Parkinson’s disease is a devastating disease that affects both motor and non-motor functions. A defining feature of PD is progressive loss of mDA neurons, yet the underlying etiology of most PD is completely unknown. Studies from animal and cellular models have shown that certain mutations in the LRRK2
gene locus can increase its kinase activity, which contributes to neurotoxicity, possibly via oxidative stress pathways (Heo et al., 2010
; MacLeod et al., 2006
; Smith et al., 2006
; West et al., 2005
). Moreover, cells expressing mutant LRRK2
have been shown to be more vulnerable to peroxide-induced cell death than LRRK2
wild-type cells (Cookson, 2009
; West et al., 2005
). Although the function of LRRK2
is currently not resolved, reduced expression of LRRK2
mRNA limits neural progenitor cell differentiation potential towards dopaminergic neurons and may enhance cell death (Milosevic et al., 2009
). These findings suggest that LRRK2 plays a pivotal role in neuronal survival and disruption in this gene can lead to PD (Lin et al., 2009
Here, we observed a subset of cardinal PD features in DA neurons derived from a patient with symptomatic PD possessing the LRRK2 p.G2019S mutation. Although, this study was limited to a single PD-iPSC line, with multiple subclones and controls, methods and results described should facilitate direct comparisons between lines carrying different LRRK2 mutations or carrying different mutations that predispose to PD. Most importantly, cardinal features that we observed included differential accumulation of α-synuclein protein, upregulation of key oxidative stress response genes, and vulnerability of PD neurons to neurotoxins, including H2O2, MG-132 and 6-OHDA, relative to unaffected neurons. Although differences are modest, given the more limited duration of experiments in vitro (relative to the time course of disease in humans), the distinction between TH-positive and –negative neurons and the increased susceptibility of diseased DA neurons from the p.G2019S LRRK2 patient suggest that central characteristics of PD are retained and detected in this model system. We believe that the differential effect would be more dramatic between the normal control and the diseased neurons if we could allow neurons to mature longer in culture. However, it is difficult to maintain DA neurons in culture, based on our modified protocol, for an extended period of time (50+ days) as cells tend to detach and die spontaneously.
West and others (West et al., 2005
) have shown that LRRK2 kinase activity is elevated by the G2019S mutation, leading many to speculate that either increased kinase activity or altered kinase target spectrum may underlie LRRK2-related PD (Cookson, 2009
). Nichols et al
. (Nichols et al., 2009
) further identified ROCK inhibitors, including Y-27632, that were able to effectively reduce kinase activity of recombinant LRRK2 G2019S. Contrary to our expectations, Y-27632, had no protective effect on patient-derived G2019S LRRK2 DA neurons. This could be due to different systems and methods used in our studies. We used hESC- and iPSC-derived DA neurons; whereas Nichols et al
. used a cell-free system.
Based on our findings, we propose that G2019S-iPSC derived neurons are more vulnerable to perturbation, caused by oxidative stress, mitochondrial impairment or defects in protein degradation. This vulnerability was selective for TH-positive neurons and accompanied by elevated α-synuclein content and a steady-state elevation in expression of several stress response genes, including activation of CASP3. In spite of the potential usefulness of using iPSC-derived neurons described here, however, there are a number of limitations that must be addressed. First, it is currently not possible to obtain a large number of DA neurons that are pure of contamination of other cell types. This means that results must be interpreted by a combination of experimental means to validate each data point, thus making high-throughput approaches difficult. Second, there is limited knowledge at this time of the endogenous programs and markers of human DA neurons, especially those with specific regionalization. Third, there is limited knowledge of the impacts of specific genetic and environmental parameters individually or in combination that trigger the onset of neurodegeneration. In spite of these limitations, the human iPSC system may provide a novel system that can be used to address specific hypotheses especially regarding the onset of PD in those with genetic determinants. Subsequently, results may be extended from genetically-defined PD to a further understanding of the ontogeny and molecular mechanisms underlying sporadic PD and possibly other neurological disorders that may share a similar etiology.