Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.     

Structural and morphological characterization of aggregated species of α-synuclein induced by docosahexaenoic acid

The interaction of brain lipids with α-synuclein may play an important role in the pathogenesis of Parkinson disease (PD). Docosahexaenoic acid (DHA) is an abundant fatty acid of neuronal membranes, and it is presents at high levels in brain areas with α-synuclein inclusions of patients with PD. In animal models, an increase of DHA content in the brain induces α-synuclein oligomer formation in vivo. However, it is not clear whether these oligomeric species are the precursors of the larger aggregates found in Lewy bodies of post-mortem PD brains. To characterize these species and to define the role of fatty acids in amyloid formation, we investigated the aggregation process of α-synuclein in the presence of DHA. We found that DHA readily promotes α-synuclein aggregation and that the morphology of these aggregates is dependent on the ratio between the protein and DHA. In the presence of a molar ratio protein/DHA of 1:10, amyloid-like fibrils are formed. These fibrils are morphologically different from those formed by α-synuclein alone and have a less packed structure. At a protein/DHA molar ratio of 1:50, we observe the formation of stable oligomers. Moreover, chemical modifications, methionine oxidations, and protein-lipid adduct formations are induced by increasing concentrations of DHA. The extent of these modifications defines the structure and the stability of aggregates. We also show that α-synuclein oligomers are more toxic if generated in the presence of DHA in dopaminergic neuronal cell lines, suggesting that these species might be important in the neurodegenerative process associated with PD.     

Single-Channel Electrophysiology Reveals a Distinct and Uniform Pore Complex Formed by α-Synuclein Oligomers in Lipid Membranes

Synucleinopathies such as Parkinson's disease, multiple system atrophy and dementia with Lewy bodies are characterized by deposition of aggregated α-synuclein. Recent findings indicate that pathological oligomers rather than fibrillar aggregates may represent the main toxic protein species. It has been shown that α-synuclein oligomers can increase the conductance of lipid bilayers and, in cell-culture, lead to calcium dyshomeostasis and cell death. In this study, employing a setup for single-channel electrophysiology, we found that addition of iron-induced α-synuclein oligomers resulted in quantized and stepwise increases in bilayer conductance indicating insertion of distinct transmembrane pores. These pores switched between open and closed states depending on clamped voltage revealing a single-pore conductance comparable to that of bacterial porins. Pore conductance was dependent on transmembrane potential and the available cation. The pores stably inserted into the bilayer and could not be removed by buffer exchange. Pore formation could be inhibited by co-incubation with the aggregation inhibitor baicalein. Our findings indicate that iron-induced α-synuclein oligomers can form a uniform and distinct pore species with characteristic electrophysiological properties. Pore formation could be a critical event in the pathogenesis of synucleinopathies and provide a novel structural target for disease-modifying therapy.     

Converse modulation of toxic α-synuclein oligomers in living cells by N′-benzylidene-benzohydrazide derivates and ferric iron

Intracellular α-synuclein (α-syn) aggregates are the pathological hallmark in several neurodegenerative diseases including Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. Recent evidence suggests that small oligomeric aggregates rather than large amyloid fibrils represent the main toxic particle species in these diseases. We recently characterized iron-dependent toxic α-syn oligomer species by confocal single molecule fluorescence techniques and used this aggregation model to identify several N′-benzylidene-benzohydrazide (NBB) derivatives inhibiting oligomer formation in vitro. In our current work, we used the bioluminescent protein-fragment complementation assay (BPCA) to directly analyze the formation of toxic α-syn oligomers in cell culture and to investigate the effect of iron and potential drug-like compounds in living cells. Similar to our previous findings in vitro, we found a converse modulation of toxic α-syn oligomers by NBB derivates and ferric iron, which was characterized by an increase in aggregate formation by iron and an inhibitory effect of certain NBB compounds. Inhibition of α-syn oligomer formation by the NBB compound 293G02 was paralleled by a reduction in cytotoxicity indicating that toxic α-syn oligomers are present in the BPCA cell culture model and that pharmacological inhibition of oligomer formation can reduce toxicity. Thus, this approach provides a suitable model system for the development of new disease-modifying drugs targeting toxic oligomer species. Moreover, NBB compounds such as 293G02 may provide useful tool compounds to dissect the functional role of toxic oligomer species in cell culture models and in vivo.     

