ROLE OF PRO-INFLAMMATORY FACTORS IN SURVIVAL AND DIFFERENTIATION OF DOPAMINERGIC PRECURSORS

SHIRLEY D WENKER

Congreso; XI Brazilian Association of Cell and Gene Therapy Meeting; 2021

Brazilian Association of Cell and Gene Therapy Meeting

Background: Parkinson?s disease (PD) is a neurodegenerative disease characterizedby the progressive loss of dopaminergic neurons (DAn) of the nigrostriatalsystem. Studies in animal models of PD have provided proof of concept thattransplantation of DA precursors can relieve parkinsonian symptoms. However, amajor limiting factor of this strategy is the poor survival rate of graftedDAn. This could be due to host inflammatory response, among other factors. Ourprevious results demonstrated a host primary response related to the graft of humandopaminergic precursors (DA14), with an increase of host-MHCII positive cells.Expression of tumor necrosis factor-alpha (TNF-alpha)was also detected on host-ED1 positive cells. We aim to study the impact of thepro-inflammatory environment on DA14 cells and the effect of a TNF-alpha inhibitorin an in vitro approach.Methods: BV2 microglialcells were treated with LPS, and nitrite and TNF alpha production weredetermined. DA14 cells were exposed to conditioned media (CM) from basal andactivated BV2 cells during 4 days. Cell morphology, cell death andimmunofluorescence for Tyrosine hydroxylase (TH) (DAn marker) was performed attwo stages: after acute exposure to a pro-inflammatory environment (DA19) andfinal stage of DA differentiation protocol (DA28).Results: Asignificant increase in cell death was observed by fluorescence microscopyafter Hoechst staining in DA14 cultures exposed with CM from activatedmicroglia. This result was in accordance to a decrease in TH positive cells inboth cell culture stages: DA19 and DA28. Moreover, a decrease in neuron likecells and neurite length of TH positive cells was detected in DA19 and DA28cultures incubated with CM from activated microglia. In order to study therelevance of TNF-alpha, DA15 cells were co-incubated with CM from basal or activatedBV2 cells and the TNF-alpha inhibitor, Etanercept. Inhibition of TNF-alpha wasable to avoid morphological alterations and diminution of DA cells. Conclusion: Our results suggest that the pro-inflammatory microenvironment has anegative impact on survival and differentiation of DA14 cells. TNF-alpha inhibitioncould be an interesting strategy in order to improve survival of DA precursors.