Protein folding is critically important for all life, from microbes to man. A bafflingly diverse set of cellular mechanisms has evolved to coordinate this exquisitely sensitive process. Not unexpectedly, problems in protein folding are the root cause of many of the most devastating diseases, which represent a major challenge to public health worldwide, especially as our population continues to age. Referred to collectively as protein-misfolding disorders, these truly disastrous neurodegenerative diseases include Alzheimer’s disease, Parkinson’s disease and ALS (Lou Gehrig’s disease). Understanding at a mechanistic level the cellular consequences of protein misfolding will help to suggest potential strategies for therapeutic intervention. We use the baker’s yeast, Saccharomyces cerevisiae, as a model system to study the cell biology underpinning protein-misfolding diseases. Since dealing with misfolded proteins is an ancient problem, we hypothesize that the mechanisms employed to cope with them are likely conserved from yeast to man. Our long-term goal is to identify the critical genes and cellular pathways affected by misfolded human disease proteins.
We don't limit ourselves to one model system or experimental approach. We start with yeast, perform genetic and chemical screens, and then move to other model systems (e.g. mammalian tissue culture, mouse, fly, zebrafish) and even work with human patient samples (tissue sections, patient-derived cells, including iPS cells, and next generation sequencing approaches to look for mutations in novel genes).
We have focused on the Parkinson’s Disease (PD) linked protein, α-synuclein: investigating both its role in pathology and its, as of yet elusive, normal function. By performing high-throughput genome-wide screens in yeast, we have identified a set of genes, many with clear human homologs, which are able to antagonize cellular toxicity associated with the accumulation of misfolded α-synuclein. Remarkably, some of these genes are also able to rescue neuron loss in animal models of PD (Cooper et al., Science 2006). A major focus of our future research will be the functional characterization of how these modifier genes interact with α-synuclein, with the goal to understand the critical cellular pathways affected by misfolded α-synuclein and how this contributes to neurodegeneration. Experiments are also underway to employ yeast cells as “living test tubes” to discover the, as of yet elusive, normal cellular function of α-synuclein. We recently found that one of the genes from our α-synuclein toxicity modifier screen is the yeast homolog of the human PARK9 gene, ATP13A2 and that yeast PARK9 functions to protect cells from manganese toxicity, an environmental risk factor for PD and PD-like syndromes (Gitler et al., Nature Genetics 2009). We are currently performing additional screens in yeast to determine the mechanism by which yeast PARK9 can function to protect cells from manganese toxicity as well as cell culture and animal model studies to test if mammalian PARK9 is also involved in manganese detoxification pathways.
Encouraged by the power of the yeast system to gain insight into α-synuclein biology, we are creating new yeast models to study additional protein-misfolding disorders, including Alzheimer’s disease and ALS. We recently developed a yeast model to study the ALS disease protein TDP-43 (Johnson et al., PNAS 2008).
We have used yeast and in vitro biochemistry (in collaboration with Jim Shorter at PENN) to analyze the effects of ALS-linked TDP-43 mutations on aggregation and toxicity (Johnson et al., JBC 2009). We are now using these models to perform high-throughput genetic and small molecule screens to elucidate the molecular pathways that regulate the function of these disease proteins and control their conversion to a pathological conformation. We are currently analyzing hits from recent high-throughput screens that identified potent modifiers of TDP-43 toxicity. We are validating these hits in cell culture, animal models (mouse, fly, and zebrafish), and human patient samples.
Interestingly, one of the hits from our yeast TDP-43 genetic modifier screen, PBP1, is the homolog of a human neurodegenerative disease protein, ataxin 2. We have validated this genetic interaction in the fly nervous system (in collaboration with Nancy Bonini at PENN), used biochemistry to show the proteins physically associate in an RNA-dependent manner.
We analyzed the ataxin 2 gene in 915 individuals with ALS and 980 healthy controls and found mutations in this gene as a common geneticrisk factor for ALS in humans. Long polyglutamine (polyQ) expansions (>34Q) in ataxin 2 cause spinocerebellar ataxia type 2 (SCA2). We found intermediate-length polyQ expansions in ataxin 2 (27-33Q) significantly associated with increased risk for ALS (Elden et al., Nature 2010). We are continuing to characterize the role of ataxin 2 in ALS as well as other neurodegenerative disease situations. Because inhibiting ataxin 2 function in yeast or fly reduces TDP-43 toxicity, we are investigating ways to disrupt the ataxin 2 / TDP-43 interaction as a potential therapeutic strategy.
We have also begun a novel functional screen in yeast to identify new human ALS disease genes. From this seemingly simple yeast screen, we were able to predict a set of ALS candidate disease genes and, remarkably, have already identified mutations in two of them in human ALS patients. We continue to sequence more genes in a large cohort of sporadic and familial ALS patients using standard as well as next generation sequencing approaches.
We also have an active interest in the genetic cancer syndrome type 1 neurofibromatosis (NF1). Affecting 1 out of 4,000 births, NF1 is caused by loss-of-function mutations in neurofibromin, a Ras GTPase activating protein. Neurofibromin homologs are present in yeast, affording the opportunity to rapidly identify modulators of neurofibromin function. Accordingly, we have initiated studies to apply yeast genetics and our high-throughput screening infrastructure to explore the cellular pathways that become dysregulated in NF1.
Finally, we are using zebrafish to explore the normal function of neurodegenerative disease proteins during development (Sun and Gitler Developmental Dynamics 2008) as well as to use live-cell imaging to visualize protein aggregation during neurodegeneration.