γ-Synuclein: seeding of α-synuclein aggregation and transmission between cells

Protein misfolding and aggregation is a ubiquitous phenomenon associated with a wide range of diseases. The synuclein family comprises three small naturally unfolded proteins implicated in neurodegenerative diseases and some forms of cancer. α-Synuclein is a soluble protein that forms toxic inclusions associated with Parkinson's disease and several other synucleinopathies. However, the triggers inducing its conversion into noxious species are elusive. Here we show that another member of the family, γ-synuclein, can be easily oxidized and form annular oligomers that accumulate in cells in the form of deposits. Importantly, oxidized γ-synuclein can initiate α-synuclein aggregation. Two amino acid residues in γ-synuclein, methionine and tyrosine located in neighboring positions (Met(38) and Tyr(39)), are most easily oxidized. Their oxidation plays a key role in the ability of γ-synuclein to aggregate and seed the aggregation of α-synuclein. γ-Synuclein secreted from neuronal cells into conditioned medium in the form of exosomes can be transmitted to glial cells and cause the aggregation of intracellular proteins. Our data suggest that post-translationally modified γ-synuclein possesses prion-like properties and may induce a cascade of events leading to synucleinopathies.     

Antibodies against alpha-synuclein reduce oligomerization in living cells

Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.     

α-Synuclein in Extracellular Vesicles: Functional Implications and Diagnostic Opportunities

Fibrillar inclusions of intraneuronal α-synuclein can be detected in certain brain areas from patients with Parkinson’s disease (PD) and other disorders with Lewy body pathology. These insoluble protein aggregates do not themselves appear to have a prominent neurotoxic effect, whereas various α-synuclein oligomers appear harmful. Although it is incompletely known how the prefibrillar species may be pathogenic, they have been detected both within and on the outside of exosomes and other extracellular vesicles (EVs), suggesting that such structures may mediate toxic α-synuclein propagation between neurons. Vesicular transfer of α-synuclein may thereby contribute to the hierarchical spreading of pathology seen in the PD brain. Although the regulation of α-synuclein release via EVs is not understood, data suggest that it may involve other PD-related molecules, such as LRRK2 and ATP13A2. Moreover, new evidence indicates that CNS-derived EVs in plasma have the potential to serve as biomarkers for diagnostic purposes. In a recent study, levels of α-synuclein were found to be increased in L1CAM-positive vesicles isolated from plasma of PD patients compared to healthy controls, and follow-up studies will reveal whether α-synuclein in EVs could be developed as a future disease biomarker. Preferentially, toxic prefibrillar α-synuclein oligomers should then be targeted as a biomarker—as evidence suggests that they reflect the disease process more closely than total α-synuclein content. In such studies, it will be essential to adopt stringent EV isolation protocols in order to avoid contamination from the abundant pool of free plasma α-synuclein in different aggregational states.     

Characterization of heparin-induced glyceraldehyde-3-phosphate dehydrogenase early amyloid-like oligomers and their implication in α-synuclein aggregation

Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of α-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote α-synuclein aggregation. Taking into account the toxicity of α-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.     

Synergistic Amyloid Switch Triggered by Early Heterotypic Oligomerization of Intrinsically Disordered α-Synuclein and Tau

Amyloidogenic intrinsically disordered proteins, α-synuclein and tau are linked to Parkinson's disease and Alzheimer's disease, respectively. A body of evidence suggests that α-synuclein and tau, both present in the presynaptic nerve terminals, co-aggregate in many neurological ailments. The molecular mechanism of α-synuclein-tau hetero-assembly is poorly understood. Here we show that amyloid formation is synergistically facilitated by heterotypic association mediated by binding-induced misfolding of both α-synuclein and tau K18. We demonstrate that the intermolecular association is largely driven by the electrostatic interaction between the negatively charged C-terminal segment of α-synuclein and the positively charged tau K18 fragment. This heterotypic association results in rapid formation of oligomers that readily mature into hetero-fibrils with a much shorter lag phase compared to the individual proteins. These findings suggested that the critical intermolecular interaction between α-synuclein and tau can promote facile amyloid formation that can potentially lead to efficient sequestration of otherwise long-lived lethal oligomeric intermediates into innocuous fibrils. We next show that a well-known familial Parkinson's disease mutant (A30P) that is known to aggregate slowly via accumulation of highly toxic oligomeric species during the long lag phase converts into amyloid fibrils significantly faster in the presence of tau K18. The early intermolecular interaction profoundly accelerates the fibrillation rate of A30P α-synuclein and impels the disease mutant to behave similar to wild-type α-synuclein in the presence of tau. Our findings suggest a mechanistic underpinning of bypassing toxicity and suggest a general strategy by which detrimental amyloidogenic precursors are efficiently sequestered into more benign amyloid fibrils.     

