Novel insights into Parkin-mediated mitochondrial dysfunction and neuroinflammation in Parkinson's disease

Mutations in PRKN cause the second most common genetic form of Parkinson's disease (PD)—a debilitating movement disorder that is on the rise due to population aging in the industrial world. PRKN codes for an E3 ubiquitin ligase that has been well established as a key regulator of mitophagy. Together with PTEN-induced kinase 1 (PINK1), Parkin controls the lysosomal degradation of depolarized mitochondria. But Parkin's functions go well beyond mitochondrial clearance: the versatile protein is involved in mitochondria-derived vesicle formation, cellular metabolism, calcium homeostasis, mitochondrial DNA maintenance, mitochondrial biogenesis, and apoptosis induction. Moreover, Parkin can act as a modulator of different inflammatory pathways. In the current review, we summarize the latest literature concerning the diverse roles of Parkin in maintaining a healthy mitochondrial pool. Moreover, we discuss how these recent discoveries may translate into personalized therapeutic approaches not only for PRKN-PD patients but also for a subset of idiopathic cases

Variants in Miro1 cause alterations of ER-mitochondria contact sites in fibroblasts from Parkinson's disease patients

Background: Although most cases of Parkinson´s disease (PD) are idiopathic with unknown cause, an increasing number of genes and genetic risk factors have been discovered that play a role in PD pathogenesis. Many of the PD‐associated proteins are involved in mitochondrial quality control, e.g., PINK1, Parkin, and LRRK2, which were recently identified as regulators of mitochondrial‐endoplasmic reticulum (ER) contact sites (MERCs) linking mitochondrial homeostasis to intracellular calcium handling. In this context, Miro1 is increasingly recognized to play a role in PD pathology. Recently, we identified the first PD patients carrying mutations in RHOT1, the gene coding for Miro1. Here, we describe two novel RHOT1 mutations identified in two PD patients and the characterization of the cellular phenotypes. Methods: Using whole exome sequencing we identified two PD patients carrying heterozygous mutations leading to the amino acid exchanges T351A and T610A in Miro1. We analyzed calcium homeostasis and MERCs in detail by live cell imaging and immunocytochemistry in patient‐derived fibroblasts. Results: We show that fibroblasts expressing mutant T351A or T610A Miro1 display impaired calcium homeostasis and a reduced amount of MERCs. All fibroblast lines from patients with pathogenic variants in Miro1, revealed alterations of the structure of MERCs. Conclusion: Our data suggest that Miro1 is important for the regulation of the structure and function of MERCs. Moreover, our study supports the role of MERCs in the pathogenesis of PD and further establishes variants in RHOT1 as rare genetic risk factors for neurodegeneration

Generation of two induced pluripotent stem cell lines and the corresponding isogenic controls from Parkinson’s disease patients carrying the heterozygous mutations c.815G>A (p.R272Q) or c.1348C>T (p.R450C) in the RHOT1 gene encoding Miro1

Fibroblasts from two Parkinson’s disease (PD) patients carrying either the heterozygous mutation c.815G>A (Miro1 p.R272Q) or c.1348C>T (Miro1 p.R450C) in the RHOT1 gene, were converted into induced pluripotent stem cells (iPSCs) using RNA-based and episomal reprogramming, respectively. The corresponding isogenic gene-corrected lines have been generated using CRISPR/Cas9 technology. These two isogenic pairs will be used to study Miro1-related molecular mechanisms underlying neurodegeneration in relevant iPSC-derived neuronal models (e.g., midbrain dopaminergic neurons and astrocytes)

Mitochondria-Endoplasmic Reticulum Contact Sites Dynamics and Calcium Homeostasis Are Differentially Disrupted in PINK1-PD or PRKN-PD Neurons

