Miro1 R272Q disrupts mitochondrial calcium handling and neurotransmitter uptake in dopaminergic neurons

Abstract

The Rho GTPase Miro1, located at the mitochondrial outer membrane is known to properly distribute mitochondria to synapses, aid calcium buffering and initiate PINK1-Parkin mediated mitophagy. Several heterozygous RHOT1/Miro1 variants were identified in sporadic Parkinson’s disease patients. Miro1 R272Q is located within a calcium binding domain, but the functional outcome of this point mutation and its contribution to the development of disease are unclear. To address this, we introduced a heterozygous RHOT1/Miro1 R272Q point mutation in healthy induced pluripotent stem cells. In dopaminergic neurons, Miro1 R272Q does not affect Miro1 protein levels, CCCP-induced mitophagy, nor mitochondrial movement yet causes the fragmentation of mitochondria with reduction of cristae and ATP5A. Inhibition of the mitochondrial calcium uniporter phenocopied Miro1 R272Q cytosolic calcium response to Thapsigargin in active neurons, a similar effect was observed during the calcium buffering phase in Miro1 knockdown neuroblastoma cells. Altered mitochondrial calcium regulation is associated with reduced mitochondrial respiration and reduced catecholamine neurotransmitter uptake. Synaptic changes are not coupled to dopamine distribution or dopamine transporters but are linked to Miro1 R272Q-related calcium handling via the mitochondria concomitant with defective dopamine regulation at the mitochondrial surface by monoamine oxidase. We conclude that the Miro1 R272Q heterozygous point mutation dampens mitochondrial-calcium regulation and mitochondrial capacity via events at the outer membrane that are sufficient to disrupt dopaminergic function.

Parkinson’s disease-related Miro1 mutation induces mitochondrial dysfunction and loss of dopaminergic neuronsin vitroandin vivo

“…However, Miro1 overexpression in animal fibroblasts also resulted in mitochondrial morphology and transport issues [71]. Similarly, we and others showed mitochondrial dysfunction in Miro1 mutant cells [6,[15][16][17]. Remarkably, all Miro1 alterations culminate in similar mitochondrial dysfunction phenotypes, suggesting that a tight regulation of Miro1 levels and/or activity is needed for maintaining mitochondrial homeostasis, with slight changes being pathogenic for cells, especially for more sensitive cell types such as dopaminergic neurons.…”

“…Miro1 is able to sense both cytosolic calcium [45] and oxidative stress [46,47], with impairments in Miro1 leading to mitophagy alterations and bioenergetics deficits [44,46,47]. A recent study in iPSC-derived dopaminergic neurons, from a healthy individual in which the heterozygous Miro1 p.R272Q mutation had been artificially introduced, showed Ca 2+ deregulation as well as altered mitochondrial morphology and basal respiration [16]. In our patient-specific PD-R272Q dopaminergic neurons, we also showed calcium handling dysregulation [15], which might contribute to mitochondrial defects observed.…”

“…However, in response to mitochondrial depolarization, the Miro1 p.R272Q mutant neurons showed a reduced ability to cope with stress, and displayed a significantly reduced MMP compared with both controls (Figure 2F). Finally, due to the nature of the Miro1 p.R272Q mutation that affects Miro1 EF-calcium binding domain [6,15,16], and the existing link between impaired cytosolic calcium handling and mitochondrial stress [29], calcium response was evaluated. Fluo-4 Direct MFI was measured over time in presence of ionomycin, which promotes a rapid influx of Ca 2+ into cells.…”

“…Moreover, in human-derived fibroblasts and iPSC-derived neurons mutations in RHOT1 have showed mitochondria-related alterations [6,[15][16][17].…”

Highly Specialized Mechanisms for Mitochondrial Transport in Neurons: From Intracellular Mobility to Intercellular Transfer of Mitochondria

“…E208K/E328K mutations in the EF hands of Miro abolish calcium entry in the matrix [ 66 ]. Curiously, a different mutation (R272Q) in the EF hands of Miro has no effect on mitochondrial movement, although it disrupts calcium handling in the mitochondria [ 67 ]. The discrepancies in the role of Miro could be explained by the use of different mutants and raise the possibility that Miro is regulated by accessory proteins bound to the EF hands, or that the regulation of the transport complex is yet to be fully addressed [ 68 ].…”