Nucleation of β-rich oligomers and β-barrels in the early aggregation of human islet amyloid polypeptide

The self-assembly of human islet amyloid polypeptide (hIAPP) into β-sheet rich amyloid aggregates is associated with pancreatic β-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of α-helical intermediates before the rapid conversion into β-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more α-helices than β-sheets, consistent with ensemble-based experimental measurements. However, in ~4–6% independent simulations, β-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These β-rich oligomers could adopt β-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these β-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. β-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.     

Hsp90 Inhibits α-Synuclein Aggregation by Interacting with Soluble Oligomers

Aggregated α-synuclein is one of the main components of the pathological Lewy bodies associated with Parkinson's disease (PD). Many other proteins, including chaperones such as Hsp90 and Hsp70, have been found co-localized with Lewy bodies and the expression levels of Hsp90 have been found to be increased in brains of PD patients. Although the role of Hsp70 in the aggregation of α-synuclein has been extensively studied, relatively little is known about the effect of Hsp90 on this process. Here, we have investigated if Hsp90 can prevent the aggregation of the A53T pathological mutant of α-synuclein in vitro. A detailed study using many biophysical methods has revealed that Hsp90 prevents α-synuclein from aggregating in an ATP-independent manner and that it forms a strong complex with the transiently populated toxic oligomeric α-synuclein species formed along the aggregation pathway. We have also shown that, upon forming a complex with Hsp90, the oligomers are rendered harmless and nontoxic to cells. Thus, we have clear evidence that Hsp90 is likely to play an important role on these processes in vivo.     

Elucidating the Aggregation Number of Dopamine-Induced α-Synuclein Oligomeric Assemblies

Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human α-synuclein, implicated in Parkinson’s disease. We show here that this approach is capable of accurately resolving mixtures of multiple distinct molecular species present in the same sample of dopamine-induced α-synuclein oligomers, and that we can determine the respective aggregation numbers of each species from a single histogram of bleaching steps. We found two distinct species with aggregation numbers of 15–19 monomers and 34–38 monomers. These results show that this single-molecule approach allows for the systematic study of the aggregation numbers of complex supramolecular assemblies formed under different aggregation conditions.     

α-Synuclein Oligomers: an Amyloid Pore?

In many human diseases, oligomeric species of amyloid proteins may play a pivotal role in cytotoxicity. Many lines of evidence indicate that permeabilization of cellular membranes by amyloid oligomers may be the key factor in disrupting cellular homeostasis. However, the exact mechanisms by which the membrane integrity is impaired remain elusive. One prevailing hypothesis, the so-called amyloid pore hypothesis, assumes that annular oligomeric species embed into lipid bilayers forming transbilayer protein channels. Alternatively, an increased membrane permeability could be caused by thinning of the hydrophobic core of the lipid bilayer due to the incorporation of the oligomers between the tightly packed lipids, which would facilitate the transport of small molecules across the membrane. In this review, we briefly recapitulate our findings on the structure of α-synuclein oligomers and the factors influencing their interaction with lipid bilayers. Our results, combined with work from other groups, suggest that α-synuclein oligomers do not necessarily form pore-like structures. The emerging consensus is that local structural rearrangements of the protein lead to insertion of specific regions into the hydrophobic core of the lipid bilayer, thereby disrupting the lipid packing.     

Cellular Uptake of α-Synuclein Oligomer-Selective Antibodies is Enhanced by the Extracellular Presence of α-Synuclein and Mediated via Fcγ Receptors

Immunotherapy targeting aggregated α-synuclein has emerged as a potential treatment strategy against Parkinson’s disease and other α-synucleinopathies. We have developed α-synuclein oligomer/protofibril selective antibodies that reduce toxic α-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective α-synuclein antibodies, linear epitope monoclonal α-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, α-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing α-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fcγ receptors were targeted and we then found that blockage of FcγRI and FcγRIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular α-synuclein and mediated via Fcγ receptors. Altogether, our finding lend further support to the belief that α-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic α-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of α-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen–antibody complexes.     