Background: It is generally believed that the pathogenesis of PINK1/parkin-related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria-endoplasmic reticulum contact sites (MERCSs). Objective: The aim of our study was to perform an in-depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin-linked PD.MethodsIn our study, we used induced pluripotent stem cell-derived dopaminergic neurons from patients with PD with loss-of-function mutations in PINK1 or PRKN. We employed a split-GFP-based contact site sensor in combination with the calcium-sensitive dye Rhod-2 AM and applied Airyscan live-cell super-resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis. Results: Our results showed that thapsigargin-induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild-type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1-PD or PRKN-PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin-induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted. Conclusions: Our results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild-type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Functional validation of a mitochondria-specific polygenic risk score in patient-based models for stratification of idiopathic Parkinson's disease

peer reviewedBackground: A large body of evidence specifically points to mitochondrial dysfunction as a major cause of Parkinson’s disease (PD) pathogenesis. Given that only ~10% of PD cases can be attributed to monogenic causes, we hypothesize that a fraction of idiopathic PD (iPD) cases may harbour a pathogenic combination of common variants in mitochondrial genes ultimately resulting in mitochondrial dysfunction. Objectives: To gain essential knowledge on the contribution of genetic variability in nuclear-encoded mitochondrial genes to iPD pathogenesis. Starting from genomic data, we aim to functionally validate mitochondrial polygenic risk profiles in patient-based cellular models, thus defining mitochondrial pathways potentially involved in neurodegeneration in subgroups of iPD patients.R-AGR-0592 - FNR - NCER-PD Phase II Coordination (01/06/2015 - 30/11/2023) - KRÜGER Rejko3. Good health and well-bein

Mitochondria interaction networks show altered topological patterns in Parkinson's disease.

Mitochondrial dysfunction is linked to pathogenesis of Parkinson's disease (PD). However, individual mitochondria-based analyses do not show a uniform feature in PD patients. Since mitochondria interact with each other, we hypothesize that PD-related features might exist in topological patterns of mitochondria interaction networks (MINs). Here we show that MINs formed nonclassical scale-free supernetworks in colonic ganglia both from healthy controls and PD patients; however, altered network topological patterns were observed in PD patients. These patterns were highly correlated with PD clinical scores and a machine-learning approach based on the MIN features alone accurately distinguished between patients and controls with an area-under-curve value of 0.989. The MINs of midbrain dopaminergic neurons (mDANs) derived from several genetic PD patients also displayed specific changes. CRISPR/CAS9-based genome correction of alpha-synuclein point mutations reversed the changes in MINs of mDANs. Our organelle-interaction network analysis opens another critical dimension for a deeper characterization of various complex diseases with mitochondrial dysregulation

Functional characterization of novel RhoT1 variants, which are associated with Parkinson's disease.

Parkinson’s disease (PD) is a common neurodegenerative disease affecting up to 2 % of the population older than 65 years. Most PD cases are sporadic with unknown cause, and about 10 % are familial inherited. PD is a progressive neurodegenerative disease characterized by loss of predominantly dopaminergic neurons, leading to typical symptoms like rigidity and tremor. Commonly involved pathogenic pathways are linked to mitochondrial dysfunction, e.g. increased oxidative stress, disruption of calcium homeostasis, decreased energy supply and mitochondrial-controlled apoptosis. The mitochondrial outer membrane protein Miro1 is important for mitochondrial distribution, quality control and maintenance. To date Miro1 is not established as risk factor for PD. Using a comprehensive mutation screening of RhoT1 in German PD patients we dissected the role of the first PD-associated mutations in RhoT1, the gene encoding for Miro1. Three mutations in RhoT1 have been identified in three PD patients with positive family history for PD. For analysis of mitochondrial phenotypes patient-derived fibroblasts from two of the three patients were available. As independent cell model served the neuroblastoma cell line M17 with stable knockdown of endogenous RhoT1 and transiently overexpression of the RhoT1 mutant variants. Investigation of yeast with knockout of endogenous Gem1 (the yeast orthologue of Miro1) and overexpression of mutant Gem1 revealed that growth on non-fermentable carbon source was impaired. These findings suggest that Miro1-mutant1 is a loss of function mutation. Interestingly, the Miro1 protein amount was significantly reduced in Miro1-mutant1 and Miro1-mutant2 fibroblast lines compared to controls. Functional analysis revealed that mitochondrial mass was decreased in Miro1-mutant2, but not in Miro1-mutant1 fibroblasts, whereas mitochondrial biogenesis was increased in Miro1-mutant2 fibroblasts, as indicated by elevation of PGC1α. A similar phenotype with reduction of mitochondrial mass was also observed in M17 cells overexpressing Miro1-mutant1 or Miro1-mutant2. Additionally, spare respiratory capacity was reduced in Miro1-mutant1 fibroblasts compared to Ctrl 1 fibroblasts. In contrast, Miro1-mutant2 fibroblasts showed increased respiratory activity compared to Ctrl 1, despite citrate synthase activity was significantly reduced. Both alterations of respiratory activity lead to mitochondrial membrane hyperpolarization in Miro1-mutant1 and Miro1-mutant2 fibroblasts, a phenotype which was also found in M17 cells with knockdown of RhoT1. Both Miro1 mutant fibroblasts lines displayed different problems with cytosolic calcium buffering: in Miro1-mutant1 fibroblasts histamine treatment increased cytosolic calcium concentration significantly compared to Ctrl 1 fibroblasts, indicating that calcium homeostasis was impaired, whereas in Miro1-mutant2 fibroblasts the buffering capacity for cytosolic calcium was impaired. The results indicate that mutations in Miro1 cause significant mitochondrial dysfunction, which are likely contributing to neurodegeneration in PD and underline the importance of Miro1 for mitochondrial maintenance

