Academic Appointments

Honors & Awards

  • Friedrich Merz Guest Professorship Award, Merz Pharma (2017)
  • R35 Research Program Award, NINDS (2016)
  • Glenn Award for Research in Biological Mechanisms of Aging, Glenn Foundation for Medical Research (2015)
  • Young Scientist Lectureship Award, International Society for Neurochemistry (2013)
  • Addgene Innovation Award, Addgene (2011)
  • Eppendorf and Science Prize for Neurobiology, Finalist, Eppendorf and Science Magazine (2011)
  • Instituto Paulo Gontijo International Medicine PG Award, Instituto Paulo Gontijo (2011)
  • Scientist to Watch, The Scientist (2010)
  • NIH Director’'s New Innovator Award, NIH (2008)
  • Pew Scholar in the Biomedical Sciences, The Pew Charitable Trusts (2008)
  • Rita Allen Foundation Scholar, The Rita Allen Foundation (2008)

Professional Education

  • Postdoctoral Fellow, Whitehead Institute for Biomedical Research, Cell biology and genetics (2007)
  • Ph.D., University of Pennsylvania, Cell and Molecular Biology (2004)
  • B.S., Penn State University, Biochemistry and Molecular Biology (2000)

Research & Scholarship

Current Research and Scholarly Interests

We use the baker’s yeast, Saccharomyces cerevisiae, as a model system to study the cell biology underpinning protein-misfolding diseases like Parkinson's disease and ALS. 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.

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. 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).

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 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.


2017-18 Courses

Stanford Advisees

Graduate and Fellowship Programs


All Publications

  • CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity. Nature genetics Kramer, N. J., Haney, M. S., Morgens, D. W., Jovičić, A., Couthouis, J., Li, A., Ousey, J., Ma, R., Bieri, G., Tsui, C. K., Shi, Y., Hertz, N. T., Tessier-Lavigne, M., Ichida, J. K., Bassik, M. C., Gitler, A. D. 2018


    Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.

    View details for DOI 10.1038/s41588-018-0070-7

    View details for PubMedID 29507424

  • Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice NATURE Becker, L. A., Huang, B., Bieri, G., Ma, R., Knowles, D. A., Jafar-Nejad, P., Messing, J., Kim, H. J., Soriano, A., Auburger, G., Pulst, S. M., Taylor, J. P., Rigo, F., Gitler, A. D. 2017; 544 (7650): 367-?


    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that is characterized by motor neuron loss and that leads to paralysis and death 2-5 years after disease onset. Nearly all patients with ALS have aggregates of the RNA-binding protein TDP-43 in their brains and spinal cords, and rare mutations in the gene encoding TDP-43 can cause ALS. There are no effective TDP-43-directed therapies for ALS or related TDP-43 proteinopathies, such as frontotemporal dementia. Antisense oligonucleotides (ASOs) and RNA-interference approaches are emerging as attractive therapeutic strategies in neurological diseases. Indeed, treatment of a rat model of inherited ALS (caused by a mutation in Sod1) with ASOs against Sod1 has been shown to substantially slow disease progression. However, as SOD1 mutations account for only around 2-5% of ALS cases, additional therapeutic strategies are needed. Silencing TDP-43 itself is probably not appropriate, given its critical cellular functions. Here we present a promising alternative therapeutic strategy for ALS that involves targeting ataxin-2. A decrease in ataxin-2 suppresses TDP-43 toxicity in yeast and flies, and intermediate-length polyglutamine expansions in the ataxin-2 gene increase risk of ALS. We used two independent approaches to test whether decreasing ataxin-2 levels could mitigate disease in a mouse model of TDP-43 proteinopathy. First, we crossed ataxin-2 knockout mice with TDP-43 (also known as TARDBP) transgenic mice. The decrease in ataxin-2 reduced aggregation of TDP-43, markedly increased survival and improved motor function. Second, in a more therapeutically applicable approach, we administered ASOs targeting ataxin-2 to the central nervous system of TDP-43 transgenic mice. This single treatment markedly extended survival. Because TDP-43 aggregation is a component of nearly all cases of ALS, targeting ataxin-2 could represent a broadly effective therapeutic strategy.

    View details for DOI 10.1038/nature22038

    View details for Web of Science ID 000399524400040

    View details for PubMedID 28405022

  • Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science Kramer, N. J., Carlomagno, Y., Zhang, Y., Almeida, S., Cook, C. N., Gendron, T. F., Prudencio, M., van Blitterswijk, M., Belzil, V., Couthouis, J., Paul, J. W., Goodman, L. D., Daughrity, L., Chew, J., Garrett, A., Pregent, L., Jansen-West, K., Tabassian, L. J., Rademakers, R., Boylan, K., Graff-Radford, N. R., Josephs, K. A., Parisi, J. E., Knopman, D. S., Petersen, R. C., Boeve, B. F., Deng, N., Feng, Y., Cheng, T., Dickson, D. W., Cohen, S. N., Bonini, N. M., Link, C. D., Gao, F., Petrucelli, L., Gitler, A. D. 2016; 353 (6300): 708-712


    An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately.

    View details for DOI 10.1126/science.aaf7791

    View details for PubMedID 27516603

  • Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nature neuroscience Jovicic, A., Mertens, J., Boeynaems, S., Bogaert, E., Chai, N., Yamada, S. B., Paul, J. W., Sun, S., Herdy, J. R., Bieri, G., Kramer, N. J., Gage, F. H., Van Den Bosch, L., Robberecht, W., Gitler, A. D. 2015; 18 (9): 1226-1229


    C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.

    View details for DOI 10.1038/nn.4085

    View details for PubMedID 26308983

    View details for PubMedCentralID PMC4552077

  • Exome sequencing to identify de novo mutations in sporadic ALS trios. Nature neuroscience Chesi, A., Staahl, B. T., Jovicic, A., Couthouis, J., Fasolino, M., Raphael, A. R., Yamazaki, T., Elias, L., Polak, M., Kelly, C., Williams, K. L., Fifita, J. A., Maragakis, N. J., Nicholson, G. A., King, O. D., Reed, R., Crabtree, G. R., Blair, I. P., Glass, J. D., Gitler, A. D. 2013; 16 (7): 851-855

    View details for DOI 10.1038/nn.3412

    View details for PubMedID 23708140

  • Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS NATURE Elden, A. C., Kim, H., Hart, M. P., Chen-Plotkin, A. S., Johnson, B. S., Fang, X., Armakola, M., Geser, F., Greene, R., Lu, M. M., Padmanabhan, A., Clay-Falcone, D., McCluskey, L., Elman, L., Juhr, D., Gruber, P. J., Rueb, U., Auburger, G., Trojanowski, J. Q., Lee, V. M., Van Deerlin, V. M., Bonini, N. M., Gitler, A. D. 2010; 466 (7310): 1069-U77


