Who/Where: Dhungel et al., 2015, Neuron, Volume 85, Issue 1, 7 January 2015, Pages 76-87
Preview of paper: www.cell.com/neuron/abstract/S0896-6273(14)01154-4
The Gitler Laboratory, Stanford University, CA, USA
Parkinson’s disease is a relatively common neurodegenerative movement disorder caused by a loss of dopamine-producing neurons from the substantia nigra region of the brain. The disease is mostly sporadic but about 10% of cases exhibit a familial pattern of inheritance and these rarer cases have led to the identification of mutations in genes that can cause Parkinson’s disease. Studying the functions of these familial Parkinson’s disease genes have led to important insights into the cellular pathways that are affected in even the more common sporadic forms of the disease. A better understanding of the pathways that contribute to Parkinson’s disease will hopefully lead to more effective therapeutic approaches.
In our laboratory we have been using a very simple experimental model system to study human diseases associated with protein misfolding and neurodegeneration. We are using the budding yeast, Saccharomyces cerevisiae. Even though human diseases like Parkinson’s disease are very complicated, we hypothesize that some of the defects that underlie the disease are associated with alterations in basic cellular pathways, and nearly all of the key cellular pathways are well-conserved from yeast to human. By using the power of yeast genetics, our laboratory aims to uncover mechanisms associated with terrible disorders such as Parkinson’s disease.
Two of the newer genes implicated in Parkinson’s disease are EIF4G1 and VPS35. EIF4G1 encodes a translation initiation factor and VPS35 is component of the retromer complex. EIF4G1 and VPS35 both have related genes in yeast (human EIF4G1 = yeast TIF4631 and TIF632 and human VPS35 = yeast VPS35). Since both of the yeast homologs of EIF4G1 and VPS35 are non-essential genes, strains of yeast with deletions of either were alive and growing well. We wanted to define the cellular functions of these genes so we decided to perform a screen to identify genes that we could delete in combination with either Tif4631 or Vps35 that would cause a growth defect. This type of a screen is called a synthetic lethal screen and is a powerful way to identify genes that function in similar cellular processes. It gives us great insight into how the genes interact with the rest of the genome, thereby giving us clues as to the pathways that are perhaps involved in Parkinson’s disease.
The yeast genome contains about 6,000 genes and about 4,850 of these are non-essential. Manipulating those many strains manually is a tremendous task that is nearly impossible and time consuming. Luckily we were able to utilize the Singer Instruments ROTOR, a High Density Array robot to conduct the monumental screens. This automated robotics method was used to combine each of the 4,850 non-essential haploid yeast deletions with either the vps35 deletion or the tif4631 deletion by mating. This generated diploid cells, which we then sporulated and finally selectively germinated haploid progeny that contained both deletions. All of these tedious tasks were taken care of by our Singer Instruments ROTOR and we did three replicates of the screens. We then used image analysis software to compare the size of colonies in order to identify double mutants that grew worse when compared to either of the single mutants. This gave us great insight into how VPS35 and EIF4G1 functioned. We were then able to take the yeast data and confirm our findings in other model organisms such as the nematode worms and mice. By using the ROTOR, we were able to conduct large genome-wide screens in less than two weeks and produce reliable, statistically testable, and repeatable data.