For more than a decade, scientists have known that α-synuclein plays an important role in Parkinson’s disease, but they are still unsure how the protein influences pathology. Reporting in two papers in the October 24 Science, scientists led by Susan Lindquist, Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, detail an unbiased yeast-to-human-cell approach to uncover α-synuclein’s dark side. They turned up toxic properties that persist in yeast, rodent-, and human-derived neurons, and found a small molecule that protects against them. “This work demonstrates the power of a simple model system to uncover something relevant to human disease,” said Aaron Gitler, Stanford University School of Medicine, California, a former postdoctoral fellow in Lindquist’s lab. Gitler was not involved with this project. While skeptics might argue that yeast hardly recreate Parkinson’s disease (PD), “the system models fundamental interactions between the protein and the cell biology that it interfaces with,” he said.

Drug developers often focus on target-based approaches, screening for compounds that modulate a protein or pathway and then testing them in appropriate disease models. Some researchers question this tactic, suggesting it has been less productive than phenotypic screens of the past (see Swinney and Anthony, 2011). This strategy tests compounds in disease models first, without any preconceived notions about the underlying dysfunction. Lindquist’s lab turned to that approach, screening for compounds that correct protein toxicity in yeast cells (see related news story and related news story).

In their first paper, first author Daniel Tardiff and colleagues described how they screened a chemical library for compounds that could protect yeast against toxic protein aggregates. An N-aryl benzimidazole (NAB) potently rescued cells against α-synuclein toxicity. The compound prevented the accumulation of α-synuclein in cytoplasmic vesicles, kept reactive oxygen species and nitrated proteins at bay, and lifted α-synuclein’s block on ER-Golgi and endocytic trafficking. The researchers then found that NAB worked similarly in other models of α-synuclein toxicity, including roundworms and rat primary neurons that overexpress human wild-type synuclein and an A53T mutant, respectively. The results hinted that the small molecule has the same mechanism of action in different species. A derivative of the small molecule, NAB2, was even more potent.

To understand how these compounds worked, the researchers tested NAB2 on libraries of yeast representing overexpression and loss of each of 5,800 genes—most of the yeast genome. NAB2 modulated a network of genes involved in ubiquitin-mediated vesicle trafficking. Rsp5, an E3 ubiquitin ligase that promotes endosomal transport, sat in the hub of this network. In wild-type yeast cells, NAB2 stimulated Rsp5-dependent trafficking, endocytosis, and degradation of substrates, whereas α-synuclein inhibited these processes. Curiously, the human homologue, Nedd4, helps ubiquitinate a variety of proteins, regulating multiple steps in vesicle trafficking. Nedd4 was reported to ubiquitinate α-synuclein (see related news story). No evidence suggested that Rsp5 directly ubiquitinates α-synuclein in this yeast model, but it could happen in other systems, said Tardiff.

Capitalizing on these yeast screens, husband-and-wife co-first authors on the second paper, Vikram Khurana and Chee Yeun Chung, uncovered toxic phenotypes in patient-derived neurons. Just as nitric oxide-related damage and endoplasmic reticulum stress surfaced in yeast that overexpressed α-synuclein, the same occurred in glutamatergic cortical neurons derived from the induced pluripotent stem cells of a person carrying the A53T synuclein mutation. NAB2 prevented both phenotypes in yeast and in neurons. All in all, the data suggest that eukaryotic cells, from yeast to humans, have conserved toxic responses to α-synuclein, a property that could be exploited for drug discovery, said Khurana. “We show that it’s possible to cross over from this yeast model not just to a worm or rat neuron, but to human neurons,” he said.

Researchers in Lindquist’s lab were careful to point out that NAB2 is not yet a drug. They are working to improve its pharmaceutical properties before they test it in animal models of PD.

“This is a fantastic way of combining techniques to speed up the drug discovery process,” said Stuart Lipton, Sanford-Burnham Medical Research Institute, La Jolla, California. It would be interesting to see whether the same α-synuclein pathways affect not just cortical, but dopaminergic neurons of patients early in PD, he said. He is conducting similar experiments to screen human induced pluripotent stem cells for compounds that protect against protein toxicity. While yeasts provide a speedier, cheaper way to screen eukaryotic cells, human neurons might reveal unique targets, he said. Lipton plans to collaborate with Lindquist’s lab.—Gwyneth Dickey Zakaib.