    The causes of amyotrophic lateral sclerosis (ALS), a devastating human neurodegenerative disease, are poorly understood, although the protein TDP-43 has been suggested to have a critical role in disease pathogenesis. Here we show that ataxin 2 (ATXN2), a polyglutamine (polyQ) protein mutated in spinocerebellar ataxia type 2, is a potent modifier of TDP-43 toxicity in animal and cellular models. ATXN2 and TDP-43 associate in a complex that depends on RNA. In spinal cord neurons of ALS patients, ATXN2 is abnormally localized; likewise, TDP-43 shows mislocalization in spinocerebellar ataxia type 2. To assess the involvement of ATXN2 in ALS, we analysed the length of the polyQ repeat in the ATXN2 gene in 915 ALS patients. We found that intermediate-length polyQ expansions (27-33 glutamines) in ATXN2 were significantly associated with ALS. These data establish ATXN2 as a relatively common ALS susceptibility gene. Furthermore, these findings indicate that the TDP-43-ATXN2 interaction may be a promising target for therapeutic intervention in ALS and other TDP-43 proteinopathies.

    View details for DOI 10.1038/nature09320

    View details for Web of Science ID 000281203600032

    View details for PubMedID 20740007

  • Genome-wide Analyses Identify KIF5A as a Novel ALS Gene. Neuron Nicolas, A., Kenna, K. P., Renton, A. E., Ticozzi, N., Faghri, F., Chia, R., Dominov, J. A., Kenna, B. J., Nalls, M. A., Keagle, P., Rivera, A. M., van Rheenen, W., Murphy, N. A., van Vugt, J. J., Geiger, J. T., Van der Spek, R. A., Pliner, H. A., Smith, B. N., Marangi, G., Topp, S. D., Abramzon, Y., Gkazi, A. S., Eicher, J. D., Kenna, A., Mora, G., Calvo, A., Mazzini, L., Riva, N., Mandrioli, J., Caponnetto, C., Battistini, S., Volanti, P., La Bella, V., Conforti, F. L., Borghero, G., Messina, S., Simone, I. L., Trojsi, F., Salvi, F., Logullo, F. O., D'Alfonso, S., Corrado, L., Capasso, M., Ferrucci, L., Moreno, C. d., Kamalakaran, S., Goldstein, D. B., Gitler, A. D., Harris, T., Myers, R. M., Phatnani, H., Musunuri, R. L., Evani, U. S., Abhyankar, A., Zody, M. C., Kaye, J., Finkbeiner, S., Wyman, S. K., LeNail, A., Lima, L., Fraenkel, E., Svendsen, C. N., Thompson, L. M., Van Eyk, J. E., Berry, J. D., Miller, T. M., Kolb, S. J., Cudkowicz, M., Baxi, E., Benatar, M., Taylor, J. P., Rampersaud, E., Wu, G., Wuu, J., Lauria, G., Verde, F., Fogh, I., Tiloca, C., Comi, G. P., Sorarù, G., Cereda, C., Corcia, P., Laaksovirta, H., Myllykangas, L., Jansson, L., Valori, M., Ealing, J., Hamdalla, H., Rollinson, S., Pickering-Brown, S., Orrell, R. W., Sidle, K. C., Malaspina, A., Hardy, J., Singleton, A. B., Johnson, J. O., Arepalli, S., Sapp, P. C., McKenna-Yasek, D., Polak, M., Asress, S., Al-Sarraj, S., King, A., Troakes, C., Vance, C., de Belleroche, J., Baas, F., Ten Asbroek, A. L., Muñoz-Blanco, J. L., Hernandez, D. G., Ding, J., Gibbs, J. R., Scholz, S. W., Floeter, M. K., Campbell, R. H., Landi, F., Bowser, R., Pulst, S. M., Ravits, J. M., MacGowan, D. J., Kirby, J., Pioro, E. P., Pamphlett, R., Broach, J., Gerhard, G., Dunckley, T. L., Brady, C. B., Kowall, N. W., Troncoso, J. C., Le Ber, I., Mouzat, K., Lumbroso, S., Heiman-Patterson, T. D., Kamel, F., Van Den Bosch, L., Baloh, R. H., Strom, T. M., Meitinger, T., Shatunov, A., Van Eijk, K. R., de Carvalho, M., Kooyman, M., Middelkoop, B., Moisse, M., McLaughlin, R. L., Van Es, M. A., Weber, M., Boylan, K. B., Van Blitterswijk, M., Rademakers, R., Morrison, K. E., Basak, A. N., Mora, J. S., Drory, V. E., Shaw, P. J., Turner, M. R., Talbot, K., Hardiman, O., Williams, K. L., Fifita, J. A., Nicholson, G. A., Blair, I. P., Rouleau, G. A., Esteban-Pérez, J., García-Redondo, A., Al-Chalabi, A., Rogaeva, E., Zinman, L., Ostrow, L. W., Maragakis, N. J., Rothstein, J. D., Simmons, Z., Cooper-Knock, J., Brice, A., Goutman, S. A., Feldman, E. L., Gibson, S. B., Taroni, F., Ratti, A., Gellera, C., Van Damme, P., Robberecht, W., Fratta, P., Sabatelli, M., Lunetta, C., Ludolph, A. C., Andersen, P. M., Weishaupt, J. H., Camu, W., Trojanowski, J. Q., Van Deerlin, V. M., Brown, R. H., van den Berg, L. H., Veldink, J. H., Harms, M. B., Glass, J. D., Stone, D. J., Tienari, P., Silani, V., Chiò, A., Shaw, C. E., Traynor, B. J., Landers, J. E. 2018; 97 (6): 1268–83.e6


    To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.

    View details for DOI 10.1016/j.neuron.2018.02.027

    View details for PubMedID 29566793

  • The epidemiology and genetics of Amyotrophic lateral sclerosis in China. Brain research Liu, X., He, J., Gao, F. B., Gitler, A. D., Fan, D. 2018


    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder associated with loss of motor neurons. Previous knowledge of the disease has been mainly based on studies from Caucasian ALS patients of European descent. Here we review the epidemiological characteristics of ALS among the Chinese population in order to compare the similarities and differences between Chinese ALS cases and those from other countries. We describe a potential lower incidence and prevalence of ALS, a younger age of onset and a lower proportion of familial ALS cases in the Chinese population. Additionally, we highlight potential genetic differences between Chinese and Caucasian ALS patients. Most notably, the frequency of GGGGCC repeat expansions in C9ORF72 in Chinese ALS is significantly lower than in Caucasians. Since some conclusions might not be consistent across all of the studies around China to date, we suggest that it is necessary to carry out a prospective population-based study and large-scale gene sequencing around to better define epidemiological and genetic features of Chinese ALS patients.

