding to Aβ oligomers. To gain molecular-level insights into curcumin's interaction with Aβ oligomers, we use all-atom molecular dynamics (MD) simulations to study the dynamics and energetics of curcumin binding to an Aβ protofibril composed of 24 peptides. Our results show that curcumin binds to specific hydrophobic sites on the protofibril surface and that binding is generally associated with the concomitant complexation of curcumin into dimers, trimers, or tetramers. Curcumin also binds to the protofibril growth axis ends but without complexation. Analysis of the energetics of the binding process revealed that curcumin complexation contributes in an additive fashion to curcumin-Aβ protofibril interactions. Favorable curcumin-protofibril binding is driven by a combination of hydrophobic interactions between curcumin and protofibril, curcumin self-aggregation, and solvation effects. These interactions are likely critical in blocking Aβ oligomer toxicity and inducing the growth of the protofibrils into "off-pathway" wormlike fibrils observed experimentally.

Blocking Oligomeric Insulin Amyloid Fibrillation via Perylenebisimides Containing Dipeptide Tentacles.

Molecular motifs that could interfere with amyloid fibrillation via non-covalent interactions are vital for aberrant protein aggregation and related human diseases. Mutual aggregation ensues in the presence of these structural motifs, and nucleation on the particle surface leads to inhibition of the fibrillization process. This modular process generates a new generation of inhibitory reagents. Oligomers are the primary toxic species that initiate pathogenic aggregation, leading to toxic β-sheet-rich structures. To inhibit these toxic oligomers, two dipeptide-linked perylenebisimide isomers (PAPAP and APPPA) are developed as selective modulators for insulin fibrillization. Early insulin aggregates are adsorbed onto the modulator surface and stabilize soluble oligomeric aggregates. Fibrillation and inhibition are examined by thioflavin T (ThT) assay in the presence and the absence of both inhibitors PAPAP and APPPA. Conformational modulation using far-UV circular dichroism studies also highlights their role as aggregation inhibitors via reduction of α-helix into β-sheet along with increased random coil contents. Moreover, the inhibitory effects were more pronounced due to the ability of these isomers to undergo varying multiple non-covalent interactions, a performance well beyond all known modulators with respect to selectivity and efficiency, with the more-aggregation-prone derivative due to higher probability of a hydrophobic encounter between the protein and the molecular modulator. These results also lead to the elucidation of an insulin fibril regulating mechanism via selective non-covalent binding and provide fundamental insights into the chemistry of peptide-based probes as tools for developing next-generation therapeutics.

Modulating Early Stage Amyloid Aggregates by Dipeptide-Linked Perylenebisimides: Structure-Activity Relationship, Inhibition of Fibril Formation in Human CSF and Aβ1-40.

Amyloid aggregation is observed in many neurodegenerative diseases, but the formation of final plaque seldom correlates to the disease severity. Early and intermediate structures such as soluble oligomers are considered as primary toxic species in protein misfolding diseases specifically linked to Aβ in Alzheimer's disease (AD). Two peptide-linked perylenebisimide isomers (PAPAP and APPPA) were developed to study the structure-activity relationship with a toxic Aβ oligomer in commercial Aβ as well as in human cerebrospinal fluid (CSF), diminish and inhibit them, and prevent them from forming toxic amyloid fibrils from an early stage. Self-aggregation of perylenebisimides enables the formation of nano/micro-objects that are used to interact with the hydrophobic regions of the peptide and direct the peptide aggregation into an "off-pathway", preventing mature fibril formation. Remarkably, one of the Ala-Phe dipeptide-linked perylenebisimide isomers (APPPA) showed a high selectivity toward an Aβ oligomer and could also cross the endothelial monolayer barrier (blood-brain barrier, BBB) more efficiently than the other derivative (PAPAP). Kinetic ThT studies and AFM imaging provided strong proof of both of the isomers being able to inhibit fibrillation of prefibrillar and oligomeric Aβ in both the commercial Aβ1-40 peptide as well as in the real human CSF sample. Further, a correlation has been built using pristine fluorescence of perylenebisimides, showing modulation and "oligo-blocking". The obtained data provides clear evidence that the mutual aggregation between the modulator and amyloid aggregate becomes predominant compared to their individual aggregation. These results reinforce the development of the structural platform design to diminish toxic oligomers, inhibit them, and prevent the formation of toxic amyloid fibrils at an early stage.