References:
Tardiff DF, Jui NT, Khurana V, Tambe MA, Thompson ML, Chung CY, Kamadurai HB, Kim HT, Lancaster AK, Caldwell KA, Caldwell GA, Rochet JC, Buchwald SL, Lindquist S. Yeast Reveal a "Druggable" Rsp5/Nedd4 Network that Ameliorates α-Synuclein Toxicity in Neurons. Science. 2013 Oct 24. Abstract

Chung CY, Khurana V, Auluck PK, Tardiff DF, Mazzulli JR, Soldner F, Baru V, Lou Y, Freyzon Y, Cho S, Mungenast AE, Muffat J, Mitalipova M, Pluth MD, Jui NT, Schüle B, Lippard SJ, Tsai LH, Krainc D, Buchwald SL, Jaenisch R, Lindquist S. Identification and Rescue of α-Synuclein Toxicity in Parkinson Patient-Derived Neurons. Science. 2013 Oct 24. Abstract

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Comments on News and Primary Papers

  1. Chung and colleagues elegantly demonstrate in this article how a familiar Parkinson mutation, the A53T variant of α-synuclein, causes nitrosative stress that affects the secretory transport of several neuronal proteins in iPS-derived neurons. This phenomenon was rescued by either expression of suppressors of endoplasmic reticulum-associated protein degradation (ERAD), like the ubiquitin ligase Hdr1, or by small compound inhibitors of NAB2. Of note, ER stress has been implicated in other neurodegenerative diseases, such as Alzheimer’s and prion disease. This study is elaborate and provides an in-depth view of the effects of nitrosative stress on core cellular functions such as protein transport and degradation.

    The questions that remain are how nitric oxide controls these events and what its targets are. In the case of PD, nitration and dityrosine modification of α-synuclein itself have been found in synucleinopathy lesions (1–3). Therefore, it is of interest to know if α-synuclein itself, or others, are modified by nitric oxide in PD.

    View all comments by Markus Kummer

References

News Citations

  1. Yeast Screen Implicates PARK9 in Synuclein Toxicity
  2. Yeast Teases Apart Huntington’s and Parkinson’s Protein Aggregation
  3. A Nod to Nedd4: Is the Synuclein Ligase Finally Found?

Paper Citations

  1. . How were new medicines discovered?. Nat Rev Drug Discov. 2011 Jul;10(7):507-19. PubMed.
  2. . Yeast Reveal a "Druggable" Rsp5/Nedd4 Network that Ameliorates α-Synuclein Toxicity in Neurons. Science. 2013 Nov 22;342(6161):979-83. PubMed.
  3. . Identification and Rescue of α-Synuclein Toxicity in Parkinson Patient-Derived Neurons. Science. 2013 Nov 22;342(6161):983-7. PubMed.

Further Reading

Papers

  1. . Development and validation of a yeast high-throughput screen for inhibitors of Aβ₄₂ oligomerization. Dis Model Mech. 2011 Nov;4(6):822-31. PubMed.
  2. . Novel suppressors of alpha-synuclein toxicity identified using yeast. Hum Mol Genet. 2008 Dec 1;17(23):3784-95. PubMed.
  3. . Phenotypic vs. target-based drug discovery for first-in-class medicines. Clin Pharmacol Ther. 2013 Apr;93(4):299-301. PubMed.
  4. . Target-based drug discovery: is something wrong?. Drug Discov Today. 2005 Jan 15;10(2):139-47. PubMed.
  5. . Yeast Reveal a "Druggable" Rsp5/Nedd4 Network that Ameliorates α-Synuclein Toxicity in Neurons. Science. 2013 Nov 22;342(6161):979-83. PubMed.
  6. . Identification and Rescue of α-Synuclein Toxicity in Parkinson Patient-Derived Neurons. Science. 2013 Nov 22;342(6161):983-7. PubMed.

Primary Papers

  1. . Yeast Reveal a "Druggable" Rsp5/Nedd4 Network that Ameliorates α-Synuclein Toxicity in Neurons. Science. 2013 Nov 22;342(6161):979-83. PubMed.
  2. . Identification and Rescue of α-Synuclein Toxicity in Parkinson Patient-Derived Neurons. Science. 2013 Nov 22;342(6161):983-7. PubMed.