Gitler Lab



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).

C9orf72 in ALS and FTD: Disease models and mechanisms

Mutations in the C9orf72 gene are the most common cause of ALS and frontotemporal dementia (FTD). The mutation is a massive hexanucleotide repeat (GGGGCC) expansion in the intron of C9orf72. The mechanism by which C9orf72 mutations cause disease has remained unclear and of intense interest. The nucleotide repeat is transcribed in the sense and antisense direction and these transcripts accumulate in the nucleus and cytoplasm of neurons of C9orf72 mutation carriers, where they might sequester important RNA-binding proteins and essential splicing factors. In collaboration with the Petrucelli laboratory we have recently identified a way to selectively inhibit the expression of both sense and antisense mutant C9orf72 transcripts, which could offer therapeutic potential (Kramer et al., Science 2016). 
In addition to the RNA accumulating, the repeat expansion is a substrate for an unconventional form of translation: repeat-associated non-ATG translation (RAN translation), which produces dipeptide repeat proteins (DPRs) from the sense and antisense transcripts (e.g., polyGlyAla; polyGlyPro; polyProAla; polyGlyArg; polyProArg). These DPRs are themselves aggregation-prone and accumulate in the brain and spinal cord of C9orf72 mutation carriers. We have developed several new yeast models to study C9orf72 mutations. These models recapitulate salient features of the human disease, including RNA foci formation and RAN translation. The arginine-rich DPRs (GlyArg and ProArg) are particularly toxic to yeast, enabling us to perform genome wide screens for modifiers. We identified genes involved in nucleoctyoplasmic transport as potent modifiers of C9orf72 DPR toxicity in yeast and have validated these results in mammalian cells and in Drosophila (Jovičić et al., Nat Neurosci 2015; Chai and Gitler, FEMS Yeast Res 2018). These results define a pathway that may be therapeutically targeted in ALS.

We are currently using yeast and mammalian cell models to define the mechanism of RAN translation and to explore the biology of additional C9orf72 DPRs. We have identified at least two genes that seem to be required for RAN translation. We hope that targeting this unconventional form of translation specifically will provide a therapeutic strategy for several neurodegenerative diseases caused by nucleotide repeat expansions (e.g., Huntington's disease and the spinocerebellar ataxias). In parallel to these yeast studies, our lab is using CRISPR/Cas9 and genome-editing to define C9orf72 mechanisms in vitro and in vivo. We have also initiated a large program in the lab to use CRISPR/Cas9 to perform genome wide screens in human cells for disease modifiers, starting with C9orf72 and extending to other neurodegenerative disease genes (Kramer et al., Nat Genet 2018). 

Parkinson's Disease and α-Synuclein

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., Nat Genet 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 (Chesi et al., PLoS One 2012).

New connections between Parkinson's disease genes

Two new Parkinson's disease genes, VPS35 and EIF4G1, have homologs in yeast and we have been using yeast genetic screens to learn more about the functions of these two genes. We recently discovered an unexpected functional interaction between VPS35 and EIF4G1 and are currently exploring this co

nnection in animal models (Dhungel et al., Neuron 2015). We are also testing how these two genes interact with α-synuclein. Finally, we are extending these studies to human and sequencing homologs of the genes from the yeast screen in Parkinson's disease patient cohorts in order to further define the genetic landscape of the disease. 

New yeast models of neurodegenerative diseases

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., Proc Natl Acad Sci USA 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., J Biol Chem 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.

These TDP-43 modifier screens are providing insight in two main ways:

1. The genes and pathways that are able to modify TDP-43 toxicity in yeast are now good candidates for evaluation as genetic contributors to ALS and related disorders in humans (e.g., see ataxin 2 below). 

2. The yeast hits and their homologs are candidate therapeutic targets, especially gene deletions (Armakola et al., Nat Genet 2012; Kim et al., Nat Genet 2014).

Ataxin-2 and ALS

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). A role for polyQ expansions in ataxin 2 in ALS and related diseases is being evaluated by us and others in independent patient populations worldwide. Click here for an updated summary of these results. 

