Parkinson's disease is a neurodegenerative disease in which there is a progressive loss of function or structure of the dopaminergic nigrostriatal neurons. Since there is no effective therapy with few unwanted effects and no precise knowledge of the pathophysiological background of the disease, cellular models are being developed. Cellular models of the disease mimic the neurodegeneration that occurs in Parkinson's disease. In our work, we investigated the expression of γ-enolase, which has neurotrophic activity, and its regulation by cathepsin X. First, we established a model of differentiation of SH-SY5Y cells into the dopaminergic subtype. We differentiated the cells with retinoic acid and a combination of retinoic acid and phorbol-12-myristate-13-acetate. We confirmed the differentiation to the dopaminergic subtype by the observation of morphological changes, the determination of the expression of dopaminergic markers and the localisation of a dopaminergic marker. We then established a model of neurodegeneration of the above-mentioned dopaminergic subtype of SH-SY5Y with the neurotoxin 6-hydroxydopamine. We determined the optimal concentration of the neurotoxin (50 and 100 µM) and treatment period (24 hours) by measuring the percentage of dead cells. Neurodegeneration was also confirmed by altered morphological characteristics of the cells. Using the neurodegeneration model, we tested the expression of γ-enolase and the co-localisation of γ-enolase and cathepsin X. The C-terminal domain of γ-enolase shows a neurotrophic-like activity by supporting neuron survival, differentiation and regeneration. We have shown that the expression of the active form of γ-enolase is higher in differentiated cells, while the higher concentration of 6-hydroxydopamine decreases its expression. γ-Enolase is strongly co-localised with cathepsin X, which regulates the concentration of γ-enolase by cleaving C-terminal dipeptide, preventing its binding to γ1-syntrophin. Without binding to γ1-syntrophin, γ-enolase is unable to translocate to the cell membrane, where it exhibits its neurotrophic activity. Treatment of differentiated cells with 6-hydroxydopamine decreases the expression of the active form of γ-enolase, which corresponds with the higher activity of cathepsin X among differentiated cells. We tested the cathepsin X inhibitor AMS36, which had neuroprotective properties against 6-hydroxydopamine-induced neurotoxic effects and potentiated the expression of the active form of γ-enolase. In our work, we have thus demonstrated the importance of the regulation of the active form of γ-enolase by the proteolytic enzyme cathepsin X in with 6-hydroxydopamine-damaged SH-SY5Y cells, differentiated to the dopaminergic subtype.
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