Membranes as modulators of amyloid protein misfolding and target of toxicity

Abnormal protein aggregation is a hallmark of various human diseases. α-Synuclein, a protein implicated in Parkinson's disease, is found in aggregated form within Lewy bodies that are characteristically observed in the brains of PD patients. Similarly, deposits of aggregated human islet amyloid polypeptide (IAPP) are found in the pancreatic islets in individuals with type 2 diabetes mellitus. Significant number of studies have focused on how monomeric, disaggregated proteins transition into various amyloid structures leading to identification of a vast number of aggregation promoting molecules and processes over the years. Inasmuch as these factors likely enhance the formation of toxic, misfolded species, they might act as risk factors in disease. Cellular membranes, and particularly certain lipids, are considered to be among the major players for aggregation of α-synuclein and IAPP, and membranes might also be the target of toxicity. Past studies have utilized an array of biophysical tools, both in vitro and in vivo, to expound the membrane-mediated aggregation. Here, we focus on membrane interaction of α-synuclein and IAPP, and how various kinds of membranes catalyze or modulate the aggregation of these proteins and how, in turn, these proteins disrupt membrane integrity, both in vitro and in vivo. The membrane interaction and subsequent aggregation has been briefly contrasted to aggregation of α-synuclein and IAPP in solution. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

GM1 oligosaccharide efficacy against α-synuclein aggregation and toxicity in vitro

Fibrillary aggregated α-synuclein represents the neurologic hallmark of Parkinson's disease and is considered to play a causative role in the disease. Although the causes leading to α-synuclein aggregation are not clear, the GM1 ganglioside interaction is recognized to prevent this process. How GM1 exerts these functions is not completely clear, although a primary role of its soluble oligosaccharide (GM1-OS) is emerging. Indeed, we recently identified GM1-OS as the bioactive moiety responsible for GM1 neurotrophic and neuroprotective properties, specifically reverting the parkinsonian phenotype both in in vitro and in vivo models.Here, we report on GM1-OS efficacy against the α-synuclein aggregation and toxicity in vitro. By amyloid seeding aggregation assay and NMR spectroscopy, we demonstrated that GM1-OS was able to prevent both the spontaneous and the prion-like α-synuclein aggregation. Additionally, circular dichroism spectroscopy of recombinant monomeric α-synuclein showed that GM1-OS did not induce any change in α-synuclein secondary structure. Importantly, GM1-OS significantly increased neuronal survival and preserved neurite networks of dopaminergic neurons affected by α-synuclein oligomers, together with a reduction of microglia activation.These data further demonstrate that the ganglioside GM1 acts through its oligosaccharide also in preventing the α-synuclein pathogenic aggregation in Parkinson's disease, opening a perspective window for GM1-OS as drug candidate.     

Comparison of aggregation enhancement and inhibition as strategies for reducing the cytotoxicity of the aortic amyloid polypeptide medin

Aortic medial amyloid (AMA) occurs as localised non-atheromatous plaques in virtually all individuals over the age of 50. The major protein component of AMA is the 50-residue polypeptide medin. Here we propose two methods of manipulating medin aggregation to reduce the cytotoxic species of medin: either by promoting formation of larger benign species or retaining small non-cytotoxic species. Medin co-localises with a variety of factors including glycosaminoglycans (GAGs). The first approach shows that the GAG heparin enhances the rate of medin aggregation and alters the morphology of the amyloid fibrils. Cellular viability measurements suggest that heparin eliminates small cytotoxic species of medin, promoting formation of benign fibrils. The second approach applies a previously successful approach of designing small peptide moieties that are complementary to the key amyloidogenic sequence but which contain modified amino acids known to disrupt hydrogen bonding and therefore prevent aggregation of the target protein. This approach also reduces cellular toxicity of medin at all stages of the aggregation process examined exhibiting a different mode of action to heparin. These results raise the question of whether enhancement of medin aggregation by GAGs is beneficial, by eliminating toxic oligomers, or has deleterious effects by reducing arterial plasticity associated with increased fibril load and whether small peptide inhibitors can be applied as drug candidates for amyloid diseases.     

The neurotransmitter serotonin interrupts α-synuclein amyloid maturation

Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.     

Soluble oligomers from a non-disease related protein mimic Abeta-induced tau hyperphosphorylation and neurodegeneration

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.