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  • PARK is a ubiquitously expressed protein and

    2018-11-08

    PARK2 is a ubiquitously expressed protein, and its ubiquitination of outer mitochondrial membrane is a prerequisite step in mitophagy-mediated removal of damaged mitochondria. However, PARK2 abnormalities in Stattic cost other than neurons fail to display the selective loss of a particular population of cells, suggesting that dysfunctional mitophagy could be compensated or delayed. Both PD patients and PARK2 KO dopaminergic neurons display upregulation of several key mitophagy-associated proteins, as determined by our array data. Similarly, in our isogenic lines, the expression of these mitophagy-related genes displayed alleleic dependency and stage specificity. We identified a number cell death-inducing genes that were upregulated in dopaminergic neurons derived from PARK2 patients and PARK2 KO lines; these include BID, BAX, BIM, BAK, PUMA, NOXA, BNIP3, and NIK (BCL-2 interacting killer). Although the mechanism by which dysfunctional mitophagy contribute to PD pathogenesis remain to be investigated, here we show that for the first time that PARK2 contributes to mitochondrial mass (volume) in dopaminergic neurons. We show that TH-positive neurons in PD patient and PARK2 KO lines have a reduced mitochondrial mass compared with controls. A decrease in population of mitochondria within these TH-positive cells would shift the balance between healthy and defective mitochondrial and render these cells more vulnerable to accumulation of damaged mitochondria. We did not observe this alteration in the absence of PARK2 mutations. Given the ubiquitous expression of PARK2 and the changes we observed in our mixed dopaminergic neuron cultures, as well as previously published reports of observable phenotypes in cell lines unrelated to neurons (da Costa et al., 2009; Tsai et al., 2003), we reasoned that a subset of these changes may be seen in other neurons other than dopaminergic neurons. We took advantage of a neuronal differentiation system that we have developed (Liu et al., 2013) and examined a pure population of neurons of PARK2 mutants. We focused our analysis on isogenic PARK2 lines as a more sensitive model of the PARK2 phenotype. Similar changes were seen, as with dopaminergic neurons. We saw a gradual decline in the number of surviving neurons in culture to approximately half of that in the isogenic control sample. This phenotypic change was consistent with previous observations in mouse models, which showed a decreased survival in response to stress (Sherer et al., 2003b; Testa et al., 2005). Comparison of the mitochondrial and cell death gene changes showed a similar but not identical profile. These results along with the lack of a phenotype in iPSC, NSC, and astrocytes highlight the importance of studying the effect in an appropriate cellular context. Our observation that the phenotype can be studied in generic neurons provides a feasible assay with additional stress using a purified population of cells that can be obtained 2- to 3-fold faster and with much less effort than authentic midbrain dopaminergic neurons.
    Experimental Procedures
    Author Contributions
    Acknowledgments
    Introduction Since their discovery and isolation in 1998, human embryonic stem cells (hESCs) have been considered a potentially valuable tool for generating replacement cells for therapeutic purposes (Lanza et al., 2009). However, despite success in numerous animal models, fears over tumorigenicity and immunogenicity, coupled with ethical concerns, and inefficiencies in differentiation methods have all contributed to delays in carrying out human clinical trials. Only one group has reported the results of the safety and possible biological activity of embryonic stem cell progeny in individuals with any disease (Schwartz et al., 2015), but these investigators only enrolled patients who were mostly Caucasian. Here, we confirmed the potential safety and efficacy of hESC-derived cells in Asian patients. Loss of the retinal pigment epithelium (RPE) is an important part of the disease process in several retinal disorders, including age-related macular degeneration (AMD) and Stargardt disease. AMD is a degenerative disease that is the leading cause of visual impairment in developed countries, with the dry (nonexudative) form of AMD accounting for 85% to 90% of cases (Age-Related Eye Disease Study Research Group, 2001). Concurrent RPE and choriocapillaris atrophy are present in severe, atrophic dry AMD, with RPE atrophy preceding choriocapillaris atrophy (Schatz and McDonald, 1989; Korte et al., 1984; Leonard et al., 1997). Stargardt macular dystrophy (SMD) is the most common form of juvenile macular degeneration that is due to the production of defective rim proteins encoded by the ABCA4 gene, leading to the accumulation of di-retinoid-pyridinium ethanolamine (A2E) in the RPE, RPE cell loss, and photoreceptor death (Glazer and Dryja, 2002). There are no known effective treatments to prevent or reverse visual loss for either disease. Since RPE loss is implicated in the pathophysiology of both disorders, RPE replacement has been suggested as a therapeutic intervention for these conditions.