Abstract:
The mitochondrial outer membrane protein Miro1 is essential for neuronal
development and maintenance, and recent research indicates that Miro1 might further
be implicated in Parkinson’s disease relevant neurodegenerative pathways. Miro1 is
targeted by and interacts with several proteins connected to familial Parkinson’s
disease, such as PINK1 and Parkin (Hsieh et al. 2016; Kazlauskaite et al. 2014;
Klosowiak et al. 2016; Shaltouki et al. 2018; Wang and Schwarz 2009; Weihofen et al.
2009). Additionally, rare variants in the encoding gene RHOT1 were identified in
sporadic Parkinson’s disease patients (Berenguer-Escuder et al. 2019; Grossmann et
al. 2019).
To better understand Miro1 involvement in Parkinson’s disease relevant pathways, I
gene-edited three Miro1 mutations into previously established, healthy human induced
pluripotent stem cells (Marrone et al. 2018): heterozygous R272Q [sporadic
Parkinson’s disease (Grossmann et al. 2019)], homozygous S156A [preventing PINK1
phosphorylation (Wang et al. 2011)], and heterozygous K572R [preventing pSer65
Parkin ubiquitination (Kazlauskaite et al. 2014; Klosowiak et al. 2016)]. To investigate
these mutations in a disease-relevant model, I differentiated neural precursor cell
intermediates derived from the gene-edited stem cells into mid-brain specific
dopaminergic neurons.
Analysis of the putative PINK1 phosphorylation site Ser156 (Wang et al. 2011) in
neurons reveals that Miro1 S156A causes a reduction of Miro1 protein levels without
affecting gene expression but impairing its degradation upon mitochondrial
depolarization. This is accompanied by loss of mitochondrial membrane potential
resulting in reduced mitochondrial respiration. These findings revealed a correlation
between Miro1 and the level of respiratory chain complexes in differentiated, postmitotic
cells.
Miro1 R272Q found in a sporadic patient (Grossmann et al. 2019) shares the reduction
of mitochondrial respiration, but links to mitochondrial calcium instead of membrane
potential. Additionally, mitochondria are more fragmented and show partial loss of
cristae structure. The data point towards Miro1 R272Q disrupting the interaction of
Miro1 with the mitochondrial calcium uniporter to modulate mitochondrial calcium
uptake thus altering cytosolic calcium handling. Consequently, decreased
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neurotransmitter uptake and monoamine oxidase activity alter dopamine handling in
neurons.
Inhibition of Parkin-mediated ubiquitination of Miro1 (Kazlauskaite et al. 2014;
Klosowiak et al. 2016) in K572R neurons results in enlarged and fragmented
mitochondria. This is concomitant with reduced membrane potential and increased
turnover while Miro1 degradation and mitophagy are unaffected.
Taken together, these novel isogenic Miro1 models shed light on domain-specific
functions of Miro1. Miro1 S156A highlights the relationship between Miro1 protein
levels and oxidative phosphorylation in different cell types and specific vulnerability of
post-mitotic neurons. In contrast, a putative mechanism in neurons for the diseaseassociated
Miro1 mutation R272Q involves mitochondrial calcium handling linking to
dopamine homeostasis. The gene-edited stem cells generated in this study serve as
powerful platform to investigate Miro1 function in any cell type and the research with
differentiated neurons provides new insight into Miro1 function in Parkinson’s disease
relevant pathways.
