Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid?

The research explores the molecular mechanisms behind the cytotoxicity of amyloid oligomers formed by horse milk lysozyme. The findings show that these oligomers, particularly larger ones, are toxic to different cell types and that the size of the oligomer affects its toxicity. This supports the idea that cytotoxicity is a common feature of protein amyloid and proposes that inhibiting oligomer formation could prevent amyloid-related diseases.

Mechanisms of Cytotoxicity of Amyloid Oligomers

• The researchers examined the cytotoxicity of amyloid oligomers formed by horse milk lysozyme, a type of protein found in the milk of mares.
• Thioflavin T and Congo Red, which are used to identify amyloid fibrils, were bound to the oligomers and protofibrils formed during incubation of the lysozyme at pH 2.0, 4.5 and a temperature of 57 degrees Celsius.
• The lysozyme structures were then analysed using atomic force microscopy, a type of scanning probe microscopy. Their morphological features and size were investigated as part of this analysis.

Amyloid Oligomers and Cell Viability

• The study found that monomeric lysozyme and its fibrils had no effect on the viability of three different cell types used in the experiments: primary murine (mouse) neurons, fibroblasts, and neuroblastoma cell line IMR-32.
• However, soluble amyloid oligomers of lysozyme were discovered to be lethal to all the cell types in question, as confirmed by flow-cytometry counting of dead cells stained with ethidium bromide, a nucleic acid stain used in cell biology.
• Primary cell cultures were also found to be more sensitive to amyloid than the neuroblastoma cell line.

Role of Amyloid Oligomer Size in Cytotoxicity

• It was observed that cytotoxicity depended on the size of the oligomeric particles, with samples containing 20-mers (formed at pH 4.5) found to be more toxic than the smaller tetramers and octamers present in the solution at pH 2.0.
• Interestingly, the soluble amyloid oligomers were found to be capable of self-assembling into doughnut-like structures. However, no correlation was seen between the quantity of these structures and cytotoxicity levels.

Implications for Amyloid Study and Treatment

• The findings substantiate the hypothesis that cytotoxicity is typical of protein amyloid, as they clearly demonstrate the cytotoxic effects of amyloid oligomers formed by lysozyme, a rather abundant protein.
• This research consequently suggests that inhibiting the formation of intermediate oligomers might be a crucial strategy in preventing the pathogeneses, or the processes that lead to diseases, associated with amyloid proteins.