Structure-Toxicity Relationship in Intermediate Fibrils from α-Synuclein Condensates.

The aberrant aggregation of α-synuclein (αS) into amyloid fibrils is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson's disease. Although the structural properties of mature amyloids of αS are currently understood, the nature of transient protofilaments and fibrils that appear during αS aggregation remains elusive. Using solid-state nuclear magnetic resonance (ssNMR), cryogenic electron microscopy (cryo-EM), and biophysical methods, we here characterized intermediate amyloid fibrils of αS forming during the aggregation from liquid-like spherical condensates to mature amyloids adopting the structure of pathologically observed aggregates. These transient amyloid intermediates, which induce significant levels of cytotoxicity when incubated with neuronal cells, were found to be stabilized by a small core in an antiparallel ß-sheet conformation, with a disordered N-terminal region of the protein remaining available to mediate membrane binding. In contrast, mature amyloids that subsequently appear during the aggregation showed different structural and biological properties, including low levels of cytotoxicity, a rearranged structured core embedding also the N-terminal region, and a reduced propensity to interact with the membrane. The characterization of these two fibrillar forms of αS, and the use of antibodies and designed mutants, enabled us to clarify the role of critical structural elements endowing intermediate amyloid species with the ability to interact with membranes and induce cytotoxicity.

Characterization of Pairs of Toxic and Nontoxic Misfolded Protein Oligomers Elucidates the Structural Determinants of Oligomer Toxicity in Protein Misfolding Diseases.

The aberrant misfolding and aggregation of peptides and proteins into amyloid aggregates occurs in over 50 largely incurable protein misfolding diseases. These pathologies include Alzheimer's and Parkinson's diseases, which are global medical emergencies owing to their prevalence in increasingly aging populations worldwide. Although the presence of mature amyloid aggregates is a hallmark of such neurodegenerative diseases, misfolded protein oligomers are increasingly recognized as of central importance in the pathogenesis of many of these maladies. These oligomers are small, diffusible species that can form as intermediates in the amyloid fibril formation process or be released by mature fibrils after they are formed. They have been closely associated with the induction of neuronal dysfunction and cell death. It has proven rather challenging to study these oligomeric species because of their short lifetimes, low concentrations, extensive structural heterogeneity, and challenges associated with producing stable, homogeneous, and reproducible populations. Despite these difficulties, investigators have developed protocols to produce kinetically, chemically, or structurally stabilized homogeneous populations of protein misfolded oligomers from several amyloidogenic peptides and proteins at experimentally ameneable concentrations. Furthermore, procedures have been established to produce morphologically similar but structurally distinct oligomers from the same protein sequence that are either toxic or nontoxic to cells. These tools offer unique opportunities to identify and investigate the structural determinants of oligomer toxicity by a close comparative inspection of their structures and the mechanisms of action through which they cause cell dysfunction.This Account reviews multidisciplinary results, including from our own groups, obtained by combining chemistry, physics, biochemistry, cell biology, and animal models for pairs of toxic and nontoxic oligomers. We describe oligomers comprised of the amyloid-ß peptide, which underlie Alzheimer's disease, and α-synuclein, which are associated with Parkinson's disease and other related neurodegenerative pathologies, collectively known as synucleinopathies. Furthermore, we also discuss oligomers formed by the 91-residue N-terminal domain of [NiFe]-hydrogenase maturation factor from E. coli, which we use as a model non-disease-related protein, and by an amyloid stretch of Sup35 prion protein from yeast. These oligomeric pairs have become highly useful experimental tools for studying the molecular determinants of toxicity characteristic of protein misfolding diseases. Key properties have been identified that differentiate toxic from nontoxic oligomers in their ability to induce cellular dysfunction. These characteristics include solvent-exposed hydrophobic regions, interactions with membranes, insertion into lipid bilayers, and disruption of plasma membrane integrity. By using these properties, it has been possible to rationalize in model systems the responses to pairs of toxic and nontoxic oligomers. Collectively, these studies provide guidance for the development of efficacious therapeutic strategies to target rationally the cytotoxicity of misfolded protein oligomers in neurodegenerative conditions.

Amyloid fibrils act as a reservoir of soluble oligomers, the main culprits in protein deposition diseases.

