One of the enduring mysteries of Parkinson disease has been the exquisite specificity of neuronal death. The movement disorder appears when a cluster of dopaminergic neurons in the substantia nigra (SN) degenerate, but at the same time their dopaminergic neighbors in the ventral tegmental area (VTA) stay alive and well. Explanations for SN neuron death have invoked dopamine, calcium, and the α-synuclein protein in turn as the pathologic players, but none of these factors on its own explains specificity. A new study from David Sulzer and colleagues at Columbia University Medical Center in New York proposes a solution, though not a simple one. Their data, appearing in the April 30 issue of Neuron, suggest that SN neurons brew up a perfect storm of dopamine plus calcium plus synuclein that leads to their selective destruction. Knocking out any one leg of the toxic tripod prevents cell death, which opens up a trio of pathways for potential new therapies.
The study centers on a novel technique, developed by first author Eugene Mosharov, to measure cytosolic dopamine concentrations in living cells (see ARF related news story). When the investigators turned their technique on SN neurons in ventral mesencephalon cultures from mice, they found that the cells have undetectable cytosolic dopamine (less than 100 nM). However, adding L-DOPA to the cells in high concentrations boosted cytosolic dopamine to measurable levels, and caused neurotoxicity. Further studies indicated that cell death was tied to cytosolic dopamine levels: Neurotoxicity depended on the conversion of L-DOPA to dopamine, and reducing cytosolic dopamine levels by overexpression of the vesicular dopamine transporter VMAT2 protected the cells from death. When the experiments were done in cells from α-synuclein knockout mice, cytosolic dopamine levels were still elevated, but toxicity was reduced. That suggested that α-synuclein was downstream of dopamine in the neurotoxic cascade. Finally, the investigators showed that blocking dopamine metabolism by inhibiting monoamine oxidase also protected the cells from death, suggesting it is not dopamine per se, but a metabolite that is the toxin. All together, the data support a model where high cytosolic dopamine leads to generation of oxidized metabolites that interact with α-synuclein to cause neurotoxicity (e.g., see Burke et al., 2008 and Martinez-Vicente et al., 2008).
Next, things became really interesting when Mosharov and colleagues compared SN cells to VTA dopaminergic neurons. They found that after treatment with L-DOPA, the SN cells developed two to three times higher levels of cytosolic dopamine than VTA cells. Along with lower levels of cytosolic dopamine, the VTA cells were more resistant to the toxic effects of L-DOPA. There was no apparent difference in uptake, storage, or degradation of dopamine between the two cell types, suggesting that elevated synthesis was to blame for the higher cytosolic dopamine levels and neurotoxic response to L-DOPA in the SN.
But why? Previous work from the lab of James Surmeier at Northwestern University in Chicago, Illinois, showed that SN neurons have an unusual physiology involving a calcium-dependent pacemaking activity that results in higher overall calcium fluxes than in VTA neurons (see ARF related news story). The calcium flows through dihydropyridine-sensitive L-type CaV1.3 channels, and the Chan paper suggests that calcium-related toxicity may explain the age-related degeneration of the cells. To test if calcium was regulating dopamine synthesis or toxicity, Mosharov and colleagues treated SN cells with inhibitors of the CaV1.2/1.3 channels and found that the L-DOPA-induced elevation in cytosolic dopamine was diminished to where it resembled that in VTA cells. The SN cells also became resistant to the neurotoxic effects of L-DOPA. The channel blockers had no effect on dopamine uptake or breakdown, leaving the likely target of regulation to be the calcium-sensitive amino acid decarboxylase enzyme, which catalyses the conversion of L-DOPA to dopamine.
“The results suggest you need a convergence of high cytosolic dopamine, synuclein, and high calcium to explain the selective sensitivity of SN neurons,” Sulzer told ARF. That makes the picture complicated, he said, but it also opens up multiple opportunities for therapies. Based on Surmeier’s previous work, clinical trials have already started on L-type calcium channel blocker isradipine for PD. The drug is attractive because of its long track record in humans, although a compound more selective for the CaV1.3 might be even better, Sulzer said. Strategies to lower α-synuclein are another possibility. A third prospect is to target cytosolic dopamine, and Sulzer and Mosharov said they are working on overexpression of the VMAT2 transporter as one approach. By loading dopamine into vesicles, VMAT2 lowers cytosolic dopamine and protects cells, as they show in the current paper. Finally, inhibitors of monoamine oxidase including selegiline and rasagiline are used to treat PD, and are being investigated for neuroprotective actions. “People are finding they work, but they don’t know why," Sulzer said. “Our paper may explain that, by suggesting that they block the metabolism of dopamine to a form that interacts with α-synuclein to make a toxic species.”
In an accompanying commentary, Surmeier ties the higher calcium flux in SN cells to the idea that mitochondria are also important in the pathogenesis of PD. He speculates that perhaps SN cells that constantly handle a higher calcium flux end up taxing their mitochondria because these organelles buffer calcium, and over the long term they have to supply the energy to maintain calcium levels. “Because aging diminishes the mitochondrial capacity to generate ATP through oxidative phosphorylation, neurons with a calcium-reliant pacemaking phenotype should be operating nearer and nearer their metabolic capacity, diminishing their ability to withstand episodic challenges,” Surmeier writes.
A related paper by Kim Tieu of the University of Rochester, New York, and Serge Przedborski of Columbia University solves another mystery by providing a missing link in a common model of PD, namely MPTP toxicity. MPTP is a contaminant in heroin that was found to cause an acute parkinsonism in some drug users, and it has become widely used in animal models to mimic the SN degeneration of PD. Researchers know that MPTP is taken up by astrocytes and converted to the mitochondrial toxin MPP+, which is selectively taken up by neurons that express the dopamine transporter. However, it was not clear how MPP+ got out of astrocytes. Tieu and Przedborski present evidence that the organic cation transporter-3 (Oct3) is expressed in astrocytes adjacent to midbrain dopaminergic neurons and is necessary for death of dopaminergic neurons in mice treated with MPTP. The bidirectional transporter may also protect cells under some conditions, the data suggest, by taking up excess extracellular dopamine or other toxins. The paper appears in the PNAS early edition this week.—Pat McCaffrey