    View details for DOI 10.1016/j.brainres.2018.02.035

    View details for PubMedID 29501653

  • Yeast screen for modifiers of C9orf72 poly(Glycine-Arginine) dipeptide repeat toxicity. FEMS yeast research Chai, N., Gitler, A. D. 2018


    A hexanucleotide repeat expansion in the C9orf72 gene has been identified as the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. The expanded hexanucleotide repeat is translated by an unconventional mechanism to produce five species of dipeptide repeat proteins (DPRs), glycine-proline (GP), glycine-alanine, glycine-arginine (GR), proline-alanine (PA), and proline-arginine (PR). Of these, the arginine-rich ones, PR and GR, are highly toxic in a variety of model systems, ranging from human cells, to Drosophila, to even the budding yeast, Saccharomyces cerevisiae. We recently performed a genetic screen in yeast for modifiers of PR toxicity and identified suppressors and enhancers, many of which function in nucleocytoplasmic transport. Whether or not GR toxicity involves similar mechanisms to PR is unresolved. Therefore, we performed a genetic screen in yeast to identify modifiers of GR toxicity and compared the results of the GR screen to results from our previous PR screen. Surprisingly, there was only a small degree of overlap between the two screens, suggesting potential for distinct toxicity mechanisms between PR and GR.

    View details for DOI 10.1093/femsyr/foy024

    View details for PubMedID 29528392

  • Unlocking the Mystery of ALS. Scientific American Petrucelli, L., Gitler, A. D. 2017; 316 (6): 46-51

    View details for DOI 10.1038/scientificamerican0617-46

    View details for PubMedID 28510559

  • Neurodegenerative disease: models, mechanisms, and a new hope DISEASE MODELS & MECHANISMS Gitler, A. D., Dhillon, P., Shorter, J. 2017; 10 (5): 499-502


    Neurodegeneration is a feature of many debilitating, incurable diseases that are rapidly rising in prevalence, such as Parkinson's disease. There is an urgent need to develop new and more effective therapeutic strategies to combat these devastating diseases. Models - from cell-based systems, to unicellular organisms, to complex animals - have proven to be a useful tool to help the research community shed light on the mechanisms underlying neurodegenerative diseases, and these advances have now begun to provide promising therapeutic avenues. In this themed issue of Disease Models & Mechanisms, a special collection of articles focused on neurodegenerative diseases is introduced. The collection includes original research articles that provide new insights into the complex pathophysiology of such diseases, revealing candidate biomarkers or therapeutic targets. Some of the articles describe a new disease model that enables deeper exploration of key mechanisms. We also present a series of reviews that highlight some of the recent translational advances made in studies of neurodegenerative diseases. In this Editorial, we summarize the articles featured in this collection, emphasizing the impact that model-based studies have made in this exciting area of research.

    View details for DOI 10.1242/dmm.030205

    View details for Web of Science ID 000400596000001

    View details for PubMedID 28468935

    View details for PubMedCentralID PMC5451177

  • Internalization, axonal transport and release of fibrillar forms of alpha-synuclein. Neurobiology of disease Bieri, G., Gitler, A. D., Brahic, M. 2017


    Intra-neuronal protein aggregates made of fibrillar alpha-synuclein (α-syn) are the hallmark of Parkinson's disease (PD). With time, these aggregates spread through the brain following axonal projections. Understanding the mechanism of this spread is central to the study of the progressive nature of PD. Here we review data relevant to the uptake, transport and release of α-syn fibrils. We summarize several cell surface receptors that regulate the uptake of α-syn fibrils by neurons. The aggregates are then transported along axons, both in the anterograde and retrograde direction. The kinetics of transport suggests that they are part of the slow component b of axonal transport. Recent findings indicate that aggregated α-syn is secreted by neurons by non-canonical pathways that may implicate various molecular chaperones including USP19 and the DnaJ/Hsc70 complex. Additionally, α-syn fibrils may also be released and transmitted from neuron-to-neuron via exosomes and tunneling nanotubes. Understanding these different mechanisms and molecular players underlying α-syn spread is crucial for the development of therapies that could halt the progression of α-syn-related degenerative diseases.

    View details for DOI 10.1016/j.nbd.2017.03.007

    View details for PubMedID 28323023

  • ATXN2 trinucleotide repeat length correlates with risk of ALS NEUROBIOLOGY OF AGING Sproviero, W., Shatunov, A., Stahl, D., Shoai, M., van Rheenen, W., Jones, A. R., Al-Sarraj, S., Andersen, P. M., Bonini, N. M., Conforti, F. L., Van Damme, P., Daoud, H., Amador, M. d., Fogh, I., Forzan, M., Gaastra, B., Gellera, C., Gitler, A. D., Hardy, J., Fratta, P., La Bella, V., Le Ber, I., Van Langenhove, T., Lattante, S., Lee, Y., Malaspina, A., Meininger, V., Millecamps, S., Orrell, R., Rademakers, R., Robberecht, W., Rouleau, G., Ross, O. A., Salachas, F., Sidle, K., Smith, B. N., Soong, B., Soraru, G., Stevanin, G., Kabashi, E., Troakes, C., Van Broeckhoven, C., Veldink, J. H., Van den Berg, L. H., Shaw, C. E., Powell, J. F., Al-Chalabi, A. 2017; 51


    We investigated a CAG trinucleotide repeat expansion in the ATXN2 gene in amyotrophic lateral sclerosis (ALS). Two new case-control studies, a British dataset of 1474 ALS cases and 567 controls, and a Dutch dataset of 1328 ALS cases and 691 controls were analyzed. In addition, to increase power, we systematically searched PubMed for case-control studies published after 1 August 2010 that investigated the association between ATXN2 intermediate repeats and ALS. We conducted a meta-analysis of the new and existing studies for the relative risks of ATXN2 intermediate repeat alleles of between 24 and 34 CAG trinucleotide repeats and ALS. There was an overall increased risk of ALS for those carrying intermediate sized trinucleotide repeat alleles (odds ratio 3.06 [95% confidence interval 2.37-3.94]; p = 6 × 10(-18)), with an exponential relationship between repeat length and ALS risk for alleles of 29-32 repeats (R(2) = 0.91, p = 0.0002). No relationship was seen for repeat length and age of onset or survival. In contrast to trinucleotide repeat diseases, intermediate ATXN2 trinucleotide repeat expansion in ALS does not predict age of onset but does predict disease risk.

