The enzyme γ-enolase is a cytoplasmic enzyme involved in metabolic degradation of glucose. For its C-terminal part has also been shown to promote neuronal survival, regeneration and differentiation when properly bound to the plasma membrane. As part of the master's thesis we experimentally investigated role of γ-enolase in the process of differentiation of SH-SY5Y cell line into an individual neuronal phenotype. Firstly, we set up an in vitro cell differentiation model. We have shown that antibiotics inhibit the differentiation process, which manifests as reduced neuritogenesis, but with addition of 0.25 % concentration of antibiotics in the medium we can still sufficiently evelute differentiation of SH-SY5Y cell line. We proved that retinoic acid (RA) independently or in combination with brain-derived neurotrophic factor (BDNF) stimulates neurite outgrowth and F-actin polymerization, which affects reorganization of actin cytoskeleton, one of the first events in the process of neurite outgrowth. On the other hand, addition of phorbol 12-myristat 13-acetate (PMA) partially inhibits RA-induced cell neuritogenesis and actin polymerization. Differentiation factors also lead to development of a specific neuronal phenotype, and we have shown that treating cells with a combination of RA and PMA increases tyrosin hydroxylase expression, thus leading to the development of a dopaminergic neuron-like phenotype. We also showed that proliferation is partialy inhibited in cells treated with RA, PMA and BDNF, at the expanse of stimulated differentiation. We further demonstrated that expression of active form of γ-enolase is increased when cells are stimulated with RA alone or in combination with BDNF. In the last part of the thesis, we demonstrated that γ-Eno peptide, which mimics C-terminal part of the enzyme and is responsible for neurotrophic activity, increases neurite outgrowth of undifferentiated SH-SY5Y cells, while addition of kinase inhibitor Trk (K252a) partially inhibits this outgrowth. Peptide γ-Eno also increases phosphorylation of serine/threonine kinase (Akt) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) in non-differentiated cells, thus stimulating activation of phosphatidilinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways, which are inhibited in the presence of K252a. Finally, we have shown that γ-Eno peptide promotes neuritogenesis of RA alone or in combination with BDNF differentiated cells. The results of thesis suggest that γ-enolase is important in the process of promoting neuritogenesis and differentiation, and thus represents a potential target for designing agents to stimulate regeneration of damaged neurons.
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