Amyloid fibril formation plays a central role in the pathogenesis of a number of neurodegenerative diseases, including Alzheimer and Parkinson diseases. Transient prefibrillar oligomers forming during the aggregation process, exhibiting a small size and a large hydrophobic surface, can aberrantly interact with a number of molecular targets on neurons, including the lipid bilayer of plasma membranes, resulting in a fatal outcome for the cells. By contrast, the mature fibrils, despite presenting generally a high hydrophobic surface, are endowed with a low diffusion rate and poorly penetrate the interior of the lipid bilayer. However, increasing evidence shows that both intracellular α-synuclein fibrils, as well and as extracellular amyloid-ß and ß2-microglobulin fibrils, can release oligomers over time that quickly diffuse to reach the membrane of the neighboring cells. The persistent leakage of harmful oligomers from fibrils triggers an ongoing cascade of events resulting in a sustained injury to neurons and glia and also provides aggregates with the ability to cross biological membranes and diffuse between cells or cellular compartments.

Effects of oligomer toxicity, fibril toxicity and fibril spreading in synucleinopathies.

Protein misfolding is a general hallmark of protein deposition diseases, such as Alzheimer's disease or Parkinson's disease, in which different types of aggregated species (oligomers, protofibrils and fibrils) are generated by the cells. Despite widespread interest, the relationship between oligomers and fibrils in the aggregation process and spreading remains elusive. A large variety of experimental evidences supported the idea that soluble oligomeric species of different proteins might be more toxic than the larger fibrillar forms. Furthermore, the lack of correlation between the presence of the typical pathological inclusions and disease sustained this debate. However, recent data show that the ß-sheet core of the α-Synuclein (αSyn) fibrils is unable to establish persistent interactions with the lipid bilayers, but they can release oligomeric species responsible for an immediate dysfunction of the recipient neurons. Reversibly, such oligomeric species could also contribute to pathogenesis via neuron-to-neuron spreading by their direct cell-to-cell transfer or by generating new fibrils, following their neuronal uptake. In this Review, we discuss the various mechanisms of cellular dysfunction caused by αSyn, including oligomer toxicity, fibril toxicity and fibril spreading.

Misfolded protein oligomers induce an increase of intracellular Ca2+ causing an escalation of reactive oxidative species.

Alzheimer's disease is characterized by the accumulation in the brain of the amyloid ß (Aß) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aß also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aß oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aß42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca2+ than ROS levels, and a lag phase in the latter process. A Ca2+-deprived cell medium prevented the increase of intracellular Ca2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca2+ causes the production of ROS in cells challenged by aberrant protein oligomers.

The Toxicity of Protein Aggregates: New Insights into the Mechanisms.

The aberrant aggregation of specific peptides and proteins is the common feature of a range of more than 50 human pathologies, collectively referred to as protein misfolding diseases [...].

Calcium Dyshomeostasis in Alzheimer's Disease Pathogenesis.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder that is characterized by amyloid ß-protein deposition in senile plaques, neurofibrillary tangles consisting of abnormally phosphorylated tau protein, and neuronal loss leading to cognitive decline and dementia. Despite extensive research, the exact mechanisms underlying AD remain unknown and effective treatment is not available. Many hypotheses have been proposed to explain AD pathophysiology; however, there is general consensus that the abnormal aggregation of the amyloid ß peptide (Aß) is the initial event triggering a pathogenic cascade of degenerating events in cholinergic neurons. The dysregulation of calcium homeostasis has been studied considerably to clarify the mechanisms of neurodegeneration induced by Aß. Intracellular calcium acts as a second messenger and plays a key role in the regulation of neuronal functions, such as neural growth and differentiation, action potential, and synaptic plasticity. The calcium hypothesis of AD posits that activation of the amyloidogenic pathway affects neuronal Ca2+ homeostasis and the mechanisms responsible for learning and memory. Aß can disrupt Ca2+ signaling through several mechanisms, by increasing the influx of Ca2+ from the extracellular space and by activating its release from intracellular stores. Here, we review the different molecular mechanisms and receptors involved in calcium dysregulation in AD and possible therapeutic strategies for improving the treatment.

Transthyretin Inhibits Primary and Secondary Nucleations of Amyloid-ß Peptide Aggregation and Reduces the Toxicity of Its Oligomers.

Alzheimer's disease is associated with the deposition of the amyloid-ß peptide (Aß) into extracellular senile plaques in the brain. In vitro and in vivo observations have indicated that transthyretin (TTR) acts as an Aß scavenger in the brain, but the mechanism has not been fully resolved. We have monitored the aggregation process of Aß40 by thioflavin T fluorescence, in the presence or absence of different concentrations of preformed seed aggregates of Aß40, of wild-type tetrameric TTR (WT-TTR), and of a variant engineered to be stable as a monomer (M-TTR). Both WT-TTR and M-TTR were found to inhibit specific steps of the process of Aß40 fibril formation, which are primary and secondary nucleations, without affecting the elongation of the resulting fibrils. Moreover, the analysis shows that both WT-TTR and M-TTR bind to Aß40 oligomers formed in the aggregation reaction and inhibit their conversion into the shortest fibrils able to elongate. Using biophysical methods, TTR was found to change some aspects of its overall structure following such interactions with Aß40 oligomers, as well as with oligomers of Aß42, while maintaining its overall topology. Hence, it is likely that the predominant mechanism by which TTR exerts its protective role lies in the binding of TTR to the Aß oligomers and in inhibiting primary and secondary nucleation processes, which limits both the toxicity of Aß oligomers and the ability of the fibrils to proliferate.

