25 Jun 2006

Mutations in the presynaptic protein α-synuclein, or even just overexpression, can cause Parkinson disease (PD) by killing off midbrain dopaminergic neurons. Despite evidence that the deadly effects of synuclein are conserved from yeast to mammals—even worms and flies develop PD-like neuron loss under the influence of human α-synuclein—researchers have been frustrated in their attempts to pin a toxic mechanism to the protein. Now, studies with yeast have yielded up a substantial insight into α-synuclein’s pathological actions. Susan Lindquist and colleagues at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, and a bevy of collaborators, show that α-synuclein overexpression causes endoplasmic reticulum (ER) stress by blocking vesicle trafficking from the ER to the Golgi. They showed that overexpression of proteins that increase forward transport, most notably the GTPase Ypt1p, released the transport bottleneck and associated pathology. Conversely, proteins that inhibited transport exacerbated α-synuclein toxicity.

The researchers demonstrated that α-synuclein-expressing human neurons had the same transport problem, and that overexpression of the human Ypt1p homolog, Rab1, blocked death of dopaminergic neurons in flies, worms, and rat primary neuron cultures expressing α-synuclein. The work, a collaboration with researchers from the University of Missouri-Kansas City, Nancy Bonini and colleagues at the University of Pennsylvania, and colleagues at several other institutions, appeared June 22 in the online edition of Science.

Lindquist’s previous work showed yeast to be an exceedingly good model for α-synuclein’s actions on cell pathways. Overexpression of the normal protein produces many of the symptoms of α-synuclein toxicity seen in mammalian systems (see ARF related news story). In this study, first authors Antony Cooper from Missouri and Aaron Gitler from Lindquist’s lab engineered the yeast with a galactose-inducible α-synuclein gene, either wild-type or the A53T mutant associated with PD, to get a look at the earliest events in cell toxicity. Within hours of turning on α-synuclein, the cells began to lose viability. ER stress, in response to the accumulation of misfolded proteins, became apparent within 4-6 hours, and all the cells died by 24 hours.

The early impairment in cell growth coincided with arrest of vesicular trafficking from the ER to the Golgi, which preceded ER stress. The authors detected the trafficking defect by following the progress of two different endogenous proteins known to travel the ER-Golgi pathway. A genome-wide screen for modifiers of α-synuclein toxicity provided corroborating evidence of the ER-Golgi involvement. After testing 3,000 genes, the researchers found 34 that suppressed toxicity and 20 that enhanced it. The largest class of genes identified in the screen all acted at the same step of membrane vesicle movement, the ER to Golgi transfer.

Together, these results suggested that promoting movement down the transport pathway might mitigate α-synuclein toxicity, and the researchers quickly proved this to be true. Overexpressing Ypt1p, a yeast Rab GTPase, increased forward trafficking of the reporter proteins and relieved α-synuclein toxicity, while blocking traffic with a dominant negative form of Ypt1p or a negative regulator exacerbated α-synuclein toxicity. In cells, Ypt1p localized with α-synuclein in cytosolic inclusions.

The same pathology applied to flies and worms, where Rab1 was as active as any α-synuclein suppressor identified so far in these systems. In rat mid-brain dopaminergic neurons, too, Rab1 expression completely reversed α-synuclein-A53T mutant toxicity.

Recent work from Thomas Sudhof’s lab showed that α-synuclein can rescue SNARE complex assembly in mice lacking the chaperone cysteine-string protein-α (CSPα, see ARF related news story). Those results implied that α-synuclein has a physiological role in synaptic vesicle transport. The current study suggests that the toxicity of overexpressed or mutant synuclein stems from the protein’s ability to interact with related proteins that function in ER-Golgi transport, and disrupt their function. In yeast, the current study shows that Ypt1p was sometimes found in synuclein cytoplasmic inclusions, consistent with the idea that synuclein might sequester Ypt1p and prevent it from doing its normal job.

The new results offer a potential explanation for the selective toxicity of α-synuclein on dopaminergic neurons, even though the protein is present throughout the brain. The authors speculate that slowdowns in the ER-Golgi early secretory pathway could hit these neurons especially hard. The diminution in vesicle traffic might lead to a shortage of secretory vesicles and a rise in cytosolic dopamine. Since dopamine, of all neurotransmitters, is particularly toxic, disruptions in its transport leading to accumulation in the cell could be the neurons’ Achilles heel.

This work, the authors write, “establishes that simple model systems can be useful for the investigation of even complex neurodegenerative disease.” All at once, the yeast PD model reveals a toxic function for α-synuclein and allows identification of a number of potential new targets for blocking its neurotoxicity, and provides a platform for rapid screening of new compounds. That’s useful, indeed.—Pat McCaffrey

Comments

1. • Bharathi Shrikanth Gadad
     Johns Hopkins Univ School of Medicine
   • Posted: 28 Jul 2006

   The role and precise function of α-synculein in Parkinson disease is still shrouded in mystery. In yeast cells and in neurons, α-synculein accumulation is cytotoxic, but little is known about its normal function or pathobiology. Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. Currently in this paper, the authors have discussed that the earliest defect following α-synculein expression in yeast was a block in endoplasmic reticulum to Golgi vesicular trafficking. In a genome-wide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab GTPase Ypt1p, which associated with cytoplasmic α-synculein inclusions. Elevated expression of Rab1, the mammalian Ypt1 homolog, protected against α-synculein-induced dopaminergic neuron loss in animal models of PD. Hence the results discussed in this article are more consistent with a role for Ypt1/Rab proteins in determining the directionality or fidelity of protein sorting and also open up new directions in understanding the role of α-synculein function and the factors that renders in regulating this process.

   References:

   Jedd G, Richardson C, Litt R, Segev N. The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway. J Cell Biol. 1995 Nov;131(3):583-90. PubMed.

   Jones S, Richardson CJ, Litt RJ, Segev N. Identification of regulators for Ypt1 GTPase nucleotide cycling. Mol Biol Cell. 1998 Oct;9(10):2819-37. PubMed.

   View all comments by Bharathi Shrikanth Gadad

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References

News Citations

1. Yeast Teases Apart Huntington’s and Parkinson’s Protein Aggregation 5 Dec 2003
2. SNAPshot—α-Synuclein Caught With Synaptic Vesicle Proteins 4 Nov 2005

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