Porphyrin Cyclodextrin Conjugates Modulate Amyloid Beta Peptide Aggregation and Cytotoxicity.

Although fibrillar amyloid beta peptide (Aβ) aggregates are one of the major hallmarks of Alzheimer's disease, increasing evidence suggests that soluble Aβ oligomers are the primary toxic species. Targeting the oligomeric species could represent an effective strategy to interfere with Aβ toxicity. In this work, the biological properties of 5[4-(6-O-β-cyclodextrin)-phenyl],10,15,20-tri(4-hydroxyphenyl)-porphyrin and its zinc complex were tested, as new molecules that interact with Aβ and effectively prevent its cytotoxicity. We found that these systems can cross the cell membrane to deliver Aβ intracellularly and promote its clearance. Our results provide evidence for the use of cyclodextrin-porphyrin derivatives as a promising strategy to target amyloid aggregation.

IKONOS Image-Based Extraction of the Distribution Area of Stellera chamaejasme L. in Qilian County of Qinghai Province, China

Stellera chamaejasme L. (S. chamaejasme) is one of the primary toxic grass species (poisonous plants) distributed in the alpine meadows of Qinghai Province, China. In this study, according to the distinctive phenological characteristics of S. chamaejasme, the spectral differences between S. chamaejasme in the full-bloom stage and other pasture grasses were analyzed and the red, blue, and near-infrared bands of IKONOS image were determined as the diagnostic bands of S. chamaejasme recognition. Feature indexes related to S. chamaejasme were established using the diagnostic bands, and \(NDVI_{blue} = (\rho_{nir} − \rho_{blue})/(\rho_{nir} + \rho_{blue})\) obtained as S. chamaejasme sensitive index based on the linear regression analysis between the indexes derived from field spectra and the actual cover fraction of S. chamaejasme communities. The distribution area of S. chamaejasme was extracted by using the index \(NDVI_{blue}\) derived from IKONOS multispectral image in Qilian County of Qinghai Province, China and the verified result reached an overall accuracy of 90.71%. The study indicated that high resolution multispectral satellite images (such as IKONOS images) had significant potential in remote sensing recognition of toxic grass species.

Amyloid fibrils are the molecular trigger of inflammation in Parkinson's disease

Parkinson's disease (PD) is an age-related movement disorder characterized by a progressive degeneration of dopaminergic neurons in the midbrain. Although the presence of amyloid deposits of α-synuclein (α-syn) is the main pathological feature, PD brains also present a severe permanent inflammation, which largely contributes to neuropathology. Although α-syn has recently been implicated in this process, the molecular mechanisms underlying neuroinflammation remain unknown. In the present study, we investigated the ability of different α-syn aggregates to trigger inflammatory responses. We showed that α-syn induced inflammation through activation of Toll-like receptor 2 (TLR2) and the nucleotide oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome only when folded as amyloid fibrils. Oligomeric species, thought to be the primary species responsible for the disease, were surprisingly unable to trigger the same cascades. As neuroinflammation is a key player in PD pathology, these results put fibrils back to the fore and rekindles discussions about the primary toxic species contributing to the disease. Our data also suggest that the inflammatory properties of α-syn fibrils are linked to their intrinsic structure, most probably to their cross-β structure. Since fibrils of other amyloids induce similar immunological responses, we propose that the canonical fibril-specific cross-β structure represents a new generic motif recognized by the innate immune system.

Heme Stabilization of α-Synuclein Oligomers during Amyloid Fibril Formation.

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases.

Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry.

There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity.

The predominant species of ionic silver in biological media is colloidally dispersed nanoparticulate silver chloride

We have investigated the behaviour of silver ions in biologically relevant concentrations (10 to 100 ppm) in different media, from physiological salt solution over phosphate-buffered saline solution to protein-containing cell culture media. The results show that the initially present silver ions are bound as silver chloride due to the presence of chloride. Only in the absence of chloride, glucose is able to reduce Ag+ to Ag0. The precipitation of silver phosphate was not observed in any case. We conclude that the predominant silver species in biological media is dispersed nanoscopic silver chloride, surrounded by a protein corona which prevents the growth of the crystals and leads to colloidal stabilization. Therefore, in cell culture experiments where dissolved silver ions are studied in the upper ppm range, in fact the effect of colloidally dispersed silver chloride is observed. We have confirmed this by cell culture experiments (human mesenchymal stem cells; T-cells; monocytes) and bacteria (S. aureus) where the cells were incubated with synthetically prepared silver chloride nanoparticles (diameter ca. 100 nm). These were easily taken up by eukaryotic cells and showed the same toxic effect at the same silver concentration as ionic silver (as silver acetate). Therefore, nanoscopic silver chloride and not free ionic silver is the primary toxic species in biological media.

