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In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57°C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.The molecular basis of the pathogenicity of amyloid aggregates is a central theme in understanding the causes of a wide range of amyloid-related diseases, including Alzheimer's, Parkinson's, prion diseases, type II diabetes, and familial amyloidotic polyneuropathy (1-4). There is a striking difference between the amounts of amyloid depositions in various types of amyloid disorders. In systemic lysozyme amyloidosis, for example, the deposits can grow to kilogram quantities in the liver (5, 6). In neurodegenerative diseases, by contrast, there is no clear correlation between the amount of amyloid deposition and the clinical severity of disease. Significant cognitive impairment of Alzheimer's patients was observed without noticeable amyloid deposits in the brain, although the level of readily soluble amyloid oligomeric assemblies in the Alzheimer's brain was found to be greatly elevated (7,8).The evidence is accumulating that prefibrillar aggregates are cytotoxic both in vivo and in vitro (4,8,9), although this question is still open to debate. Moreover, aggregates of the proteins, which are not related to clinical amyloidoses, such as human ␣-lactalbumin (10), SH3 domain, or HypF-N protein, have also been found to be cytotoxic, which implies a common mechanism for cytotoxicity of misfolded proteins (11). By contrast, it has been shown that the mature amyl...

Peripheral immune responses can be sensitive indicators of disease pathology. We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of 26 Parkinson's disease (PD) patients compared with controls by using ELISA. We found a statistically significant increase of the autoimmune responses to both antigens in PD patients compared with controls with a mean increase of 70% and 50% in the autoimmune reactions towards insulin and S100B, respectively. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in PD patients, indicating that an amyloid-specific conformational epitope is not involved in immune recognition of this amyloid type, while sequential epitope of native insulin is hidden within the amyloid structures. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD.

We have found an increased level of serum antibodies to the prefibrillar structures of both Aβ_(25–35) peptide and human lysozyme in Alzheimer’s disease (AD) patients compared to age-matched controls, indicating that autoimmunity is implicated in AD. In the serum of AD patients with a long-term duration (>15 years) the titer of serum antibodies to aggregates of Aβ_(25–35) peptide increased by approximately 5-fold, whilst the antibody titer to lysozyme protofilaments decreased by approximately 8-fold compared to patients with AD duration of <5 years. The content of immunoglobulins of the A, G and M types declined, particularly in AD duration of >15 years. An increase in the concentration of immune complexes and higher lysozyme activity was detected in the serum of all patients and this was suggestive of an inflammatory reaction. We propose that the autoimmune response to different amyloid structures in AD can be viewed as a clearance pathway targeting amyloid development. Autoimmune response can be exploited as a marker of ongoing protein aggregation and hence be used as a diagnostic feature of AD.