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Investigating the role of mitochondrial dysfunction in the pathogenesis of Parkinsons´s disease using patient-specific derived astrocytes

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Universitat de Barcelona. Facultat de Biologia

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[eng] Parkinson’s disease (PD) is an incurable, chronically progressive disorder of old age leading to premature invalidity and death. Clinically, PD is characterized by classical motor syndrome linked to a progressive loss of dopamine-containing neurons (DAn) in the substantia nigra pars compacta, and disabling non-motor symptoms related to extranigral lesions. The identification of several genes associated to familiar PD have brought considerable insight into underlying pathogenic mechanisms. However, the unknown etiology of the sporadic forms (90% of patients) and the emerging view that non-neuronal cells could be also implicated in the pathophysiology of the disease, greatly impact on the development of accurate models and on the discovery of a cure. Here, I investigate the role of astrocytes in disease pathogenesis using a human iPSC-based model of Parkinson’s disease. First, I introduce Parkinson’s disease, its pathological hallmarks and the progression of the symptoms, and discuss genetic and environmental influences. Then, I elaborate on the different mechanisms involved in PD including mitochondrial dysfunction, oxidative stress and autophagy as well as on the inflammatory phenotypes observed in the disease and recent work describing the role of inflammation in PD animal models and post-mortem brain tissue. Subsequently, I examine the association of astrocytic dysfunctions with neuronal morphological and functional abnormalities that contribute to the progression of several neurodegenerative including Parkinson’s disease and the recent data showing an astrocyte-autonomous process mediating PD-associated degeneration of dopaminergic neurons, mainly via intracellular accumulation of α-synuclein aggregates in astrocytes and subsequent propagation of such toxic aggregates to surrounding neurons. In the results section, I describe the generation and characterization of iPSC- derived astrocytes of LRRK2-PD patients (LRRK2G2019S PD), healthy individual (Ctrl) and CRISPR/Cas9 gene edited isogenic control. I show that LRRK2G2019S PD astrocytes exhibited extensive perinuclear accumulation of fragmented mitochondria and a significant increase in DRP1 phosphorylation compared to control astrocytes. Fragmented mitochondria accumulated in LRRK2G2019S PD astrocytes was due to a defective mitophagy leading to an increase in oxidative stress. I also show that oxygen consumption rate, ATP production and mitochondrial membrane potential were significantly decreased in LRRK2G2019S PD astrocytes indicating altered mitochondrial function in PD astrocytes and that LRRK2G2019S PD astrocytes exhibited lower expression levels of mitochondrial biogenesis-related genes compared to control astrocytes. Importantly, correction of G2019S mutation in the LRRK2 gene by CRISPR-Cas9 gene editing normalized mitochondria morphology, clearance and function to those of control astrocytes. Then, I describe the effects of Urolithin A, a mitophagy activator drug, that was able to rescue mitochondrial fragmentation and accumulation in LRRK2G2019S PD astrocytes by inducing mitophagy, promoting expression of mitochondrial biogenesis-related genes and reducing ROS production in those astrocytes. Finally, in the last chapter, I show that, in a co-culture system established between LRRK2G2019S PD astrocytes and healthy DA neurons, the treatment with Urolithin A, prevented neuronal cell death, suggesting a potential astrocyte- targeted therapeutic. In conclusion, our findings provide the advantage for using iPSC-based modeling for assessing the consequences of mitochondrial dysfunctions in astrocytes and dissecting the initial mechanisms that lead to neuronal cell loss in PD. The present modeling has uncovered mitophagy dysfunction as a relevant altered mechanism in PD astrocytes whose activation might represent an interesting therapeutic option for counteracting PD-related neurodegeneration.

# metadata.dc.title[].value
Investigating the role of mitochondrial dysfunction in the pathogenesis of Parkinsons´s disease using patient-specific derived astrocytes