Role of pro-inflammatory factors on the survival and differentiation of dopaminergic precursors

Abstract

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons (DAn) of the nigrostriatal system. Studies in animal models of PD have provided proof of concept that transplantation of DA precursors can relieve parkinsonian symptoms. However, a major limiting factor of this strategy is the poor survival rate of grafted DAn. This could be due to host inflammatory response, among other factors. Our previous results demonstrated a host primary response related to the graft of human dopaminergic precursors (DA14), with an increase of host-MHCII positive cells. Expression of tumor necrosis factor-alpha (TNF-alpha) was also detected on hostED1 positive cells. We aim to study the impact of the proinflammatory environment on DA14 cells and the effect of a TNFalpha inhibitor on an in vitro approach. DA14 cells were exposed to conditioned media (CM) from basal or activated BV2 microglial cells during 4 days. A significant increase in cell death was observed by fluorescence microscopy after Hoechst staining in DA14 cultures exposed with CM from activated microglia (p<0.05). This result was in accordance to a decrease in the number of Tyrosine hydroxylase (TH) positive cells detected by immunofluorescence (p<0.01). Neurite length measurement was performed to evaluate the differentiation process. A decrease in neurite length of TH positive cells was detected in DA14 cultures incubated with CM from activated microglia (p<0.05). In order to study the relevance of TNFalpha, DA14 cells were co-incubated with CM from basal or activated BV2 cells and the TNF-alpha inhibitor, Etanercept. Inhibition of TNF-alpha was able to avoid morphological alterations (p<0.05) and diminution of DA cells (p<0.05). Our results suggest that the pro-inflammatory microenvironment has a negative impact on survival and differentiation of DA14 cells. TNF-alpha inhibition could be an interesting strategy in order to improve survival of DA precursors.