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Partial Failure of Proteostasis Systems Counteracting TDP-43 Aggregates in Neurodegenerative Diseases.

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are progressive and fatal neurodegenerative disorders showing mislocalization and cytosolic accumulation of TDP-43 inclusions in the central nervous system. The decrease in the efficiency of the clearance systems in aging, as well as the presence of genetic mutations of proteins associated with cellular proteostasis in the familial forms of TDP-43 proteinopathies, suggest that a failure of these protein degradation systems is a key factor in the aetiology of TDP-43 associated disorders. Here we show that the internalization of human pre-formed TDP-43 aggregates in the murine neuroblastoma N2a cells promptly resulted in their ubiquitination and hyperphosphorylation by endogenous machineries, mimicking the post-translational modifications observed in patients. Moreover, our data identify mitochondria as the main responsible sites for the alteration of calcium homeostasis induced by TDP-43 aggregates, which, in turn, stimulates an increase in reactive oxygen species and, finally, caspase activation. The inhibition of TDP-43 proteostasis in the presence of selective inhibitors against the proteasome and macroautophagy systems revealed that these two systems are both severely involved in TDP-43 accumulation and have a strong influence on each other in neurodegenerative disorders associated with TDP-43.

Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using both immunofluorescence and Western blotting analysis, and nuclear functional TDP-43 was measured by monitoring the sortilin 1 mRNA splicing activity. Various degrees of TDP-43 cytoplasmic accumulation and nuclear TDP-43 depletion were achieved and the resulting cellular viability was evaluated, leading to a quantitative global analysis on the relative effects of LOF and GOF on the overall cytotoxicity. These were found to be ∼55% and 45%, respectively, in both cell lines and using both readouts of cell toxicity, showing that these two mechanisms are likely to contribute apparently equally to the pathologies of ALS and FTLD-U.

Single molecule experiments emphasize GM1 as a key player of the different cytotoxicity of structurally distinct Aß1-42 oligomers.

It is well established that cytotoxic Aß oligomers are the key factor that triggers the initial tissue and cell modifications eventually culminating in the development of Alzheimer's disease. Aß1-42 oligomers display a high degree of polymorphism, and several structurally different oligomers have been described. Amongst them, two types, recently classified as A+ and A-, have been shown to possess similar size but distinct toxic properties, as a consequence of their biophysical and structural differences. Here, we have investigated by means of single molecule tracking the oligomer mobility on the plasma membrane of living neuroblastoma cells and the interaction with the ganglioside GM1, a component of membrane rafts. We have found that A+ and A- oligomers display a similar lateral diffusion on the plasma membrane of living cells. However, only the toxic A+ oligomers appear to interact and alter the mobility of GM1. We have also studied the lateral diffusion of each kind of oligomers in cells depleted or enriched in GM1. We found that the content of GM1 influences the diffusion of both types of oligomer, although the effect of the increased levels of GM1 is higher for the A+ type. Interestingly, the content of GM1 also affects significantly the mobility of GM1 molecules themselves.

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations.

Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 µm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

Nucleophosmin contains amyloidogenic regions that are able to form toxic aggregates under physiological conditions.

Nucleophosmin (NPM)-1 is a multifunctional protein involved in a variety of biologic processes and has been implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments in NPM1 activities, we dissected the C-terminal domain (CTD) into its helical fragments. In this study, we observed the unexpected structural behavior of the peptide fragment corresponding to helix (H)2 (residues 264-277). This peptide has a strong tendency to form amyloidlike assemblies endowed with fibrillar morphology and ß-sheet structure, under physiologic conditions, as shown by circular dichroism, thioflavin T, and Congo red binding assays; dynamic light scattering; and atomic force microscopy. The aggregates are also toxic to neuroblastoma cells, as determined using 3-(4;5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and Ca(2+) influx assays. We also found that the extension of the H2 sequence beyond its N terminus, comprising the connecting loop with H1, delayed aggregation and its associated cytotoxicity, suggesting that contiguous regions of H2 have a protective role in preventing aggregation. Our findings and those in the literature suggest that the helical structures present in the CTD are important in preventing harmful aggregation. These findings could elucidate the pathogenesis of acute myeloid leukemia (AML) caused by NPM1 mutants. Because the CTD is not properly folded in these mutants, we hypothesize that the aggregation propensity of this NPM1 region is involved in the pathogenesis of AML. Preliminary assays on NPM1-Cter-MutA, the most frequent AML-CTD mutation, revealed its significant propensity for aggregation. Thus, the aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology.

Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers.

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.

Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro.

Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aß fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aß42). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aß42 oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability.

Extracellular chaperones prevent Aß42-induced toxicity in rat brains.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by cognitive decline, formation of the extracellular amyloid ß (Aß42) plaques, neuronal and synapse loss, and activated microglia and astrocytes. Extracellular chaperones, which are known to inhibit amyloid fibril formation and promote clearance of misfolded aggregates, have recently been shown to reduce efficiently the toxicity of HypF-N misfolded oligomers to immortalised cell lines, by binding and clustering them into large species. However, the role of extracellular chaperones on Aß oligomer toxicity remains unclear, with reports often appearing contradictory. In this study we microinjected into the hippocampus of rat brains Aß42 oligomers pre-incubated for 1h with two extracellular chaperones, namely clusterin and α2-macroglobulin. The chaperones were found to prevent Aß42-induced learning and memory impairments, as assessed by the Morris Water Maze test, and reduce Aß42-induced glia inflammation and neuronal degeneration in rat brains, as probed by fluorescent immunohistochemical analyses. Moreover, the chaperones were able to prevent Aß42 colocalisation with PSD-95 at post-synaptic terminals of rat primary neurons, suppressing oligomer cytotoxicity. All such effects were not effective by adding pre-formed oligomers and chaperones without preincubation. Molecular chaperones have therefore the potential to prevent the early symptoms of AD, not just by inhibiting Aß42 aggregation, as previously demonstrated, but also by suppressing the toxicity of Aß42 oligomers after they are formed. These findings elect them as novel neuroprotectors against amyloid-induced injury and excellent candidates for the design of therapeutic strategies against AD.

Membrane lipid composition and its physicochemical properties define cell vulnerability to aberrant protein oligomers.

Increasing evidence suggests that the interaction of misfolded protein oligomers with cell membranes is a primary event resulting in the cytotoxicity associated with many protein-misfolding diseases, including neurodegenerative disorders. We describe here the results of a study on the relative contributions to toxicity of the physicochemical properties of protein oligomers and the cell membrane with which they interact. We altered the amount of cholesterol and the ganglioside GM1 in membranes of SH-SY5Y cells. We then exposed the cells to two types of oligomers of the prokaryotic protein HypF-N with different ultrastructural and cytotoxicity properties, and to oligomers formed by the amyloid-ß peptide associated with Alzheimer's disease. We identified that the degree of toxicity of the oligomeric species is the result of a complex interplay between the structural and physicochemical features of both the oligomers and the cell membrane.

A single-domain antibody detects and neutralises toxic Aß42 oligomers in the Alzheimer's disease CSF.

BACKGROUND: Amyloid-ß42 (Aß42) aggregation consists of a complex chain of nucleation events producing soluble oligomeric intermediates, which are considered the major neurotoxic agents in Alzheimer's disease (AD). Cerebral lesions in the brain of AD patients start to develop 20 years before symptom onset; however, no preventive strategies, effective treatments, or specific and sensitive diagnostic tests to identify people with early-stage AD are currently available. In addition, the isolation and characterisation of neurotoxic Aß42 oligomers are particularly difficult because of their transient and heterogeneous nature. To overcome this challenge, a rationally designed method generated a single-domain antibody (sdAb), named DesAb-O, targeting Aß42 oligomers. METHODS: We investigated the ability of DesAb-O to selectively detect preformed Aß42 oligomers both in vitro and in cultured neuronal cells, by using dot-blot, ELISA immunoassay and super-resolution STED microscopy, and to counteract the toxicity induced by the oligomers, monitoring their interaction with neuronal membrane and the resulting mitochondrial impairment. We then applied this approach to CSF samples (CSFs) from AD patients as compared to age-matched control subjects. RESULTS: DesAb-O was found to selectively detect synthetic Aß42 oligomers both in vitro and in cultured cells, and to neutralise their associated neuronal dysfunction. DesAb-O can also identify Aß42 oligomers present in the CSFs of AD patients with respect to healthy individuals, and completely prevent cell dysfunction induced by the administration of CSFs to neuronal cells. CONCLUSIONS: Taken together, our data indicate a promising method for the improvement of an early diagnosis of AD and for the generation of novel therapeutic approaches based on sdAbs for the treatment of AD and other devastating neurodegenerative conditions.