SUMMARY Mitochondrial movements are tightly controlled to maintain energy homeostasis and prevent oxidative stress. Miro is an outer mitochondrial membrane protein that anchors mitochondria to microtubule motors, and is removed to stop mitochondrial motility as an early step in clearance of dysfunctional mitochondria. Here, using human iPSC-derived neurons and other complementary models, we build on a previous connection of Parkinson’s disease (PD)-linked PINK1 and Parkin to Miro, by showing that a third PD-related protein, LRRK2, promotes Miro removal via forming a complex with Miro. Pathogenic LRRK2G2019S disrupts this function, delaying the arrest of damaged mitochondria and consequently slowing the initiation of mitophagy. Remarkably, partial reduction of Miro levels in LRRK2G2019S human neuron and Drosophila PD models rescues neurodegeneration. Miro degradation and mitochondrial motility are also impaired in sporadic PD patients. We reveal that prolonged retention of Miro, and the downstream consequences that ensue, may constitute a central component of PD pathogenesis.

Astrocytes can be generated from various tissue sources including human pluripotent stem cells (PSC). In this manuscript, we describe a chemically defined xeno-free medium culture system for rapidly generating astrocytes from neural stem cells derived from PSC. We show that astrocyte development in vitro, mimics normal development in vivo, and also passes through a CD441 astrocyte precursor stage. Astrocytes generated by our method display similar gene expression patterns, morphological characteristics and functional properties to primary astrocytes, and they survive and integrate after xenotransplantation. Whole genome expression profiling of astrocyte differentiation was performed at several time points of differentiation, and the results indicate the importance of known regulators and identify potential novel regulators and stage-specific lineage markers. STEM CELLS 2013;31:941-952 Disclosure of potential conflicts of interest is found at the end of this article.

Alpha-synuclein is a component of Lewy bodies, the pathological hallmark of Parkinson’s disease (PD), and is also mutated in familial PD. Here, by extensively analyzing PD patient brains and neurons, and fly models, we show that alpha-synuclein accumulation results in upregulation of Miro protein levels. Miro is a motor/adaptor on the outer mitochondrial membrane that mediates mitochondrial motility, and is removed from damaged mitochondria to facilitate mitochondrial clearance via mitophagy. PD patient neurons abnormally accumulate Miro on the mitochondrial surface leading to delayed mitophagy. Partial reduction of Miro rescues mitophagy phenotypes and neurodegeneration in human neurons and flies. Upregulation of Miro by alpha-synuclein requires an interaction via the N-terminus of alpha-synuclein. Our results highlight the importance of mitochondria-associated alpha-synuclein in human disease, and present Miro as a novel therapeutic target.

SummaryIn this study, we used patient-specific and isogenic PARK2-induced pluripotent stem cells (iPSCs) to show that mutations in PARK2 alter neuronal proliferation. The percentage of TH+ neurons was decreased in Parkinson’s disease (PD) patient-derived neurons carrying various mutations in PARK2 compared with an age-matched control subject. This reduction was accompanied by alterations in mitochondrial:cell volume fraction (mitochondrial volume fraction). The same phenotype was confirmed in isogenic PARK2 null lines. The mitochondrial phenotype was also seen in non-midbrain neurons differentiated from the PARK2 null line, as was the functional phenotype of reduced proliferation in culture. Whole genome expression profiling at various stages of differentiation confirmed the mitochondrial phenotype and identified pathways altered by PARK2 dysfunction that include PD-related genes. Our results are consistent with current model of PARK2 function where damaged mitochondria are targeted for degradation via a PARK2/PINK1-mediated mechanism.

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.