11 November 2010. Scientists have long wondered why Parkinson’s disease selectively kills a small group of dopamine-generating neurons. A case is building that these vulnerable neurons, found in the substantia nigra pars compacta (SNc), are under high levels of oxidative stress due to the influx of calcium. In yesterday’s Nature, researchers led by James Surmeier at Northwestern University in Chicago, Illinois, detail some of the mechanisms behind this vulnerability and show how mutations in the DJ-1 gene, which is associated with familial Parkinson’s disease, can exacerbate it. Their results strengthen the case for testing a class of calcium channel blockers, dihydropyridines, in Parkinson’s patients. Some of these drugs are already FDA-approved for treating high blood pressure. Two Phase 2 safety and tolerability trials of the dihydropyridine isradipine for PD are currently enrolling volunteers.
Midbrain dopaminergic neurons need to fire in a rhythmic, pacemaking pattern to maintain steady concentrations of dopamine in their target neurons. In the vulnerable SNc neurons, this electrical pacemaking activity is associated with large influxes of calcium through L-type calcium channels. This contrasts with dopaminergic neurons in the ventral tegmental area (VTA), which do not depend on calcium for activation and are much less affected in PD. SNc cells must expend energy to remove excess calcium, which puts more stress on their mitochondria. Since mitochondria produce energy through oxidative processes, more toxic oxygen species are created in the overworked organelles, leading to cellular damage over time, the theory goes. Previous work by Surmeier and others in the field implicated calcium toxicity in PD-related SNc cell death (see ARF related news story on Chan et al., 2007 and ARF related news story on Mosharov et al., 2009). Intriguingly, however, Surmeier’s group recently showed that calcium entry is not essential for pacemaking activity in SNc neurons (see Guzman et al., 2009). This lends hope to the idea that blocking calcium channels could be a safe therapeutic strategy for people with Parkinson’s.
To take a closer look at the consequences of calcium dependence in SNc neurons, first author Jaime Guzman generated transgenic mice that expressed a redox-sensitive green fluorescent protein (roGFP) under the control of the tyrosine hydroxylase promoter, which limited expression of the reporter to dopaminergic neurons, including those in the SNc and VTA. The roGFP also included a sequence that targeted the protein to mitochondria. Since roGFP lights up when oxidized, the protein provided a way to directly visualize the oxidation state of mitochondria. Guzman and colleagues examined brain slices from the transgenic mice with two-photon laser scanning microscopy, and confirmed that SNc neurons had higher levels of fluorescence, and therefore of oxidative stress, than VTA neurons. Without affecting pacemaking activity, isradipine reduced the redox state of SNc mitochondria to the levels seen in the VTA.
The authors used a different fluorescent dye to measure mitochondrial membrane potential. With this reporter, Guzman and colleagues observed rapidly rising and falling fluorescence in SNc mitochondria, while VTA mitochondrial fluorescence was stable. The flickering fluorescence corresponded to small depolarizations of the mitochondrial membrane, and seemed to occur in response to oxidative stress, suggesting it was a protective mechanism. Depolarization was caused by the opening of two mitochondrial ion channels, uncoupling proteins 4 and 5 (UCP4 and UCP5). Uncoupling proteins, which allow electrons to freely flow down and reduce components of the respiratory chain without concomitant production of ATP, were previously shown to lower the production of reactive oxygen species in mitochondria (see Echtay et al., 2002). The authors found that blocking these channels reduced flickering and also increased oxidative stress in SNc neurons, confirming their protective role.
To tie these observations to Parkinson’s disease, Guzman and colleagues crossed the roGFP transgenics with DJ-1 knockout mice. DJ-1 mutations cause a rare form of early-onset PD, and are believed to have a role in mounting cellular defenses against oxidative stress (see Kahle et al., 2009). As expected, the lack of DJ-1 led to higher levels of oxidation in SNc neurons, but had no effect in VTA neurons. DJ-1 knockouts showed less mitochondrial flickering, suggesting this protective mechanism was impaired. The knockouts had less UCP4 and UCP5 mRNA, implying that one of the roles of DJ-1 is to promote the production of the protective ion channels.
The paper helps explain the role of DJ-1 in dopaminergic neurons and “is quite a nice step forward,” said Mark Cookson at the National Institute on Aging in Bethesda, Maryland. Cookson is particularly intrigued by the roGFP reporter molecule, saying it provides a novel system for looking at the functions of proteins, such as DJ-1, in intact animals. He said in future studies, it would be interesting to nail down exactly how DJ-1 works. Is it a transcription factor, or does it act indirectly to increase mRNA levels, perhaps by stabilizing transcripts?
Although many unanswered questions about DJ-1 and UCPs remain, Surmeier said the question that most interests him is whether the mechanism they have described is at work in all forms of Parkinson’s and in all affected cell types. Several other groups of neurons besides SNc neurons die in PD, Surmeier said. His group is in the process of generating new strains of roGFP mice that will express the reporter in other vulnerable cell populations, such as the cholinergic neurons of the dorsal motor nucleus. They will look to see if these neurons also have high calcium influx and oxidative stress. In addition, Surmeier said, only a small number of PD patients have mutations in DJ-1. Do other genetic linkages interact in the same way with mitochondrial stress? “While the phenomenon fits very nicely with most of the prevailing theories about the pathogenesis of PD, many of which point to dysfunctions in mitochondria as being causal,” Surmeier said, “we are not certain that this is the major mechanism in most forms of PD.”
The only way to definitively test how widespread this mechanism is in human disease, Surmeier suggested, is to try dihydropyridine therapy in people with PD and see if it helps. There is already epidemiological evidence that people taking dihydropyridines have a lower risk of developing Parkinson’s (see Becker et al., 2008 and Ritz et al., 2010). Two clinical trials of isradipine are currently in the works—a small Phase 2 safety trial with about 20 participants, led by Northwestern University, and a larger Phase 2 trial with about 100 participants. The larger trial involves numerous institutions, including Northwestern, and is funded by the Michael J. Fox Foundation. Surmeier said the main purpose of the trials, which will conclude next year, is to determine the best dosage of isradipine. Once that is known, “we plan to take that to a Phase 3 clinical trial.” Surmeier expects to submit the Phase 3 proposal to the National Institutes of Health in 2012.
Surmeier sees more far-reaching implications to these findings. “This paper provides an explanation for why age is the number one risk factor in Parkinson’s disease. There is a low level of oxidative stress [in SNc neurons] that does not kill cells overnight, but that creates wear and tear on the mitochondria that may take decades to manifest itself. Ultimately, this may be a clue not just to what is going on in PD, but to what is going on in some of the other aging-related diseases like Alzheimer’s disease as well.”—Madolyn Bowman Rogers.
Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT, Surmeier DJ. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature. 2010 November 10. Abstract