Generation and differentiation of dopaminergic neurons from mouse multipotent and human induced pluripotent stem cells to study parkinson’s disease

Abstract

Parkinson’s disease (PD) is mainly characterized by the degeneration of DA neurons of the substantia nigra pars compacta, of largely unknow etiology although mutations in the NURR1 and GBA1 genes, among others, are genetic risk factors for this human disease. Thus, studying the dopaminergic (DA) system and establishing cellular models to study this disease might be very useful to understand its etiology and for therapeutic purposes. Consequently, in this thesis we have generated two cellular models, a mouse model based on NURR1 overexpression in neural stem cells, and a human model based on induced pluripotent cell (iPSC) generated by reprogramming fibroblasts isolated from PD patients with GBA1 mutations and controls. The human model has allowed us to study cellular, molecular and functional phenotypes related with this disease. The transcription factor NURR1 is involved in progenitor cell differentiation, and the maturation, survival and maintenance of DA neurons. Its influence on the generation of DA neurons in cells isolated from the olfactory bulb region remained unclear. Thus, in this thesis we have overexpressed NURR1 in embryonic mouse olfactory bulb stem cells (eOBSCs) and we have found a marked inhibition of cell proliferation concomitant with an upregulation of the dopaminergic transcripts Th and Dat; and of the fibroblast growth factor receptor-2 (Fgfr2), suggesting a role of this receptor in regulating DA neuron generation. Moreover, NURR1 overexpression triggered the generation of two populations of DA neurons: a major mesencephalic-like DA population and an OB-like DA subpopulation. Some of the neurons obtained expressed molecular markers of mature DA neurons, synaptic proteins, responded to dopaminergic stimulation, and released dopamine indicating that they acquired functional properties. Mutations in the glucocerebrosidase1 (GBA1) in heterozygosis are considered the strongest genetic risk factor for PD development. In this thesis we have established a cellular model to study PD associated with GBA1 mutations. Fibroblasts isolated from control subjects and PD patients with N370S/wt and L444P/wt GBA1 mutations have been reprogrammed to human iPSCs, using Sendai viral vectors, which have been differentiated into DA neurons. After 17-18 days of differentiation, the hiPSC-derived cells started to express mesencephalic dopaminergic markers. Later, we have detected dopamine release after 33-59 days and electrophysiological properties after 80-92 days of differentiation. Notably, we have found effects of the GBA1 mutations on the expression of the chaperone CRYAB, dopamine release and excitability of the neurons generated.