Understanding Amyloid Fibril Nucleation and Aβ Oligomer/Drug Interactions from Computer Simulations

Evolution has fine-tuned proteins to accomplish a variety of tasks. Yet, with aging, some proteins assemble into harmful amyloid aggregates associated with neurodegenerative diseases, such as Alzheimer's disease (AD), which presents a complex and costly challenge to our society. Thus, far, drug after drug has failed to slow the progression of AD, characterized by the self-assembly of the 39-43 amino acid β-amyloid (Aβ) protein into extracellular senile plaques that form a cross-β structure. While there is experimental evidence that the Aβ small oligomers are the primary toxic species, standard tools of biology have failed to provide structures of these transient, inhomogeneous assemblies. Despite extensive experimental studies, researchers have not successfully characterized the nucleus ensemble, the starting point for rapid fibril formation. Similarly scientists do not have atomic data to show how the compounds that reduce both fibril formation and toxicity in cells bind to Aβ42 oligomers. In this context, computer simulations are important tools for gaining insights into the self-assembly of amyloid peptides and the molecular mechanism of inhibitors. This Account reviews what analytical models and simulations at different time and length scales tell us about the dynamics, kinetics, and thermodynamics of amyloid fibril formation and, notably, the nucleation process. Though coarse-grained and mesoscopic protein models approximate atomistic details by averaging out unimportant degrees of freedom, they provide generic features of amyloid formation and insights into mechanistic details of the self-assembly process. The thermodynamics and kinetics vary from linear peptides adopting straight β-strands in fibrils to longer peptides adopting in parallel U shaped conformations in fibrils. In addition, these properties change with the balance between electrostatic and hydrophobic interactions and the intrinsic disorder of the system. However, simulations suggest that the critical nucleus size might be on the order of 20 chains under physiological conditions. The transition state might be characterized by a simultaneous change from mixed antiparallel/parallel β-strands with random side-chain packing to the final antiparallel or parallel states with the steric zipper packing of the side chains. Second, we review our current computer-based knowledge of the 3D structures of inhibitors with Aβ42 monomer and oligomers, a prerequisite for developing new drugs against AD. Recent extensive all-atom simulations of Aβ42 dimers with known inhibitors such as the green tea compound epigallocatechin-3-gallate and 1,4-naphthoquinon-2-yl-l-tryptophan provide a spectrum of initial Aβ42/inhibitor structures useful for screening and drug design. We conclude by discussing future directions that may offer opportunities to fully understand nucleation and further AD drug development.

Therapeutic potential of a novel cannabinoid agent CB52 in the mouse model of experimental autoimmune encephalomyelitis

Multiple Sclerosis (MS) is a demyelinating disease which causes inflammation, demyelination, and axonal injury. Currently, there is no cure for the disease. The endocannabinoid system has recently emerged as a promising therapeutic target for MS. The protective mechanisms of cannabinoids are thought to be mediated by the activation of the cannabinoid type 1 (CB1) and type 2 (CB2) receptors expressed primarily in neurons and immune cells, respectively. However, the molecular mechanisms and the contribution of each receptor in ameliorating disease progression are still debatable. Although CB1 and CB2 receptors are expressed in oligodendrocytes, the myelin producing cells in the central nervous system, the role of cannabinoids in oligodendrocyte survival has not been well investigated. Using primary cultures of mature oligodendrocytes, we tested the effect of a novel synthetic cannabinoid CB52 on oligodendrocyte toxicity induced by peroxynitrite, the primary toxic species released by microglia. Interestingly, we found that CB52 is more potent than a number of broad and selective CB1 and CB2 agonists in protecting oligodendrocytes against peroxynitrite-induced toxicity. The protection provided by CB52 is likely due to its reduction of ERK1/2 phosphorylation and reactive oxygen species (ROS) generation in these cells. Using experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we found that CB52 reduces microglia activation, nitrotyrosine formation, T cell infiltration, oligodendrocyte toxicity, myelin loss and axonal damage in the mouse spinal cord white matter and alleviates the clinical scores when given either before or after disease onset. These effects are reversed by the CB1 receptor antagonist, but not by the CB2 receptor antagonist, suggesting that the activation of CB1 receptors contributes significantly to the anti-inflammatory and neuroprotective effects of cannabinoids on MS.

