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

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.

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  1. I found this paper quite intriguing and very much relevant to PD. I believe it provides a very plausible model that tries to address the long-standing question of the fairly selective neurodegenerative process seen in PD (i.e. dopaminergic neuronal vulnerability), and it convincingly links the metabolisms of dopamine and α-synuclein to each other and to a neurotoxic process. That the work was carried out in cultured human neurons no doubt brings this to a new level. Peter Lansbury's team has addressed this in an in-vitro system (see related news item).

    If I were to be the reviewer, I would raising the following points:

    1. I think there is a significant element of confusion in the terminology—not the actual data—in this article. The authors speak of a "soluble" synuclein form/complex. However, they mean "solubilized", as their regimen to study proteins in cells and tissue employs several membrane-disrupting detergents that clearly release α-synuclein and other proteins from membrane structures. This makes sense, then, with what they describe in the nigra from PD patients, where people have shown repeatedly an α-synuclein increase in detergent-requiring extracted material.

    2. The data provided on the association of 14-3-3 and α-synuclein is less convincing than they write when one looks at Figure 6e. There is a significant component of non-specificity, which should be clarified. Nevertheless, there are already other reports (Ostrerova et al, 1999; Perez et al., 2002, and two neuropathological studies) that show an interaction of α-synuclein with 14-3-3.

    3. The authors did not discuss that, in PD, a significantly affected area is that of Meynert's basal nucleus, a group of cholinergic neurons that use the same principal neurotransmitter, i.e. acetylcholine, as the human cortical neurons used as controls in their study.

    This paper does not directly address the glycosylated species of α-synuclein that we found in human brain (see related news item). However, the work is consistent with our findings in that Xu et al. do not identify parkin associated with their higher Mr complex that contains regular, non-glycosylated α-synuclein both in cellular and brain extracts (we also don't see this.

    References:

    . alpha-Synuclein shares physical and functional homology with 14-3-3 proteins. J Neurosci. 1999 Jul 15;19(14):5782-91. PubMed.

    . A role for alpha-synuclein in the regulation of dopamine biosynthesis. J Neurosci. 2002 Apr 15;22(8):3090-9. PubMed.

  2. We need to remember that many studies, such as this one by Yankner, involve in vitro systems that are not sustained by a complex network, as is the case in vivo in transgenic mice. This is also relevant to Alzheimer disease models.

    Specifically, activation of caspase pathways is more easily triggered in vitro than in vivo. In general, we have a very difficult time triggering these cascades in transgenic mice. However--and this goes for humans, as well--synapses are very sensitive and vulnerable even in vivo, and apoptotic pathways that are activated in the whole cell in vitro are activated at the synaptic site in vivo. In the AβPP/synuclein-transgenic models, we observe widespread synaptic damage accompanied by focal activation of caspases (unpublished). Greg Cole and Mark Mattson call this process "synaptosis." It probably reflects more closely what happens in human brains and mice than do in vitro models.

    It is interesting that the authors observed that α-synuclein is toxic to dopaminergic neurons while being rather protective for cortical neurons. Indeed, some papers have published cytotoxic effects of α-synuclein, whereas others have report on protective effects of α-synuclein against oxidative stress. As we recently reported (Hashimoto et al., 2002), such selectivity of the response against oxidative stress might reflect the interaction of α-synuclein with components of stress signaling pathways, such as JIP.

    Finally, a series of biochemical experiments by J. Kim. and colleagues clearly showed that α-synuclein behaves as a high molecular weight molecule (~around 53 kD) under non-denaturing conditions, such as HPLC using size-exclusion columns. This seems to be due to the unique elongated structure of α-synuclein. Therefore, I wonder if the 54-83 kD immunoreactivity of α-synuclein, which was detectable by size-exclusive chromatography in the paper, might be due to neither α-synuclein oligomerization nor complex formation with other molecules, such as 14-3-3. More scrutiny will be required on this point.

    References:

    . alpha-Synuclein protects against oxidative stress via inactivation of the c-Jun N-terminal kinase stress-signaling pathway in neuronal cells. J Biol Chem. 2002 Mar 29;277(13):11465-72. PubMed.

