As part of the Master's thesis, we wanted to test whether cytotoxic fragment A affects the expression of the (pro) cathepsin X gene in PC-12 cell line. The PC-12 cell line represents a good model for studying neurodegeneration, as it also expresses the inactive form of the enzyme - procathepsin X in lysosomes. Undifferentiated PC-12 cells were incubated with A at eight time intervals (each 3 hours apart), the quantity of (pro) cathepsin X was determined by an ELISA assay and the amount of active cathepsin X was measured by kinetic method of enzymatic activity in cell lysates. The results were normalized to the total amount of proteins, quantified by the Lowry method.
We found that the amount of total cathepsin X (pro and active form) increases statistically significantly within 3 h of incubation with Aβ(25-35) and remains elevated until 12 h, which could be due to the non-specific stress response of cells to the entry of the amyloid peptide into the cell. Exposure to Aβ(25-35) does not have a statistically significant effect on the amount of active cathepsin X in PC-12 cells. In spite of increased expression of the (pro) cathepsin X gene, the amount of active cathepsin X within the cells remains more or less constant. The possible excretion of active cathepsin X from cells into the culture medium could be confirmed by measuring the enzymatic activity of cathepsin X in the culture medium itself. The PC-12 model cells therefore respond to the presence of Aβ (25-35) in the culture medium by overexpressing intracellularly measured procathepsin X, which could represent a novel molecular mechanism in the defense against amyloid.
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