Papel de la esterasa diana de neuropátia (nte) y su gen codificante (pnpla6) en el desarrollo y la diferenciación celular in vitro

Resum

Role of neuropathy target esterase and its encoding gene (pnpla6) in in vitro cell development and differentiation abstract neuropathy target esterase (nte) was initially identified as the target of some organophosphorus compounds which cause the so-called organophosphorus-induced delayed neuropathy. Some in vivo studies indicate that this protein could also perform an important function in embryonic development and, therefore, in cell differentiation. This thesis aims to study the role of nte and the gene encoding it, pnpla6, in cell differentiation. D3 and r1 mouse embryonic stem cells (escs) and human embryonic tumour cells derived from ntera2/cl.D1 (hnt2) expressed nte enzymatic activity. Furthermore, d3 and hnt2 cells and human h9 escs also expressed gene pnpla6, even in its more undifferentiated state. D3 and h9 escs increased the pnpla6 expression by 4-5 times during early spontaneous differentiation stages, and the gene expression returned to the undifferentiated cells¿ level on around day 5 of the process. Nonetheless, the pnpla6 gene expression in hnt2 cells progressively increased with time as cells differentiated to neuronal and glial cells to reach a maximum expression, which was towards day 35 about 200 times higher than the undifferentiated cells¿ level. Pnpla6 silencing in d3 escs during spontaneous differentiation with specific silencing rna (sirna) lowered the gene expression by 80% and nte enzymatic activity by 50% after 48 h and 30 h of treatment, respectively. This silencing also altered the expression of 545 genes at 96 h after sirna exposure. The analyses of the most altered processes after pnpla6 silencing showed that the most affected pathways were those implied in organism development, cell assembly, cell organization and nervous system development, and suggest that pnpla6 could play an important role in lipid homeostasis and in vasculogenesis. Pnpla6 silencing in hnt2 during differentiation directed towards the neuroectoderm lowered the gene expression by 97% and nte enzymatic activity by 60% at 24 h and 96 h of sirna treatment, respectively. At these same differentiation points (days 1 and 4 of the process), 28 and 394 genes were altered by silencing, respectively. Altered functional pathways were related with epithelium formation, neurogenesis and development processes. Moreover, the number of neurons found in the silenced hnt2 cells cultures after 13 days of neurodifferentiation dropped by 80% if compared with control cultures, and major morphological alterations were observed in mature nerve cells after 66 days of differentiation. Chronic nte inhibition by neuropathic organophosphorus mipafox in hnt2 cells during neurodifferentiation caused no important genotypic or phenotypic alterations. To conclude, it has been demonstrated that nte and pnpla6 are implicated in differentiation and embryonic development as they are essential for nervous and vascular systems formation and for placenta development.