The Emerging Role of RHOT1/Miro1 in the Pathogenesis of Parkinson's Disease.

The expected increase in prevalence of Parkinson's disease (PD) as the most common neurodegenerative movement disorder over the next years underscores the need for a better understanding of the underlying molecular pathogenesis. Here, first insights provided by genetics over the last two decades, such as dysfunction of molecular and organellar quality control, are described. The mechanisms involved relate to impaired intracellular calcium homeostasis and mitochondrial dynamics, which are tightly linked to the cross talk between the endoplasmic reticulum (ER) and mitochondria. A number of proteins related to monogenic forms of PD have been mapped to these pathways, i.e., PINK1, Parkin, LRRK2, and α-synuclein. Recently, Miro1 was identified as an important player, as several studies linked Miro1 to mitochondrial quality control by PINK1/Parkin-mediated mitophagy and mitochondrial transport. Moreover, Miro1 is an important regulator of mitochondria-ER contact sites (MERCs), where it acts as a sensor for cytosolic calcium levels. The involvement of Miro1 in the pathogenesis of PD was recently confirmed by genetic evidence based on the first PD patients with heterozygous mutations in RHOT1/Miro1. Patient-based cellular models from RHOT1/Miro1 mutation carriers showed impaired calcium homeostasis, structural alterations of MERCs, and increased mitochondrial clearance. To account for the emerging role of Miro1, we present a comprehensive overview focusing on the role of this protein in PD-related neurodegeneration and highlighting new developments in our understanding of Miro1, which provide new avenues for neuroprotective therapies for PD patients

Generation of two induced pluripotent stem cell lines and the corresponding isogenic controls from Parkinson’s disease patients carrying the heterozygous mutations c.815G > A (p.R272Q) or c.1348C > T (p.R450C) in the RHOT1 gene encoding Miro1

Fibroblasts from two Parkinson’s disease (PD) patients carrying either the heterozygous mutation c.815G > A (Miro1 p.R272Q) or c.1348C > T (Miro1 p.R450C) in the RHOT1 gene, were converted into induced pluripotent stem cells (iPSCs) using RNA-based and episomal reprogramming, respectively. The corresponding isogenic gene-corrected lines have been generated using CRISPR/Cas9 technology. These two isogenic pairs will be used to study Miro1-related molecular mechanisms underlying neurodegeneration in relevant iPSC-derived neuronal models (e.g., midbrain dopaminergic neurons and astrocytes)

Generation of two induced pluripotent stem cell lines and the corresponding isogenic controls from Parkinson’s disease patients carrying the heterozygous mutations c.1290A > G (p.T351A) or c.2067A > G (p.T610A) in the RHOT1 gene encoding Miro1

Primary skin fibroblasts from two Parkinson’s disease (PD) patients carrying distinct heterozygous mutations in the RHOT1 gene encoding Miro1, namely c.1290A > G (Miro1 p.T351A) and c.2067A > G (Miro1 p.T610A), were converted into induced pluripotent stem cells (iPSCs) by episomal reprogramming. The corresponding isogenic gene-corrected lines have been generated using CRISPR/Cas9 technology. Here, we provide a comprehensive characterization and quality assurance of both isogenic pairs, which will be used to study Miro1-related molecular mechanisms underlying neurodegeneration in iPSC-derived neuronal models (e.g., midbrain dopaminergic neurons and astrocytes)