Of importance was our finding of changes in other PD

Of importance was our finding of changes in other PD-associated genes, including SNCA (Krüger et al., 1998; Polymeropoulos et al., 1997; Singleton et al., 2003; Zarranz et al., 2004). SNCA is a presynaptic protein and function in regulating synaptic vesicle and neurotransmitter release. Aggregates of SNCA have been identified within the cytoplasmic inclusions (Lewy bodies) along with PARK2 in the brains of PD patients. It has been suggested that PARK2-mediated ubiquitination regulates SNCA assembly into ubiquitin-positive cytosolic inclusions, lending support for absence of these inclusions in PD patients with PARK2 mutation (Chung et al., 2001). Here, we report an increase in SNCA protein levels in patient-derived neurons concomitant with a decrease in TH-positive cells. Similar correlation was reported previously to be associated with the aging process (Chu and Kordower, 2007). Although, some cellular and tissue studies in PD patients argue against the excess of SNCA in pathogenesis of PD (Dächsel et al., 2007), recent studies reaffirm the increase in SNCA protein levels in iPSC-derived neurons from patient with PARK2 mutation (Imaizumi et al., 2012). It is possible that this increase in SNCA expression is an early event in the disease process and that patients with a late stage of PD do not display this phenotype. We acknowledge that there are many differences between a cell culture model and what may be seen in a culture dish. One operating assumption is that loss of PARK2 may lead to reduced processing of SNCA-associated proteins in particular SYNPHILIN; other data suggest that it is a nonclassical pathway (Chung et al., 2001; Lim et al., 2005; Sherer et al., 2003b; Zhang et al., 2013). We believe that altered ratios of interacting proteins may lead to either an increase or decrease depending on the stage of the disease. However, it is difficult to mimic the exact disease stage in culture just as it has been hard to do so in rodents in vivo. Nevertheless, our data were consistent in all PARK2 patient lines in vitro. 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 cytokine receptor 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.