Fibrillar seeds alleviate amyloid-β cytotoxicity by omitting formation of higher-molecular-weight oligomers

Amyloid-β (Aβ) peptides can exist in distinct forms including monomers, oligomers and fibrils, consisting of increased numbers of monomeric units. Among these, Aβ oligomers are implicated as the primary toxic species as pointed by multiple lines of evidence. It has been suggested that toxicity could be rendered by the soluble higher-molecular-weight (high-n) Aβ oligomers. Yet, the most culpable form in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Moreover, the potential interaction among the insoluble fibrils that have been excluded from the responsible aggregates in AD development, Aβ monomers and high-n oligomers is undetermined. Here, we report that insoluble Aβ fibrillar seeds can interact with Aβ monomers at the stoichiometry of 1:2 (namely, each Aβ molecule of seed can bind to two Aβ monomers at a time) facilitating the fibrillization by omitting the otherwise mandatory formation of the toxic high-n oligomers during the fibril maturation. As a result, the addition of exogenous Aβ fibrillar seeds is seen to rescue neuronal cells from Aβ cytotoxicity presumably exerted by high-n oligomers, suggesting an unexpected protective role of Aβ fibrillar seeds.

Accumulation of insoluble forms of FUS protein correlates with toxicity in Drosophila

Recently, the fused in sarcoma/translated in liposarcoma (FUS) protein has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patients with frontotemporal lobar degeneration or amyotrophic lateral sclerosis. The molecular mechanisms underlying FUS toxicity are currently not understood. To address aspects of FUS pathogenesis in vivo, we have generated new Drosophila transgenic models expressing a full-length wild-type isoform of human FUS protein. We found that when expressed in retinal cells, FUS proteins are mainly recovered as soluble forms, and their overexpression results in a mild eye phenotype, with malformed interommatidial bristles and the appearance of ectopic extensions. On the other hand, when FUS proteins are specifically targeted to adult differentiated neurons, they are mainly recovered as insoluble forms, and their overexpression drastically reduces fly life span. Importantly, FUS neurotoxicity occurs regardless of inclusion formation. Lastly, we showed that molecular chaperones reduce FUS toxicity by modulating protein solubility. Altogether, our data indicate that accumulation of insoluble non-aggregated FUS forms might represent the primary toxic species in human FUS proteinopathies.

Key residues for the oligomerization of Aβ42 protein in Alzheimer’s disease

Deposition of amyloid fibrils consisting of amyloid β (Aβ) protein as senile plaques in the brain is a pathological hallmark of Alzheimer’s disease. However, a growing body of evidence shows that soluble Aβ oligomers correlate better with dementia than fibrils, suggesting that Aβ oligomers may be the primary toxic species. The structure and oligomerization mechanism of these Aβ oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of Aβ42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of Aβ sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8M urea at pH 10, is unfavorable for Aβ42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that Aβ42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS–PAGE shows that Aβ42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, Aβ40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of Aβ42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions between these residues are most critical for Aβ42 oligomerization.

Development and validation of a yeast high-throughput screen for inhibitors of Aβ42 oligomerization

SUMMARYRecent reports point to small soluble oligomers, rather than insoluble fibrils, of amyloid β (Aβ), as the primary toxic species in Alzheimer’s disease. Previously, we developed a low-throughput assay in yeast that is capable of detecting small Aβ42 oligomer formation. Specifically, Aβ42 fused to the functional release factor domain of yeast translational termination factor, Sup35p, formed sodium dodecyl sulfate (SDS)-stable low-n oligomers in living yeast, which impaired release factor activity. As a result, the assay for oligomer formation uses yeast growth to indicate restored release factor activity and presumably reduced oligomer formation. We now describe our translation of this assay into a high-throughput screen (HTS) for anti-oligomeric compounds. By doing so, we also identified two presumptive anti-oligomeric compounds from a sub-library of 12,800 drug-like small molecules. Subsequent biochemical analysis confirmed their anti-oligomeric activity, suggesting that this form of HTS is an efficient, sensitive and cost-effective approach to identify new inhibitors of Aβ42 oligomerization.