31 May 2002. In tomorrow's Nature Medicine, Bruce Yankner at Children's Hospital in Boston offers up a partial explanation for one of the abiding questions in Parkinson's research: Why do neurons die in such a selective pattern? A similar question looms large in Alzheimer's research. The answer, first author Jin Xu et al. propose, is that accumulating α-synuclein becomes neurotoxic in a way that is dependent on the affected cells' neurotransmitter, dopamine, and its link to oxidative stress. The study implicates an α-synuclein complex containing 14-3-3, a protein chaperone known to tie up pro-apoptotic signals. The authors suggest that this complex sequesters this cell death inhibitor, leaving its normal binding partners free to promote cell death.
Xu et al. established cultures enriched in human fetal dopaminergic neurons, as well as non-dopaminergic human cortical neurons as controls. Expressing high levels of human α-synuclein led to apoptosis in the dopaminergic cells but, curiously, appeared to extend survival of other neuron types. Notably, there was no clear difference between the human wildtype or the A53T or A30P mutant forms of α-synuclein, which cause early-onset Parkinson's. This neurotoxic effect disappeared when the scientists blocked dopamine production, even when the neurons were put under oxidative stress. Further, the scientists found that transfected α-synuclein increased production of reactive oxygen species. They suggest that accumulating α-synuclein potentiates the generation of oxygen radicals that is known to occur with dopamine metabolism (Hastings et al., 1996).
What is the physical nature of the neurotoxic α-synuclein? The authors report finding the protein in the soluble compartment after fractionation and centrifugation of cell lysates, within a detection limit of 2 percent of total a-synuclein. Based on size-exclusion chromatography and immunoprecipitation experiments, the authors implicate a 54-83 kD complex as the neurotoxic species, which contains 14-3-3 protein. The study also includes chromatography of a-synuclein isolated from substantia nigra of PD patients, and suggests that a 14-3-3/a-synuclein complex of similar molecular weight accumulates in this affected area. The precise composition of the complex remains unclear.
Taken together, the paper advances the hypothesis that accumulating α-synuclein sequesters 14-3-3, which releases pro-apoptotic proteins and in turn increases the cell's vulnerability to endogenous reactive oxygen species generated by dopamine.
In an accompanying News and Views, Kevin Welch and Junying Yuan of Harvard Medical School write that, if true, this points to a treatment dilemma long known to neurologists: While clearly relieving symptoms, dopamine might also hasten an underlying neurotoxic process.-Gabrielle Strobel.
Xu J, Kao SY, Lee FJS, Song W, Jin LW, Yankner B. Dopamine-dependant neurotoxicity of a-synuclein: A mechanism for selective neurodegeneration in Parkinson disease. Nature Medicine. 1 June 2002; 8(6):600-06. Abstract
Welch K, Yuan J. Releasing the nerve cell killers. Nature Medicine. 1 June 2002;8(6):564-565. Abstract
Q&A with Bruce Yankner
Q: The toxic 54-83 kD complex, what is in it? Does it contain dimers/trimers of α-synuclein together with 14-3-3? Or α-synuclein monomers with 14-3-3? I wonder because of the debate about what is the toxic species, monomers, oligomers, protofibrils, mature fibrils…
A: Yes, the toxic form is in a complex that at least in part contains 14-3-3. We cannot comment yet on the stoichiometry of the components. There is no evidence of fibrils or protofibrils in our in-vitro system. Regarding the controversy, it is important to note that protofibrils have never been observed in vivo either in AD or PD. They may be too unstable to accumulate. A minor point is that protofibrils have traditionally been defined as small fibrillar intermediates. Some have recently used this term to define any oligomer-a practice I think is confusing as it is unclear whether these oligomers progress to fibrils.
Q: In PD, why would this happen primarily in the substantia nigra and not in other dopaminergic regions, i.e. tegmentum? The selectivity question then becomes why does α-synuclein accumulate selectively in striatum, right?
A: Synuclein accumulation in nigral neurons may reflect a high level of vulnerability to oxidative stress. It is unclear why other dopaminergic populations do not exhibit the same levels of α-synuclein accumulation or neurodegeneration.
Q: Why don't we see death of dopaminergic cells in α-synuclein overexpressing transgenics? What's missing in the mouse models? (Increased oxidative stress?)
A: Dopaminergic cell death has been observed in an adenovirus-mediated model of synuclein overexpression in the rat brain (see related news item), and in the Drosophila model. And the Masliah transgenic mouse showed loss of dopaminergic nerve terminals in striatum. Murine neurons may be more resistant than human dopaminergic neurons, which were used in our study. The mouse endogenously expresses the A53T α-synuclein mutation that causes familial PD in humans. Since mice don't get PD, there has to be a basic difference.
Q: How could you prove that your work on cultured fetal DA neurons is relevant to PD?
A: An important step would be to show that reducing endogenous dopamine levels prevents α-synuclein-related neurodegeneration in animal models of PD. The role of dopamine in the disease mechanism is an important issue because it bears on the question of whether L-DOPA therapy could accelerate neurodegeneration while transiently relieving extrapyramidal symptoms. Our study does not provide any information on this issue. However, this question is being addressed in clinical trials.
Q: Finally, how does Aβ fit into your system with regard to the Lewy-body variant of AD?
A: Aβ, like dopamine, can induce the generation of reactive oxygen species. The accumulation of α-synuclein in the Lewy-body variant may potentiate Aβ toxicity, similar to the potentiation of endogenous dopamine toxicity. However, this may be due to the accumulation of soluble α-syn-14-3-3 complexes. Lewy bodies may be a secondary effect.