    View details for DOI 10.1016/j.neurobiolaging.2016.11.010

    View details for Web of Science ID 000397168600022

    View details for PubMedID 28017481

  • Raise the Roof: Boosting the Efficacy of a Spinal Muscular Atrophy Therapy. Neuron Kramer, N. J., Gitler, A. D. 2017; 93 (1): 3-5


    Spinal muscular atrophy is the most common genetic killer of infants. A therapy shows promise in the clinic, but there is a potential limit to its efficacy. In this issue of Neuron, d'Ydewalle et al. (2017) devise a new way to make it more effective.

    View details for DOI 10.1016/j.neuron.2016.12.029

    View details for PubMedID 28056344

  • Distinct repertoires of microRNAs present in mouse astrocytes compared to astrocyte-secreted exosomes. PloS one Jovicic, A., Gitler, A. D. 2017; 12 (2)


    Astrocytes are the most abundant cell type in the central nervous system (CNS) and secrete various factors that regulate neuron development, function and connectivity. microRNAs (miRNAs) are small regulatory RNAs involved in posttranslational gene regulation. Recent findings showed that miRNAs are exchanged between cells via nanovesicles called exosomes. In this study, we sought to define which miRNAs are contained within exosomes secreted by astrocytes. We also explored whether astroglial miRNA secretion via exosomes is perturbed in a mouse model of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease where astrocytes play a crucial role in driving disease progression.By isolating and profiling the expression of miRNAs from primary mouse astrocytes and from the exosomes that astrocytes secrete, we compared miRNA expression in the cells and secreted vesicles. We established that miRNA expression profiles of astrocytes and their exosomes are vastly different. In addition, we determined that exosomal miRNA expression in astrocytes is not significantly perturbed in a mouse model of ALS.Astrocytes secrete numerous miRNAs via exosomes and miRNA species contained in exosomes are considerably different from miRNAs detectable in astrocytes, suggesting the existence of a mechanism to select certain miRNAs for inclusion or exclusion from exosomes. The exosomal miRNA profiling dataset we have generated will provide a resource to aid in the investigation of this selection mechanism. Finally, the miRNA expression profile in astrocyte-secreted exosomes is not perturbed by expression of mutant SOD1-G93A.

    View details for DOI 10.1371/journal.pone.0171418

    View details for PubMedID 28152040

  • Glycolytic Enzymes Coalesce in G Bodies under Hypoxic Stress. Cell reports Jin, M., Fuller, G. G., Han, T., Yao, Y., Alessi, A. F., Freeberg, M. A., Roach, N. P., Moresco, J. J., Karnovsky, A., Baba, M., Yates, J. R., Gitler, A. D., Inoki, K., Klionsky, D. J., Kim, J. K. 2017; 20 (4): 895–908


    Glycolysis is upregulated under conditions such as hypoxia and high energy demand to promote cell proliferation, although the mechanism remains poorly understood. We find that hypoxia in Saccharomyces cerevisiae induces concentration of glycolytic enzymes, including the Pfk2p subunit of the rate-limiting phosphofructokinase, into a single, non-membrane-bound granule termed the "glycolytic body" or "G body." A yeast kinome screen identifies the yeast ortholog of AMP-activated protein kinase, Snf1p, as necessary for G-body formation. Many G-body components identified by proteomics are required for G-body integrity. Cells incapable of forming G bodies in hypoxia display abnormal cell division and produce inviable daughter cells. Conversely, cells with G bodies show increased glucose consumption and decreased levels of glycolytic intermediates. Importantly, G bodies form in human hepatocarcinoma cells in hypoxia. Together, our results suggest that G body formation is a conserved, adaptive response to increase glycolytic output during hypoxia or tumorigenesis.

    View details for DOI 10.1016/j.celrep.2017.06.082

    View details for PubMedID 28746874

    View details for PubMedCentralID PMC5586494

  • Efficient Prevention of Neurodegenerative Diseases by Depletion of Starvation Response Factor Ataxin-2. Trends in neurosciences Auburger, G., Sen, N. E., Meierhofer, D., Başak, A. N., Gitler, A. D. 2017; 40 (8): 507–16


    Ataxin-2 (ATXN2) homologs exist in all eukaryotic organisms and may have contributed to their origin. Apart from a role in endocytosis, they are known for global effects on mRNA repair and ribosomal translation. Cell size, protein synthesis, and fat and glycogen storage are repressed by ATXN2 via mTORC1 signaling. However, specific liver mitochondrial matrix enzymes and the mitochondrial repair factor PINK1 require ATXN2 abundance. During periods of starvation, ATXN2 is transcriptionally induced and localized to cytosolic stress granules, where nuclear factors dock to compensate RNA pathology. These physiological actions were now revealed to be crucial for human neurodegenerative diseases, given that ATXN2 depletion is surprisingly efficient in preventing motor neuron and cerebellar atrophy, as demonstrated in mouse models, flies, and yeast.

    View details for DOI 10.1016/j.tins.2017.06.004

    View details for PubMedID 28684172

  • ERAD defects and the HFE-H63D variant are associated with increased risk of liver damages in Alpha 1-Antitrypsin Deficiency. PloS one Joly, P., Vignaud, H., Di Martino, J., Ruiz, M., Garin, R., Restier, L., Belmalih, A., Marchal, C., Cullin, C., Arveiler, B., Fergelot, P., Gitler, A. D., Lachaux, A., Couthouis, J., Bouchecareilh, M. 2017; 12 (6): e0179369


    The most common and severe disease causing allele of Alpha 1-Antitrypsin Deficiency (1ATD) is Z-1AT. This protein aggregates in the endoplasmic reticulum, which is the main cause of liver disease in childhood. Based on recent evidences and on the frequency of liver disease occurrence in Z-1AT patients, it seems that liver disease progression is linked to still unknown genetic factors.We used an innovative approach combining yeast genetic screens with next generation exome sequencing to identify and functionally characterize the genes involved in 1ATD associated liver disease.Using yeast genetic screens, we identified HRD1, an Endoplasmic Reticulum Associated Degradation (ERAD) associated protein, as an inducer of Z-mediated toxicity. Whole exome sequencing of 1ATD patients resulted in the identification of two variants associated with liver damages in Z-1AT homozygous cases: HFE H63D and HERPUD1 R50H. Functional characterization in Z-1AT model cell lines demonstrated that impairment of the ERAD machinery combined with the HFE H63D variant expression decreased both cell proliferation and cell viability, while Unfolded Protein Response (UPR)-mediated cell death was hyperstimulated.This powerful experimental pipeline allowed us to identify and functionally validate two genes involved in Z-1AT-mediated severe liver toxicity. This pilot study moves forward our understanding on genetic modifiers involved in 1ATD and highlights the UPR pathway as a target for the treatment of liver diseases associated with 1ATD. Finally, these findings support a larger scale screening for HERPUD1 R50H and HFE H63D variants in the sub-group of 1ATD patients developing significant chronic hepatic injuries (hepatomegaly, chronic cholestasis, elevated liver enzymes) and at risk developing liver cirrhosis.