  3. Xu et al., present very interesting data that will improve our understanding of the relationship between α-synuclein and the degeneration seen in PD patients. Their work is in support of an interaction of dopamine and α-synuclein to mediate toxic effects specifically in dopaminergic cells of the substantia nigra pars compacta, while α-synuclein in the absence of dopamine may, in contrast, be neuroprotective. Secondly, they show that α-synuclein in these cells remains in a detergent-extractable fraction, possibly bound to 14-3-3 protein. It remains to be determined whether this is the toxic species in the human disease, or inversely, whether the formation of inclusions is protective.

    Regarding the apparent similarity of human wildtype versus mutant a-synuclein proteins in their in-vitro and our in-vivo experiments: The difference between these two may be more obvious if the expression level is low. An indication of this sort is in the Xu et al. paper. In our material we think we have about a 10-fold induction above the normal levels, which may be well above where you can detect this difference (see ARF news story).

    If their hypothesis is true, then overexpression of 14-3-3 protein should be helpful in reversing the apoptotic changes and, by analogy, should reduce the cell death seen in our rat model. In addition it would be worth trying whether antioxidants protect against α-synuclein toxicity in vivo.

    The notion that dopamine accelerates the neurodegenerative process while treating symptoms is interesting. We are currently testing the interaction of α-synuclein and dopamine in various in-vivo models where we overexpress α-synuclein using the AAV vectors, but it is too early to give any results on that.

  4. I appreciated reading the above discussion of Xu et al.’s paper regarding a potential role for α-synuclein, 14-3-3, and dopamine in the pathogenesis of Parkinson's disease. I agree that α-synuclein and dopamine are a toxic mix. I do, however, want to point out our paper in the April 15th Journal of Neuroscience (Perez et al., 2002), which was the first to report a function for α-synuclein that is specific to dopamine neurons. Our data show that α-synuclein, in addition to 14-3-3, is an important regulator of tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis.

    Our data clearly show that dopaminergic cells stably overexpressing α-synuclein are healthy, maintain high-level tyrosine hydroxylase expression, co-localize tyrosine hydroxylase with α-synuclein, and have dramatically reduced dopamine synthesis. First reported at the Society for Neuroscience meeting in 2000, our findings prompted us to speculate even then that α-synuclein likely contributes to macromolecular damage underlying the selective loss of dopamine neurons in Parkinson's disease, and that it does so by an interaction with 14-3-3 that regulates dopamine with potential toxic consequences. Elegant work by Peter Lansbury's group (see related news item) also implicates α-synuclein and dopamine in Parkinson's disease pathogenesis, if by another mechanism.

    The findings of Xu et al. are intriguing. It is curious, however, that nigral neurons overexpressing α-synuclein were not protected against dopamine-related toxicity, as would be expected since a-synuclein downregulates dopamine synthesis. It will be important to elucidate the mechanism(s) by which α-synuclein affects dopaminergic cell viability. Nonetheless, ours is the first report to identify a function for α-synuclein that is specific to dopaminergic neurons."

    References:

    . A role for alpha-synuclein in the regulation of dopamine biosynthesis. J Neurosci. 2002 Apr 15;22(8):3090-9. PubMed.

  5. Reply by Bruce Yankner

    The paper by Perez et al. is quite interesting because it suggests that α-synuclein can downregulate tyrosine hydroxylase activity in a stably transfected dopaminergic cell line. However, this is a cell line that has been selected for stable α-synuclein overexpression. The cells probably survive because they have downregulated dopamine production. It remains to be determined whether primary neurons can do the same thing. Our report suggests that α-synuclein-induced degeneration of dopaminergic neurons is dependent on dopamine production. Thus, primary neurons may not have the same capacity for TH downregulation as a transformed cell line. Interestingly, we also have been able to select for stably transfected SH-SY5Y cell lines that overexpress α-synuclein and survive. However, during this selection process most cells die. It is only a small subpopulation that can survive and be expanded. And although these cells survive, they exhibit increased vulnerability to exogenous dopamine.

References

News Citations

  1. Viral Transgenic Techniques Pay Off In Parkinson's Models

External Citations

  1. Hastings et al., 1996

Further Reading

Primary Papers

  1. . Dopamine-dependent neurotoxicity of alpha-synuclein: a mechanism for selective neurodegeneration in Parkinson disease. Nat Med. 2002 Jun;8(6):600-6. PubMed.
  2. . Releasing the nerve cell killers. Nat Med. 2002 Jun;8(6):564-5. PubMed.