Axel CHEMLA

Analyzing the role of Parkinson’s disease-associated RHOT1/Miro1 variants in vitro and in vivo
Background:
Many studies highlighted the involvement of the protein Miro1 in regulating mitochondrial quality control, transport, and homeostasis as well as cellular calcium handling, which are all mechanisms associated with Parkinson’s Disease (PD) pathogenesis. Both fibroblasts and neurons from PD patients display impaired Miro1 degradation, confirming Miro1 role in neurodegeneration (Hsieh et al. 2016, 2019). Accordingly, Miro1 removal from damaged mitochondria by triggering its proteasomal degradation (using a “Miro1 reducer” compound) decreased stress-induced neurodegeneration, strengthening Miro1 potential as a therapeutic target in PD (Hsieh et al. 2019) . We were the first group to identify PD patients carrying heterozygous mutations in the RHOT1 gene (encoding Miro1), named R272Q and R450C, as cause of neurodegeneration. We demonstrated that fibroblasts from these patients and induced-pluripotent stem cells (iPSC) derived dopaminergic (DA) neurons from the R272Q patient had an impaired calcium homeostasis as well as dysregulated cellular and mitochondrial quality control mechanisms (Grossmann et al. 2019; Berenguer-Escuder et al. 2020). However, Miro1 mutations’ impact on mitochondrial bioenergetics and the link between mitochondrial dysfunction and neurodegeneration observed in PD patients carrying these Miro1 variants have not been elucidated yet.
Objectives:
This project’s goal is to identify disease-associated cellular phenotypes in our Miro1 mutant models and find novel targets to correct potential defects. To this end, we defined 3 objectives: 1) in vitro characterization of Miro1 variants’ impact on mitochondrial function, bioenergetics and dynamics, 2) in vivo assessment of Miro1 mutation’s impact on neuronal physiology, 3) identification of cellular phenotypes and molecular targets that can be rescued by drug treatment.
Methods:
Primary skin fibroblasts and iPSC from both healthy controls and PD patients carrying either the Miro1 R272Q or the R450C mutation were used in this study. To identify cellular phenotypes that could be targeted by selected drugs, we analyzed Miro1 removal from damaged mitochondria (following CCCP treatment, by western blot) in skin fibroblasts from PD patients carrying the Miro1 R272Q and R450C mutations as well as from sporadic cases. Isogenic, gene-corrected, iPSC lines were generated from the Miro mutant lines and characterized to 1) confirm expression of pluripotency markers (by immunostaining and RT-qPCR) and 2) verify their ability to differentiate into the three germ layers (differentiation into meso-, ecto- endoderm and confirmed expression of specific markers by immunostaining). To characterize Miro1 mutations impact on mitochondrial function in vitro, we assessed mitochondrial bioenergetics in iPSC-derived neurons and organoids by using the Seahorse technology.
To study pathophysiological consequences of Miro1 mutations in vivo, we generated a novel CRISPR-Cas9 engineered mouse model harboring the orthologue of the R272Q mutation in humans, namely R285Q. To assess the Nigrostriatal pathway’s integrity, we performed immunohistofluorescent stainings of Tyrosine Hydroxylase (TH) and Dopamine Transporter (DAT) and measured alterations in the dopaminergic terminals in the striatum as well as the neurons in the Substantia Nigra pars compacta (SNpc). Dopamine levels in the striatum of aged mice were measured by gas chromatography. Dopamine levels in striatum of old mice were measured by gas chromatography.
Results:
Native fibroblasts from the R272Q and R450C mutants behave differently following CCCP incubation. The R450C mutant efficiently degrades Miro1, while Miro1 degradation in the R272Q mutant is much less pronounced. IPSC characterization revealed no chromosomal aberrations while confirming expression of stemness markers, as well as capacity to differentiate into the 3 germ layers. This will help us to shed light on cellular defects associated to RHOT1 variants, irrespective of patients’ genetic background. Seahorse experiments performed in different cellular models consistently demonstrate that Miro1-R272Q mutants have a significantly decreased basal and maximal respiration, as well as oxygen consumption linked to ATP production, compared to healthy controls, indicating that the R272Q Miro1 mutation has a significant impact on energy metabolism.
In vivo, the R285Q mutation does not affect the structural integrity of the striatum or neurons quantity in the SNpc in 3, 6 and 15 months old mice. The striatal dopamine concentration of 15 months old homozygous Miro1-R285Q is comparable to wild type littermates.
Conclusions:
Native fibroblasts with the R272Q and R450C mutations degrade Miro1 with significantly different efficiency compared to each other. The reduced degradation of Miro1 in the R272Q mutant might be a cause of the phenotypes previously highlighted. This impaired degradation could bring the project to a translational phase, by testing a “Miro1 reducer” compound in R272Q mutant neurons and fibroblasts to correct harmful phenotypes.
We highlighted that the R272Q PD mutation causes alterations in energy metabolism of iPSC-derived neurons in vitro in 2D and 3D cultures. ROS production and mitochondrial membrane potential were unchanged in basal conditions.
In vivo, the R285Q mutant does not seem to cause DA neurons loss. However, the aforementioned experiments were all done in basal conditions, suggesting Miro triggers a response to PD-associated neurodegeneration. Thus, this mutation could regulate the sensibility of the nigrostriatal pathway and exacerbates the effects of acute (6-OHDA) or chronic (Crossing with DJ-1 k.o, Synuclein lines) PD challenges.
Outlook:
As a next step, Miro degradation in iPSC-derived neurons of mutants and their isogenic controls will be characterized, to know whether the Miro mutants behave the same way as the native fibroblasts, and might be marked by impairments in Miro degradation. If this is the case, a compound aiming at reducing Miro levels will be tested to see if the detrimental phenotype found in mitochondrial bioenergetics (seahorse) can be rescued both in native fibroblasts and in neurons. All relevant readouts will be performed with the isogenic controls.
Miro mouse characterization will be completed with behavioral and electrophysiological analysis thanks to external collaborators. In the future, this model could be treated with 6-OHDA, to trigger neurodegeneration, and see if the Miro mutant develops stronger symptoms.

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