This document summarizes a thesis that uses induced pluripotent stem cells (iPSCs) as an in vitro model of Parkinson's disease. The thesis reprogrammed fibroblasts from patients with a genetic form of Parkinson's caused by SNCA gene duplication and from healthy donors into iPSCs. It then differentiated these iPSCs into dopaminergic neurons to evaluate differences in phenotype between healthy and affected cells. In particular, the thesis aims to investigate autophagy in these neurons under stress conditions, as autophagy has been linked to the clearance of alpha-synuclein, which seems impaired in Parkinson's disease.

1. UNIVERSITA' VITA-SALUTE SAN RAFFAELE
Facoltà di Medicina e Chirurgia
Corso di Laurea in Biotecnologie Mediche e Farmaceutiche
Elaborato finale di: Martina Tagliaferri
Relatore: Sonia Levi
Correlatore: Vania Broccoli
NEURONS DERIVED FROM PATIENT-SPECIFIC IPSCS AS AN IN VITRO MODEL OF
PARKINSON’S DISEASE
Parkinson’s Disease (PD) is a neurodegenerative disorder which is initially triggered by the loss of
dopaminergic neurons in substantia nigra pars compacta Commonly patients exhibit an array of
neurological symptoms including bradikinesia, rigidity and tremor, but some variablity in the
pathological manifestation is also pesent.The pathogenetic mechanisms that underly the
neurodegeneration process is still unclear but oxidative stress, protein aggregation, mitochondrial
dysfunctions and impaired autophagy have been shown to contribute. PD can be classified as
idiopathic, which happens in most of the cases (95%) or genetic,when one or few genes are
mutated. Up to now, 8 loci have been linked to genetic PD, among which 6 have been well studied
and the corresponding genes are known. Genetic forms are definitively less common but they can
be extremely useful to cast light on the molecular processes involved in the disorder. To evaluate
the role of genes involved in diseases, animal models have been generally resulted strongly
informative. However, despite a large effort, PD animal models have not been recapitulating the
hallmark of the disease and dopaminergic neuronal cell death is even not occurring. On the other
hand, in vitro cellular models can only partially provide meaningful results to what is generally
occurring in diseased dopaminergic neurons. To overcome these hurdles, in this thesis work, I chose
iPSCs-derived neurons as an innovative in vitro model of PD. Thus, iPSCs were generated by
somatic cell reprogramming to a pluripotent state. Many reprogramming approaches have been
described. Among them, we chose a cocktail of retroviruses expressing the three transcription
factors (OCT4, SOX2 and KLF4) to reprogram both affected patient fibroblasts with SNCA gene
duplication (a genetic abnormality that gives rise a dominant inherited form of PD), and matched
healthy donor fibroblasts (control cells). Generation and culture of these cells overcome ethical
problems related to the manipulation of embryonic stem cell as they share with them many
properties. However, some debate on the reliability of iPSCs is still open because they have been
demonstrated to differ in genetic expression from embryonic stem cells and to be very
heterogeneous depending on experimental approach and somatic cell of origin. Anyway, iPSCs are
a useful tool in the context of PD since can differentiate in any type of somatic cell, and therefore
generate patient-specific neurons. In this thesis work I differentiated both iPSCs lines in
dopaminergic neurons to evaluate their in vitro phenotype. Recently, some studies reported that α-
synuclein is cleared through a specific autophagy process that seems to be impaired in PD. This
thesis, as a part of a larger project, aims to investigate differences between healthy and affected
iPSC- derived dopaminergic neurons with particular attention to autophagy in those stress
conditions, which normally emphasize the pathological phenotype in vitro.