    View details for DOI 10.1371/journal.pone.0179369

    View details for PubMedID 28617828

    View details for PubMedCentralID PMC5472284

  • Defects in trafficking bridge Parkinson's disease pathology and genetics NATURE Abeliovich, A., Gitler, A. D. 2016; 539 (7628): 207-216


    Parkinson's disease is a debilitating, age-associated movement disorder. A central aspect of the pathophysiology of Parkinson's disease is the progressive demise of midbrain dopamine neurons and their axonal projections, but the underlying causes of this loss are unclear. Advances in genetics and experimental model systems have illuminated an important role for defects in intracellular transport pathways to lysosomes. The accumulation of altered proteins and damaged mitochondria, particularly at axon terminals, ultimately might overwhelm the capacity of intracellular disposal mechanisms. Cell-extrinsic mechanisms, including inflammation and prion-like spreading, are proposed to have both protective and deleterious functions in Parkinson's disease.

    View details for DOI 10.1038/nature20414

    View details for Web of Science ID 000387318500031

    View details for PubMedID 27830778

  • Semisynthetic and in Vitro Phosphorylation of Alpha-Synuclein at Y39 Promotes Functional Partly Helical Membrane-Bound States Resembling Those Induced by PD Mutations. ACS chemical biology Dikiy, I., Fauvet, B., Jovicic, A., Mahul-Mellier, A., Desobry, C., El-Turk, F., Gitler, A. D., Lashuel, H. A., Eliezer, D. 2016; 11 (9): 2428-2437


    Alpha-synuclein is a presynaptic protein of poorly understood function that is linked to both genetic and sporadic forms of Parkinson's disease. We have proposed that alpha-synuclein may function specifically at synaptic vesicles docked at the plasma membrane, and that the broken-helix state of the protein, comprising two antiparallel membrane-bound helices connected by a nonhelical linker, may target the protein to such docked vesicles by spanning between the vesicle and the plasma membrane. Here, we demonstrate that phosphorylation of alpha-synuclein at tyrosine 39, carried out by c-Abl in vivo, may facilitate interconversion of synuclein from the vesicle-bound extended-helix state to the broken-helix state. Specifically, in the presence of lipid vesicles, Y39 phosphorylation leads to decreased binding of a region corresponding to helix-2 of the broken-helix state, potentially freeing this region of the protein to interact with other membrane surfaces. This effect is largely recapitulated by the phosphomimetic mutation Y39E, and expression of this mutant in yeast results in decreased membrane localization. Intriguingly, the effects of Y39 phosphorylation on membrane binding closely resemble those of the recently reported disease linked mutation G51D. These findings suggest that Y39 phosphorylation could modulate functional aspects of alpha-synuclein and perhaps influence pathological aggregation of the protein as well.

    View details for DOI 10.1021/acschembio.6b00539

    View details for PubMedID 27356045

  • There has been an awakening: Emerging mechanisms of C9orf72 mutations in FTD/ALS BRAIN RESEARCH Gitler, A. D., Tsuiji, H. 2016; 1647: 19-29


    The discovery of C9orf72 mutations as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has awakened a surge of interest in deciphering how mutations in this mysterious gene cause disease and what can be done to stop it. C9orf72 harbors a hexanucleotide repeat, GGGGCC, in a non-coding region of the gene and a massive expansion of this repeat causes ALS, FTD, or both (FTD/ALS). Many questions lie ahead. What does this gene normally do? What is the consequence of an enormous GGGGCC repeat expansion on that gene's function? Could that hexanucleotide repeat expansion have additional pathological actions unrelated to C9orf72 function? There has been tremendous progress on all fronts in the quest to define how C9orf72 mutations cause disease. Many new experimental models have been constructed and unleashed in powerful genetic screens. Studies in mouse and human patient samples, including iPS-derived neurons, have provided unprecedented insights into pathogenic mechanisms. Three major hypotheses have emerged and are still being hotly debated in the field. These include (1) loss of function owing to decrease in the abundance of C9orf72 protein and its ability to carryout its still unknown cellular role; (2) RNA toxicity from bidirectionally transcribed sense (GGGGCC) and antisense (GGCCCC) transcripts that accumulate in RNA foci and might sequester critical RNA-binding proteins; (3) proteotoxicity from dipeptide repeat proteins produced by an unconventional form of translation from the expanded nucleotide repeats. Here we review the evidence in favor and against each of these three hypotheses. We also suggest additional experiments and considerations that we propose will help clarify which mechanism(s) are most important for driving disease and therefore most critical for considering during the development of therapeutic interventions. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.

    View details for DOI 10.1016/j.brainres.2016.04.004

    View details for Web of Science ID 000383315200003

    View details for PubMedID 27059391

  • Nuclear transport dysfunction: a common theme in amyotrophic lateral sclerosis and frontotemporal dementia. Journal of neurochemistry Jovicic, A., Paul, J. W., Gitler, A. D. 2016; 138: 134-144


    Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with overlapping genetic factors and pathology. On the cellular level, a majority of ALS and FTD cases are characterized by nuclear clearance and cytoplasmic aggregation of otherwise nuclear proteins, TAR DNA-binding protein 43 (TDP-43), or fused in sarcoma. Recent studies investigating cellular pathways perturbed by genetic risk factors for ALS/FTD converge on nucleocytoplasmic transport dysfunction as a mechanism leading to disease pathophysiology. We propose that mutations in FUS and hexanucleotide expansions in C9orf72 and aging all converge on the impairment of nucleocytoplasmic transport, which results in the hallmark pathological feature of ALS/FTD - cytoplasmic aggregation of TDP-43 or FUS.

    View details for DOI 10.1111/jnc.13642

    View details for PubMedID 27087014

  • Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis. Neuron Lee, S., Shang, Y., Redmond, S. A., Urisman, A., Tang, A. A., Li, K. H., Burlingame, A. L., Pak, R. A., Jovicic, A., Gitler, A. D., Wang, J., Gray, N. S., Seeley, W. W., Siddique, T., Bigio, E. H., Lee, V. M., Trojanowski, J. Q., Chan, J. R., Huang, E. J. 2016; 91 (1): 41-55


    Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1(G93A), activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1(G93A) mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH-tTA/tetO-hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. VIDEO ABSTRACT.

