The basic structure of the central nervous system is the neuron. Numerous in vitro cellular models are used to study processes in neurons, including differentiated SH-SY5Y cells. Neurotrophic factors play an important role in the differentiation of neurons, and the glycolytic enzyme γ-enolase also shows similar activity. The neurotrophic activity of γ-enolase is regulated by the cysteine peptidase cathepsin X, which proteolytically cleaves the last two amino acids from the C-terminal domain of γ-enolase. In this master's thesis, we determined the levels of protein expression and activity of cathepsin X and protein expression of enolase isoforms in phenotypically different populations of SH-SY5Y cells, which consist of three distinct phenotypes: epithelial S-type, neuronal N-type, and intermediate I-type SH-SY5Y cells. We first established a protocol for the differentiation and successful enrichment of individual populations of SH-SY5Y cells by modifying the cell culture plate coating, culture medium composition and cell growth time, which we monitored using a light inverted microscope. A 10-day cell culture growing in reduced serum medium content and the addition of 5 µM retinoic acid was found to be successful in obtaining enriched populations of S- and N-type cells. Through the assessment of marker expression using Western blotting and confocal microscopy, we observed that the expression of the epithelial cell marker vimentin was elevated in the S-type cell population, while the expression of neuronal markers, specifically tyrosine hydroxylase and growth-associated protein 43, was elevated in the N-type cell population. In the enriched populations of S- and N-type cells, we examined the expression levels of cathepsin X and its activity, as well as the expression levels of the target of cathepsin X in neuronal cells, enolase. We demonstrated that the highest expression of the cathepsin X protein was found in N-type cells, while the highest activity of cathepsin X was observed in S-type cells. The expression of the α-enolase isoform was independent of the individual SH-SY5Y cell populations, while a higher level of the γ-enolase isoform was characteristic of N-type SH-SY5Y cells, as evaluated by Western blotting and enzyme-linked immunosorbent assay. The expression of the active form of γ-enolase was elevated in both populations of SH-SY5Y cells compared to the control cells, nevertheless the increase was more noticeable in N-type SH-SY5Y cells. Using confocal microscopy, we demonstrated stronger co-localization of γ-enolase and cathepsin X in N-type cells, particularly in the cell extensions, so-called neurites, which suggests a role for γ-enolase in the process of neuronal growth and differentiation, and its regulation by cathepsin X.
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