Functional and Transcriptional Studies of Human Dopaminergic Neurons

Abstract: Parkinson’s Disease (PD) is the most common movement disorder and second most common neurodegenerative disease. The principal hallmark of the pathology is represented by a loss of mesencephalic Dopaminergic neurons (mesDA) that reside in the Substantia Nigra pars compacta (SNpc). Another feature of the disease is represented by formation of abnormal protein aggregates, known as Lewy Bodies (LBs), mainly composed by the a-synuclein protein. The etiology of mesDA death is still unknown, however LBs formation could represent one of the factor contributing to neuronal mesDA death and PD progression.Cell Replacement Therapy for PD aims at restoring the function of the dopaminergic neurons through the transplantation of the lost cells in the brain. Recently, cell sources derived from stem cells such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) have been investigated and implicated in clinical trials for PD. Another route for generating neurons is represented by the direct reprogramming of terminally differentiated cells. With the overexpression of specific transcription factors (TFs) and/or micro RNA (miRNA) is possible to target somatic cells in vitro or resident brain cells in vivo for reprogramming into mesDA neurons.The overall aim of my thesis has been to study functional and transcriptional profile of newly generated mesDA neurons in vitro and in vivo for cell-based therapies of PD. Indeed the transplantation outcome depends on the ability to generate mesDA neurons that are as similar as possible to the endogenous DA neurons. However, our knowledge of human DA neurons is limited by the inaccessibility of developing and adult brain tissues. In the first part of my thesis I focused on studying the properties of directly reprogrammed cells to determine their phenotypic and functional profile. In the second part of this thesis, I performed an extensive molecular, transcriptional and functional analysis of human fetal mesDA neurons to increase our understanding of DA neurons. Lastly, I focused on establishing a stem cell derived organoid system that allowed for the generation of authentic human DA neurons.