    View details for DOI 10.1016/j.neuron.2016.05.021

    View details for PubMedID 27321923

    View details for PubMedCentralID PMC4938715

  • Axonal transport and secretion of fibrillar forms of a-synuclein, Aß42 peptide and HTTExon 1. Acta neuropathologica Brahic, M., Bousset, L., Bieri, G., Melki, R., Gitler, A. D. 2016; 131 (4): 539-548


    Accruing evidence suggests that prion-like behavior of fibrillar forms of α-synuclein, β-amyloid peptide and mutant huntingtin are responsible for the spread of the lesions that characterize Parkinson disease, Alzheimer disease and Huntington disease, respectively. It is unknown whether these distinct protein assemblies are transported within and between neurons by similar or distinct mechanisms. It is also unclear if neuronal death or injury is required for neuron-to-neuron transfer. To address these questions, we used mouse primary cortical neurons grown in microfluidic devices to measure the amounts of α-synuclein, Aβ42 and HTTExon1 fibrils transported by axons in both directions (anterograde and retrograde), as well as to examine the mechanism of their release from axons after anterograde transport. We observed that the three fibrils were transported in both anterograde and retrograde directions but with strikingly different efficiencies. The amount of Aβ42 fibrils transported was ten times higher than that of the other two fibrils. HTTExon1 was efficiently transported in the retrograde direction but only marginally in the anterograde direction. Finally, using neurons from two distinct mutant mouse strains whose axons are highly resistant to neurodegeneration (Wld(S) and Sarm1(-/-)), we found that the three different fibrils were secreted by axons after anterograde transport, in the absence of axonal lysis, indicating that trans-neuronal spread can occur in intact healthy neurons. In summary, fibrils of α-synuclein, Aβ42 and HTTExon1 are all transported in axons but in directions and amounts that are specific of each fibril. After anterograde transport, the three fibrils were secreted in the medium in the absence of axon lysis. Continuous secretion could play an important role in the spread of pathology between neurons but may be amenable to pharmacological intervention.

    View details for DOI 10.1007/s00401-016-1538-0

    View details for PubMedID 26820848

    View details for PubMedCentralID PMC4789229

  • Analysis of COPII Vesicles Indicates a Role for the Emp47-Ssp120 Complex in Transport of Cell Surface Glycoproteins. Traffic Margulis, N. G., Wilson, J. D., Bentivoglio, C. M., Dhungel, N., Gitler, A. D., Barlowe, C. 2016; 17 (3): 191-210


    Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium-binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47-Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway.

    View details for DOI 10.1111/tra.12356

    View details for PubMedID 26650540

  • Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD SCIENTIFIC REPORTS Boeynaems, S., Bogaertl, E., Michiels, E., Gijselinck, I., Sieben, A., Jovicic, A., De Baets, G., Scheveneels, W., Steyaert, J., Cuijt, I., Verstrepen, K. J., Callaerts, P., Rousseau, F., Schymkowitz, J., Cruts, M., Van Broeckhoven, C., Van Damme, P., Gitler, A. D., Robberecht, W., Van Den Bosch, L. 2016; 6

    View details for DOI 10.1038/srep20877

    View details for Web of Science ID 000369936600001

  • Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD. Scientific reports Boeynaems, S., Bogaert, E., Michiels, E., Gijselinck, I., Sieben, A., Jovicic, A., De Baets, G., Scheveneels, W., Steyaert, J., Cuijt, I., Verstrepen, K. J., Callaerts, P., Rousseau, F., Schymkowitz, J., Cruts, M., Van Broeckhoven, C., Van Damme, P., Gitler, A. D., Robberecht, W., Van Den Bosch, L. 2016; 6: 20877-?


    Hexanucleotide repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) (c9ALS/FTD). Unconventional translation of these repeats produces dipeptide repeat proteins (DPRs) that may cause neurodegeneration. We performed a modifier screen in Drosophila and discovered a critical role for importins and exportins, Ran-GTP cycle regulators, nuclear pore components, and arginine methylases in mediating DPR toxicity. These findings provide evidence for an important role for nucleocytoplasmic transport in the pathogenic mechanism of c9ALS/FTD.

    View details for DOI 10.1038/srep20877

    View details for PubMedID 26869068

    View details for PubMedCentralID PMC4751451

  • Regrowing axons with alternative splicing. eLife Kramer, N. J., Gitler, A. D. 2016; 5


    The regeneration of axons relies on a previously unknown mechanism that involves the regulation of alternative splicing by CELF proteins.

    View details for DOI 10.7554/eLife.18707

    View details for PubMedID 27420813

    View details for PubMedCentralID PMC4946881

  • CCNF mutations in amyotrophic lateral sclerosis and frontotemporal dementia. Nature communications Williams, K. L., Topp, S., Yang, S., Smith, B., Fifita, J. A., Warraich, S. T., Zhang, K. Y., Farrawell, N., Vance, C., Hu, X., Chesi, A., Leblond, C. S., Lee, A., Rayner, S. L., Sundaramoorthy, V., Dobson-Stone, C., Molloy, M. P., van Blitterswijk, M., Dickson, D. W., Petersen, R. C., Graff-Radford, N. R., Boeve, B. F., Murray, M. E., Pottier, C., Don, E., Winnick, C., McCann, E. P., Hogan, A., Daoud, H., Levert, A., Dion, P. A., Mitsui, J., Ishiura, H., Takahashi, Y., Goto, J., Kost, J., Gellera, C., Gkazi, A. S., Miller, J., Stockton, J., Brooks, W. S., Boundy, K., Polak, M., Muñoz-Blanco, J. L., Esteban-Pérez, J., Rábano, A., Hardiman, O., Morrison, K. E., Ticozzi, N., Silani, V., de Belleroche, J., Glass, J. D., Kwok, J. B., Guillemin, G. J., Chung, R. S., Tsuji, S., Brown, R. H., García-Redondo, A., Rademakers, R., Landers, J. E., Gitler, A. D., Rouleau, G. A., Cole, N. J., Yerbury, J. J., Atkin, J. D., Shaw, C. E., Nicholson, G. A., Blair, I. P. 2016; 7: 11253-?


    Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase complex (SCF(Cyclin F)). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCF(Cyclin F) substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.

