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See detailTHE PARKINSON’S DISEASE ASSOCIATED PINK1-PARKIN PATHWAY IN PATHOLOGY AND DEVELOPMENT
Jarazo, Javier UL

Doctoral thesis (2018)

Parkinson’s disease (PD) has an aetiology not completely understood. One of the hypothesis in the field is that many neurodegenerative diseases are influenced by developmental disorders. The underlying ... [more ▼]

Parkinson’s disease (PD) has an aetiology not completely understood. One of the hypothesis in the field is that many neurodegenerative diseases are influenced by developmental disorders. The underlying concept is that already during brain development some processes are deregulated producing a higher degree of susceptibility for neurodegeneration during aging. Two hereditary early onset forms of PD are caused by recessive mutations in PTEN-induced putative kinase 1 (PINK1) and Parkin genes that regulate mitochondrial function and morphology, quarantining damaged mitochondria before their degradation as well as triggering the process of mitophagy. Our hypothesis is that alterations of the Pink1-Parkin pathway have an impact in mitochondrial physiology tempering the differentiation ability of neuroepithelial stem cells into dopaminergic neurons. For evaluating this hypothesis we reprogramed patients’ fibroblasts carrying PINK1 mutations, as well as from healthy individuals, to human induced pluripotent stem cells. We developed a streamlined technique of gene editing (FACE) by using the CRISPR/Cas9 system combined with a composite of fluorescent proteins in the donor template for biallelic gene targeting. Isogenic controls were generated using this technique that allowed us to analyze the contribution of corrected patients’ mutations in the cellular defects observed. Human iPSCs were differentiated into a neuroepithelial stem cell state (NESC) from where the cells were further differentiated into neurons. We established different algorithms for pattern recognition and applied them for image analysis of different features such as mitochondrial morphology, proliferation capacity, apoptosis and differentiation. Patient’s derived cells presented an impaired differentiation efficiency into dopaminergic neurons as well as an imbalanced cell renewal that can be linked to the mitochondrial differences. Using 3D cultures, such as microfluidics and organoids, we were able to recapitulate this differentiation impairment in a system that mimics better the context of an in vivo environment. We evaluated the energetic capabilities of the NESCs and the firing activity of differentiated neurons, which also showed a dysregulation in patient cells. We introduced a new system for large-scale analysis of the autophagy and mitophagy pathways by the combination of stably integrated Rosella constructs in different patients’ lines and an image analysis script for classification of the different subcellular structures involved in these pathways activities. This revealed that the basal activity as well as the response against stressors of these pathways are altered in cells derived from patients having different mutations causative of PD. We performed a screen of repurposed drugs as well as of novel compounds to evaluate their impact in this altered developmental transition identifying a potential candidate to be further analysed in an in vivo context. [less ▲]

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