PATHOGENIC ROLE OF PARKINSON’S DISEASE-ASSOCIATED MIRO1 MUTATIONS IN THE MITOCHONDRIAL-ENDOPLASMIC RETICULUM INTERPLAY

Doctoral thesis (2019)

Parkinson´s disease (PD) is a chronic neurodegenerative disorder, in which only 5-10% of the cases are caused by genetic mutations. One of the main pathological hallmarks of PD is the loss of midbrain ... [more ▼]

Parkinson´s disease (PD) is a chronic neurodegenerative disorder, in which only 5-10% of the cases are caused by genetic mutations. One of the main pathological hallmarks of PD is the loss of midbrain dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of diseased brains. These DA neurons require large amounts of energy for the maintenance of their pace-making activity and their complex dendritic and axonal arborizations, features that force them to rely on a fully functional mitochondrial network. In this regard, mitochondrial dyshomeostasis is a central factor in PD pathophysiology. Mitochondria are considered the powerhouse of the cells, and they are extremely dynamic organelles that are distributed throughout the entire neuronal body to meet the cellular energy demands. The maintenance of mitochondrial function requires their interaction with other cellular organelles, in particular, the endoplasmic reticulum (ER). Overwhelming evidence indicates that the mitochondrial-ER interface is a potential target of growing importance for the investigation of PD. Several PD-related proteins were found to be involved in the structural maintenance and signaling regulation of mitochondrial-ER contact sites (MERCs). In recent years, myriad studies have identified the mitochondrial GTPase Miro1 as a crucial player in PD pathology. Miro1 protein is not only an adaptor for mitochondrial transport, but also acts as a cytosolic calcium sensor and as an ubiquitination target for the mitochondrial quality control machinery. Moreover, Miro1 can localize to MERCs, where it functions as a regulator of the calcium exchange between both organelles. To date, no genetic link between Miro1 and PD has been identified, and the influence of Miro1 in the regulation of MERCs within the context of neurodegeneration is still underestimated. This current study explored the damaging effect of novel PD-associated heterozygous mutations in RHOT1, the gene encoding Miro1 protein, in a diseased genetic background. We first obtained skin fibroblasts from the affected PD patients harboring Miro1 mutations, which we further differentiated into iPSC-derived neurons. The characterization of the mutations in both patient-derived cellular models unveiled important impairments in mitochondrial calcium homeostasis and sensitivity to calcium stress, associated with alterations in the abundance and functionality of the MERCs. Consequently, downstream pathways to these mechanisms were affected, such as autophagy flux and mitochondrial clearance. From our results, we can conclude that PD-associated mutant Miro1 leads to crucial alterations in MERCs, consequently affecting downstream mechanisms such as calcium homeostasis and mitophagy. These dysregulations might lead to an increased sensitivity to stress and finally cell death. Our findings strongly support the key role of MERCs in the progress of neurodegeneration and establish RHOT1 as a rare genetic risk factor in PD. [less ▲]