    View details for DOI 10.1038/ncomms11253

    View details for PubMedID 27080313

    View details for PubMedCentralID PMC4835537

  • Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation HUMAN MOLECULAR GENETICS Coyne, A. N., Yamada, S. B., Siddegowda, B. B., Estes, P. S., Zaepfel, B. L., Johannesmeyer, J. S., Lockwood, D. B., Pham, L. T., Hart, M. P., Cassel, J. A., Freibaum, B., Boehringer, A. V., Taylor, J. P., Reitz, A. B., Gitler, A. D., Zarnescu, D. C. 2015; 24 (24): 6886-6898

    View details for DOI 10.1093/hmg/ddv389

    View details for Web of Science ID 000368372600003

  • Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways SCIENCE Cirulli, E. T., Lasseigne, B. N., Petrovski, S., Sapp, P. C., Dion, P. A., Leblond, C. S., Couthouis, J., Lu, Y., Wang, Q., Krueger, B. J., Ren, Z., Keebler, J., Han, Y., Levy, S. E., Boone, B. E., Wimbish, J. R., Waite, L. L., Jones, A. L., Carulli, J. P., Day-Williams, A. G., Staropoli, J. F., Xin, W. W., Chesi, A., Raphael, A. R., McKenna-Yasek, D., Cady, J., de Jong, J. M., Kenna, K. P., Smith, B. N., Topp, S., Miller, J., Gkazi, A., Al-Chalabi, A., Van den Berg, L. H., Veldink, J., Silani, V., Ticozzi, N., Shaw, C. E., Baloh, R. H., Appel, S., Simpson, E., Lagier-Tourenne, C., Pulst, S. M., Gibson, S., Trojanowski, J. Q., Elman, L., McCluskey, L., Grossman, M., Shneider, N. A., Chung, W. K., Ravits, J. M., Glass, J. D., Sims, K. B., Van Deerlin, V. M., Maniatis, T., Hayes, S. D., Ordureau, A., Swarup, S., Landers, J., Baas, F., Allen, A. S., Bedlack, R. S., Harper, J. W., Gitler, A. D., Rouleau, G. A., Brown, R., Harms, M. B., Cooper, G. M., Harris, T., Myers, R. M., Goldstein, D. B. 2015; 347 (6229): 1436-1441


    Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

    View details for DOI 10.1126/science.aaa3650

    View details for Web of Science ID 000352136400031

    View details for PubMedID 25700176

  • Parkinson's Disease Genes VPS35 and EIF4G1 Interact Genetically and Converge on a-Synuclein. Neuron Dhungel, N., Eleuteri, S., Li, L., Kramer, N. J., Chartron, J. W., Spencer, B., Kosberg, K., Fields, J. A., Stafa, K., Adame, A., Lashuel, H., Frydman, J., Shen, K., Masliah, E., Gitler, A. D. 2015; 85 (1): 76-87


    Parkinson's disease (PD) is a common neurodegenerative disorder. Functional interactions between some PD genes, like PINK1 and parkin, have been identified, but whether other ones interact remains elusive. Here we report an unexpected genetic interaction between two PD genes, VPS35 and EIF4G1. We provide evidence that EIF4G1 upregulation causes defects associated with protein misfolding. Expression of a sortilin protein rescues these defects, downstream of VPS35, suggesting a potential role for sortilins in PD. We also show interactions between VPS35, EIF4G1, and α-synuclein, a protein with a key role in PD. We extend our findings from yeast to an animal model and show that these interactions are conserved in neurons and in transgenic mice. Our studies reveal unexpected genetic and functional interactions between two seemingly unrelated PD genes and functionally connect them to α-synuclein pathobiology in yeast, worms, and mouse. Finally, we provide a resource of candidate PD genes for future interrogation.

    View details for DOI 10.1016/j.neuron.2014.11.027

    View details for PubMedID 25533483

    View details for PubMedCentralID PMC4289081

  • Targeted Exon Capture and Sequencing in Sporadic Amyotrophic Lateral Sclerosis PLOS GENETICS Couthouis, J., Raphael, A. R., Daneshjou, R., Gitler, A. D. 2014; 10 (10)
  • Targeted exon capture and sequencing in sporadic amyotrophic lateral sclerosis. PLoS genetics Couthouis, J., Raphael, A. R., Daneshjou, R., Gitler, A. D. 2014; 10 (10)


    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive degeneration of motor neurons, ultimately leading to paralysis and death. Approximately 10% of ALS cases are familial, with the remaining 90% of cases being sporadic. Genetic studies in familial cases of ALS have been extremely informative in determining the causative mutations behind ALS, especially as the same mutations identified in familial ALS can also cause sporadic disease. However, the cause of ALS in approximately 30% of familial cases and in the majority of sporadic cases remains unknown. Sporadic ALS cases represent an underutilized resource for genetic information about ALS; therefore, we undertook a targeted sequencing approach of 169 known and candidate ALS disease genes in 242 sporadic ALS cases and 129 matched controls to try to identify novel variants linked to ALS. We found a significant enrichment in novel and rare variants in cases versus controls, indicating that we are likely identifying disease associated mutations. This study highlights the utility of next generation sequencing techniques combined with functional studies and rare variant analysis tools to provide insight into the genetic etiology of a heterogeneous sporadic disease.

    View details for DOI 10.1371/journal.pgen.1004704

    View details for PubMedID 25299611

  • The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of a-synuclein, and enhances its secretion and nuclear localization in cells. Human molecular genetics Fares, M., Ait-Bouziad, N., Dikiy, I., Mbefo, M. K., Jovicic, A., Kiely, A., Holton, J. L., Lee, S., Gitler, A. D., Eliezer, D., Lashuel, H. A. 2014; 23 (17): 4491-4509


    A novel mutation in the α-Synuclein (α-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of α-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates α-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, α-Syn(G51D) behaves similarly to α-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where α-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated α-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of α-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of α-Syn aggregation.

    View details for DOI 10.1093/hmg/ddu165

    View details for PubMedID 24728187

  • Congenital muscular dystrophy and generalized epilepsy caused by GMPPB mutations. Brain research Raphael, A. R., Couthouis, J., Sakamuri, S., Siskind, C., Vogel, H., Day, J. W., Gitler, A. D. 2014; 1575: 66-71


    The alpha-dystroglycanopathies are genetically heterogeneous muscular dystrophies that result from hypoglycosylation of alpha-dystroglycan (α-DG). Alpha-dystroglycan is an essential link between the extracellular matrix and the muscle fiber sarcolemma, and proper glycosylation is critical for its ability to bind to ligands in the extracellular matrix. We sought to identify the genetic basis of alpha-dystroglycanopathy in a family wherein the affected individuals presented with congenital muscular dystrophy, brain abnormalities and generalized epilepsy. We performed whole exome sequencing and identified compound heterozygous GMPPB mutations in the affected children. GMPPB is an enzyme in the glycosylation pathway, and GMPPB mutations were recently linked to eight cases of alpha-dystroglycanopathy with a range of symptoms. We identified a novel mutation in GMPPB (p.I219T) as well as a previously published mutation (p.R287Q). Thus, our work further confirms a role for GMPPB defects in alpha-dystroglycanopathy, and suggests that glycosylation may play a role in the neuronal membrane channels or networks involved in the physiology of generalized epilepsy syndromes. This article is part of a Special Issue entitled RNA Metabolism 2013.