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 found that lowering levels of ataxin 2 in mouse, either by knockout or with antisense oligonucleotides (ASOs) can markedly extend survival and reduce pathology in TDP-43 transgenic mice (Becker et al., Nature 2017). We are extending these studies to additional mouse models and testing effects of ataxin 2 lowering in human cell models. 

We found that ALS cases harboring intermediate-length ataxin 2 polyQ expansions have distinct TDP-43 pathology compared to ALS cases with normal length ataxin 2 (Hart et al., Acta Neuropathol 2012). We have also found that intermediate-length, but not normal or SCA2-length ataxin 2 polyQ causes stress-induced caspase activation, TDP-43 cleavage and phosphorylation (Hart and Gitler J Neurosci 2012). Since ataxin 2 plays a key role in stress granule formation and function, we are currently exploring a role for ataxin 2 in integrating stress signals and how such pathways, if dysregulated, may converge in disease pathologies (Kim et al., Nat Genet 2014).

New ALS Disease Genes

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.
    Since TDP-43 and FUS are both RNA-binding proteins linked to ALS, and our lab has used the yeast system to define key aspects of their aggregation and toxicity properties (Johnson et al., Proc Natl Acad Sci USA 2008; Johnson et al., J Biol Chem 2009; Sun et al., PLoS Biol 2011), we reasoned that additional human RNA-binding proteins with properties similar to TDP-43 and FUS (e.g. aggregation and toxicity when expressed in yeast) might also contribute to ALS. We recently performed an unbiased yeast screen for such genes. 
We also used bioinformatics, in collaboration with Oliver King (BBRI) and Jim Shorter (PENN) to
predict prion-like domains in a subset of these RNA-binding proteins (Cushman et al., J Cell Sci 2010; Gitler and Shorter, Prion 2011).
 We are currently sequencing some of these genes in our collection of ALS patient samples and controls, and have recently identif
ied missense variants in the TAF15 
gene (Couthouis et al., Proc Natl Acad Sci USA 2011). We are continuing to characterize the effects of these variants on TAF15 and to determine if additional RNA-binding proteins, with aggregation-prone properties, also contribute to ALS (Couthouis et al., Hum Mol Genet 2012). We hypothesize that TDP-43 and FUS might be the tip of the iceberg (King et al., Brain Res 2012) and that this class of proteins could contribute very broadly to ALS and related neurodegenerative diseases (e.g. FTLD, IBMPFD (Kim et al., Nature 2013). 

Stress Granules as the Crucibles of ALS Pathogenesis

In addition to defining the role of aggregation-prone RNA-binding proteins in disease, we are actively investigating the normal cellular functions of these proteins, especially their remarkable ability to self-assemble to form functional RNA granules (e.g., P-bodies, stress granules, and neuronal RNA transport granules; Li et al., J Cell Biol 2013). We aim to define additional components of 
RNA granules using a combination
of genetic and biochemical approaches.
We have recently identified a novel role of the ALS disease protein profilin 1 in stress granule assembly and dynamics (Figley et al., J Neurosci 2014). Elucidating the complex cellular functions of stress granules
, their constituents, and their regulators will hopefully allow us to therapeutically modulate these structures in disease situations (e.g., Kim et al., Nat Genet 2014). 

Next Generation DNA Sequencing to Define Novel Neurodegenerative Disease Genes

We have several ongoing projects in the lab to use exome and whole genome sequencing to define new genetic contributors to neurodegenerative diseases (Couthouis et al., PLoS Genet 2014; Cirulli et al., Science 2015). We recently sequenced the exomes of 47 sporadic ALS trios (ALS patient and both unaffected biological parents) to
 determine the role of de novo mutations in ALS. These studies suggest a potential
role of chromatin regulator genes, including the neuronal chromatin
remodeling complex (nBAF) component SS18L1/CREST in ALS (Chesi et al., Nat Neurosci 2013).
We also have several active collaborations with neurologists at Stanford Hospital and the Veterans Administration Hospital in Palo Alto to define new disease genes for inherited neuromuscular and neurodegenerative diseases (Couthouis et al., Neuromuscul Disord 2014; Raphael et al., Brain Res 2014; Couthouis et al., PLoS Genet 2014).