FUNCTIONAL CHARACTERIZATION OF NEURODEGENERATION IN CELLULAR AND MOUSE MODELS OF PARKINSON’S DISEASE CARRYING PATHOGENIC VARIANTS IN THE RHOT1 GENE ENCODING MIRO1

Parkinson’s disease (PD) is the fastest growing neurological disorder, and the first most common neurodegenerative movement disorder, with patient number expected to double in 2040 (Dorsey et al., 2018b). While most PD cases are sporadic, approximately 10% of patients develop PD due to genetic causes. Interestingly, many of these PD causing genes are related to mitochondrial function, which is in line with the main pathological hallmark of PD, namely the selective death of the dopaminergic (DA) neurons in the Substantia Nigra Pars Compacta (SNpc) of the brain. Indeed, DA neurons heavily rely on adenosine triphosphate (ATP) production via mitochondrial oxidative phosphorylation, to sustain their pace-making activity. The last few years saw an increasing interest in the Mitochondrial Rho GTPase 1 (Miro1) protein, a mitochondria-anchored cytosolic calcium sensor involved in the regulation of mitochondria-ER contact sites (MERCs), mitochondrial transport, and mitophagy. Our team previously demonstrated in fibroblasts that four different Miro1 mutations found in PD patients pathologically affected calcium homeostasis, mitochondrial quality control, and MERCs formation (Berenguer-Escuder et al., 2019a; Grossmann et al., 2019a). Moreover, Miro1 p.R272Q mutant induced-pluripotent stem cells (iPSCs)-derived neurons, display a significant impairment of cytosolic calcium handling compared to age/gender matched controls, similarly to fibroblast from this patient (Berenguer-Escuder et al., 2020a). This phenotype was accompanied by MERCs levels dysregulation as well as mitophagy and autophagy impairment, supporting the role of Miro1 as a rare genetic risk factor for PD (Berenguer-Escuder et al., 2020a). Moreover, recent studies revealed a pathological retention of Miro1 upon mitophagy induction, thus delaying mitophagy in cells from genetic PD as well as in a significant proportion of sporadic PD patients (Hsieh et al., 2016a, 2019a; Shaltouki et al., 2018a). In this thesis, we first generated and characterized iPSCs and isogenic controls lines from the four aforementioned PD patients. We then explored the pathogenic effect of the Miro1 p.R272Q mutation in three different models, namely iPSC-derived neurons, midbrain organoids (MO), and Miro1 p.R285Q knockin (KI) mice. We first confirmed the exacerbated sensitivity to calcium stress in vitro in neurons, and unveiled that it was also accompanied by mitochondrial bioenergetics impairment (lower ATP levels) and elevated reactive oxygen species (ROS) production in both 2D and 3D models, finally resulting in DA neurons death in MO. This was accompanied by elevated SNCA mRNA expression in both models, as well as higher α-synuclein protein amounts in neurons, which was already found in post-mortem samples from PD patients (Shaltouki et al., 2018a). Lastly, our mouse model displayed significant neuronal loss in its SNpc, as well as impaired motor learning, recapitulating two PD signs found in patients. Taken together, these results support the involvement of Miro1 in PD pathogenesis, and highlights the potential of Miro1 variants to be used as novel, promising models for PD in vitro and in vivo.