Generation of new models to study propagation and pathology of alpha-synuclein in PArkinson's disease and multiple system atrophy, by Staffan Holmqvist (2016).

Abstract

Synucleinopthies are neurodegenerative diseases characterised by the formation of α-synuclein-rich intracellular inclusions in neurons and glia. Traditionally, animal models and immortalized cell lines have been used to investigate why and how these inclusions form, and how they impact on cellular function. Recent advances in cellular reprogramming and directed differentiation have enabled the generation of neurons and glia, providing new models to study alpha-synuclein biology in patienttailored brain cells. The work presented in this thesis aimed to establish a platform of novel models to further address questions relevant to α-synucleinopathies. We created a library of human induced pluripotent stem cell lines from patients diagnosed with familial Parkinson’s disease (PD) and multiple system atrophy (MSA), as well as healthy controls, which we extensively characterized. Using these new cellular models, we generated defined regionalized cellular subtypes relevant for modelling PD and MSA, such as dopaminergic neurons, oligodendrocytes and astrocytes, using efficient differentiation protocols. In contrast to previous studies, we found that α-synuclein is transiently expressed in oligodendrocytes during development and in the adult human brain. We also devised a transgenic strategy for generating reporter lines, from which pure populations of astrocytes could be obtained. These human astrocytes were capable of releasing cytokines and chemokines in response to stressors, and readily took up α-synuclein from their surroundings, demonstrating their relevance in modelling of synucleinopathies. Braak’s hypothesis suggests that the pathology starts in the peripheral nervous system and progresses to the central nervous system (CNS), based on clinical observations of Lewy pathology distribution. We found that following injection into the intestinal wall of rats, α-synuclein was transported via the vagal nerve to the brain, thereby strengthening the hypothesis postulated by Braak. The models and cell systems presented in this thesis have provided unprecedented possibilities to address key questions relevant to the initiation and progression of α-synucleinopathies PD and MSA.

Stem Cell-based models to study cellular alterations in Parkinson's disease, by Margarita Chumarina (2020).

Abstract

Parkinson’s Disease (PD) is the most prevalent neurodegenerative movement disorder. Despite investigative efforts into the pathogenesis of the disease, the aetiology and mechanism of PD remains unknown, with multiple factors and cellular pathways known to play a part. But the genetic component of the disease, as well as enhanced vulnerability of the midbrain dopaminergic neurons of the Substantia nigra pars compacta (SNpc) make PD a suitable candidate for stem cell-based studies of the disease initiation and progression. The work presented in this thesis aims to generate and utilize PD patient-derived induced pluripotent stem cells (iPSCs), making it possible to assess cellular alterations among patients with different genetic disease backgrounds and disease phenotypes. We also highlight the potential role of other brain cell types, particularly glial cells, in the PD pathogenesis, and employ the same iPSC-based strategies to derive oligodendrocytes, a cell type not well-studied in relation to PD. Our work demonstrated that as oligodendrocytes express synuclein-alpha (SNCA), the expression of PD-associated genetic variant p.A53T impairs oligodendrocyte maturation. The same effect was observed using mouse embryonic stem cell (mESC)-derived oligodendrocytes from a transgenic mouse model overexpressing human SNCA variant p.A53T. These findings validate the use stem cells to further investigate oligodendrocyte pathology and its possible implications in the pathogenesis of PD. We also obtained iPSCs from a patient with a novel, not previously characterised variant p.Q811R in the POLG1, a risk factor for PD. Differentiation of these iPSCs into midbrain dopaminergic neuron containing spheroids (MDNS) enabled for the first time to assess the neuronal phenotype of POLG1-related PD, and identified several disease phenotypes, associated with PD, including SNCA pathology, disturbance of dopamine homeostasis and impaired energy metabolism. Using omics approaches for investigation of key altered networks in MDNS in POLG1^Q811R and SNCA^A53Tlines revealed distinct altered pathways, highlighting the heterogeneity and multifactorial nature of PD.

Oligodendrocyte (dys)function in alpha-synucleinopathies, by Carla Azevedo (2020).

Abstract

The complexity of a-synucleinopathies, which include multiple system atrophy (MSA) and Parkinson’s disease (PD), is not entirely understood. It is known that pathological accumulation of a-synuclein (a-syn) into proteinaceous aggregates is a cellular hallmark of these diseases. a-Syn positive aggregates appear in neurons in PD and dementia with Lewy bodies (DLB), and in oligodendrocytes in MSA. Much research has focused on using animal models that pathocopy and phenocopy PD and MSA to investigate the disease pathogenesis. However, as no genetic link has been associated with MSA, it is possible that current transgenic models do not fully reflect the human pathology. The discovery of induced pluripotent stem cells (hiPSCs) changed life science, and allowed for the first time to conduct large scale experimental work using patient cells. Since these cells are embryonic pluripotent-like, their use can help unravel early disease mechanisms, as they can be differentiated into young brain cells. Despite intense research efforts made to understand the pathogenesis of PD and MSA, many questions remain, in particular the origin of a-syn in oligodendrocytes and possible toxicity to these cells. The work presented in this thesis aims to 1) generate patient iPSC- based models, 2) develop efficient protocols to generate dopaminergic neurons and oligodendrocytes from iPSCs, and 3) study the role of oligodendrocytes in synucleinopathies, to gain insights into oligodendrocyte (dys)function in PD and MSA. Here, we report that during oligodendrocyte development and in the human brain, the SNCA gene encoding for a-syn is differentially expressed in oligodendrocytes. Since neurons expressing pathogenic forms of a-syn exhibit cellular alterations, we hypothesized that oligodendrocytes having the same genetic background should also be affected.For the first time, we show that iPSC-derived oligodendrocytes generated from PD patients carrying the variation p.A53T in SNCA or a triplication of the SNCA locus, and from MSA patients, display impaired differentiation and maturation. This was further supported by observations from experiments involving mouse embryonic stem cell (mESC)-derived oligodendrocytes generated from the M83 transgenic mouse model of PD. Moreover, we demonstrate that p.A53T SNCA and MSA oligodendrocytes exhibit a deviation in their phenotype, adopting an immune-reactive phenotype and not myelinating oligodendroglia. Finally, our transcriptomic data further reveal alterations in innate inflammatory components, with differential expression of complement proteins, MHC-class and immune-proteasome genes in p.A53T SNCA and MSA oligodendrocytes.This thesis is composed of a unique set of studies addressing crucial questions related to the origin of a-syn in oligodendroglia, and focuses on elucidating the cellular alterations in oligodendrocytes in PD and MSA