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

Reduced Basal Autophagy and Impaired Mitochondrial Dynamics Due to Loss of Parkinson's Disease-Associated Protein DJ-1

BACKGROUND: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2. CONCLUSIONS/SIGNIFICANCE: We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease

The use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of Parkinson's disease.

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 (1). Because ageing is the most important risk factor, cases of PD will increase during the next decades (2). Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD (3-11). After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease. Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD

Converging environmental and genetic pathways in the pathogenesis of Parkinson's disease.

As a prototypic neurodegenerative disorder Parkinson's disease (PD) is characterized by the progressive loss of specific neuronal subpopulations leading to a late-onset movement disorder. Based on familial forms of PD, to date a significant number of genes were identified that allowed first insight into the molecular pathogenesis of this common movement disorder. These pathways include impaired protein degradation and subsequent aggregation within neuronal cells and impaired mitochondrial function followed by energy depletion due to oxidative stress leading to cell death. The respective disease models were supported by pathoanatomical and biochemical findings in brains of sporadic PD patients without apparent genetic contribution to pathogenesis. Indeed recent genetic and epidemiological studies hint to a complex interplay of genetic susceptibility factors and environmental risk factors to converge to processes of pathological protein accumulation and mitochondrial damage that trigger neurodegeneration in PD. Therefore large-scale geneticoepidemiological studies combining genetic whole genome approaches with a detailed ascertainment of environmental exposures are expected to provide important clues to decipher the complexity of neurodegeneration of this most frequent neurodegenerative movement disorder

Balance is the challenge--the impact of mitochondrial dynamics in Parkinson's disease.

Impaired mitochondrial function has been implicated in neurodegeneration in Parkinson's disease (PD) based on biochemical and pathoanatomical studies in brains of PD patients. This observation was further substantiated by the identification of exogenic toxins, i.e. complex I inhibitors that directly affect mitochondrial energy metabolism and cause Parkinsonism in humans and various animal models. Recently, insights into the underlying molecular signalling pathways leading to alterations in mitochondrial homeostasis were gained based on the functional characterization of mitoprotective genes identified in rare forms of inherited PD. Using in vitro and in vivo loss of function models of the Parkin, PINK1, DJ-1 and Omi/HtrA2 gene, the emerging field of mitochondrial dynamics in PD was established as being critical for the maintenance of mitochondrial function in neurons. This underscored the concept that mitochondria are highly dynamic organelles, which are tightly regulated to continuously adapt shape to functional and anatomical requirements during axonal transport, synaptic signalling, organelle degradation and cellular energy supply. The dissection of pathways involved in mitochondrial quality control clearly established the PINK1/Parkin-pathway in the clearance of dysfunctional mitochondria by autophagy and hints to a complex interplay between PD-associated proteins acting at the mitochondrial interface. The elucidation of this mitoprotective signalling network may help to define novel therapeutic targets for PD via molecular modelling of mitochondria and/or pharmacological modulation of mitochondrial dynamics

Identification of ASCL1 as a determinant for human iPSC-derived dopaminergic neurons

During cellular specification, transcription factors orchestrate cellular decisions through gene regulation. By hijacking these transcriptional networks, human pluripotent stem cells (hPSCs) can be specialized into neurons with different molecular identities for the purposes of regenerative medicine and disease modeling. However, molecular fine tuning cell types to match their in vivo counterparts remains a challenge. Directing cell fates often result in blended or incomplete neuron identities. A better understanding of hPSC to neuron gene regulation is needed. Here, we used single cell RNA sequencing to resolve some of these graded molecular identities during human neurogenesis from hPSCs. Differentiation platforms were established to model neural induction from stem cells, and we characterized these differentiated cell types by 10x single cell RNA sequencing. Using single cell trajectory and co-expression analyses, we identified a co-regulated transcription factor module expressing achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1) and neuronal differentiation 1 (NEUROD1). We then tested the function of these transcription factors in neuron subtype differentiation by gene knockout in a novel human system that reports the expression of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. ASCL1 was identified as a necessary transcription factor for regulating dopaminergic neurotransmitter selection