    View details for DOI 10.1016/j.brainres.2014.04.028

    View details for PubMedID 24780531

  • A Cellular System that Degrades Misfolded Proteins and Protects against Neurodegeneration MOLECULAR CELL Guo, L., Giasson, B. I., Glavis-Bloom, A., Brewer, M. D., Shorter, J., Gitler, A. D., Yang, X. 2014; 55 (1): 15-30


    Misfolded proteins compromise cellular function and cause disease. How these proteins are detected and degraded is not well understood. Here we show that PML/TRIM19 and the SUMO-dependent ubiquitin ligase RNF4 act together to promote the degradation of misfolded proteins in the mammalian cell nucleus. PML selectively interacts with misfolded proteins through distinct substrate recognition sites and conjugates these proteins with the small ubiquitin-like modifiers (SUMOs) through its SUMO ligase activity. SUMOylated misfolded proteins are then recognized and ubiquitinated by RNF4 and are subsequently targeted for proteasomal degradation. We further show that PML deficiency exacerbates polyglutamine (polyQ) disease in a mouse model of spinocerebellar ataxia 1 (SCA1). These findings reveal a mammalian system that removes misfolded proteins through sequential SUMOylation and ubiquitination and define its role in protection against protein-misfolding diseases.

    View details for DOI 10.1016/j.molcel.2014.04.030

    View details for Web of Science ID 000340632900004

    View details for PubMedID 24882209

  • Profilin 1 Associates with Stress Granules and ALS-Linked Mutations Alter Stress Granule Dynamics JOURNAL OF NEUROSCIENCE Figley, M. D., Bieri, G., Kolaitis, R., Taylor, J. P., Gitler, A. D. 2014; 34 (24): 8083-8097


    Mutations in the PFN1 gene encoding profilin 1 are a rare cause of familial amyotrophic lateral sclerosis (ALS). Profilin 1 is a well studied actin-binding protein but how PFN1 mutations cause ALS is unknown. The budding yeast, Saccharomyces cerevisiae, has one PFN1 ortholog. We expressed the ALS-linked profilin 1 mutant proteins in yeast, demonstrating a loss of protein stability and failure to restore growth to profilin mutant cells, without exhibiting gain-of-function toxicity. This model provides for simple and rapid screening of novel ALS-linked PFN1 variants. To gain insight into potential novel roles for profilin 1, we performed an unbiased, genome-wide synthetic lethal screen with yeast cells lacking profilin (pfy1Δ). Unexpectedly, deletion of several stress granule and processing body genes, including pbp1Δ, were found to be synthetic lethal with pfy1Δ. Mutations in ATXN2, the human ortholog of PBP1, are a known ALS genetic risk factor and ataxin 2 is a stress granule component in mammalian cells. Given this genetic interaction and recent evidence linking stress granule dynamics to ALS pathogenesis, we hypothesized that profilin 1 might also associate with stress granules. Here we report that profilin 1 and related protein profilin 2 are novel stress granule-associated proteins in mouse primary cortical neurons and in human cell lines and that ALS-linked mutations in profilin 1 alter stress granule dynamics, providing further evidence for the potential role of stress granules in ALS pathogenesis.

    View details for DOI 10.1523/JNEUROSCI.0543-14.2014

    View details for Web of Science ID 000338338700004

    View details for PubMedCentralID PMC4051967

  • A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories. Journal of medical genetics Akimoto, C., Volk, A. E., van Blitterswijk, M., Van den Broeck, M., Leblond, C. S., Lumbroso, S., Camu, W., Neitzel, B., Onodera, O., van Rheenen, W., Pinto, S., Weber, M., Smith, B., Proven, M., Talbot, K., Keagle, P., Chesi, A., Ratti, A., van der Zee, J., Alstermark, H., Birve, A., Calini, D., Nordin, A., Tradowsky, D. C., Just, W., Daoud, H., Angerbauer, S., DeJesus-Hernandez, M., Konno, T., Lloyd-Jani, A., De Carvalho, M., Mouzat, K., Landers, J. E., Veldink, J. H., Silani, V., Gitler, A. D., Shaw, C. E., Rouleau, G. A., Van den Berg, L. H., Van Broeckhoven, C., Rademakers, R., Andersen, P. M., Kubisch, C. 2014; 51 (6): 419-424


    The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories.The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference.Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9-100%), and the mean specificity was 98.0% (87.5-100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories.Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.

    View details for DOI 10.1136/jmedgenet-2014-102360

    View details for PubMedID 24706941

    View details for PubMedCentralID PMC4033024

  • Exome sequencing identifies a DNAJB6 mutation in a family with dominantly-inherited limb-girdle muscular dystrophy. Neuromuscular disorders Couthouis, J., Raphael, A. R., Siskind, C., Findlay, A. R., Buenrostro, J. D., Greenleaf, W. J., Vogel, H., Day, J. W., Flanigan, K. M., Gitler, A. D. 2014; 24 (5): 431-435


    Limb-girdle muscular dystrophy primarily affects the muscles of the hips and shoulders (the "limb-girdle" muscles), although it is a heterogeneous disorder that can present with varying symptoms. There is currently no cure. We sought to identify the genetic basis of limb-girdle muscular dystrophy type 1 in an American family of Northern European descent using exome sequencing. Exome sequencing was performed on DNA samples from two affected siblings and one unaffected sibling and resulted in the identification of eleven candidate mutations that co-segregated with the disease. Notably, this list included a previously reported mutation in DNAJB6, p.Phe89Ile, which was recently identified as a cause of limb-girdle muscular dystrophy type 1D. Additional family members were Sanger sequenced and the mutation in DNAJB6 was only found in affected individuals. Subsequent haplotype analysis indicated that this DNAJB6 p.Phe89Ile mutation likely arose independently of the previously reported mutation. Since other published mutations are located close by in the G/F domain of DNAJB6, this suggests that the area may represent a mutational hotspot. Exome sequencing provided an unbiased and effective method for identifying the genetic etiology of limb-girdle muscular dystrophy type 1 in a previously genetically uncharacterized family. This work further confirms the causative role of DNAJB6 mutations in limb-girdle muscular dystrophy type 1D.

    View details for DOI 10.1016/j.nmd.2014.01.014

    View details for PubMedID 24594375

    View details for PubMedCentralID PMC4013999

  • Evaluating noncoding nucleotide repeat expansions in amyotrophic lateral sclerosis. Neurobiology of aging Figley, M. D., Thomas, A., Gitler, A. D. 2014; 35 (4): 936 e1-4


    Intermediate-length polyglutamine expansions in ataxin 2 are a risk factor for amyotrophic lateral sclerosis (ALS). The polyglutamine tract is encoded by a trinucleotide repeat in a coding region of the ataxin 2 gene (ATXN2). Noncoding nucleotide repeat expansions in several genes are also associated with neurodegenerative and neuromuscular diseases. For example, hexanucleotide repeat expansions located in a noncoding region of C9ORF72 are the most common cause of ALS. We sought to assess a potential larger role of noncoding nucleotide repeat expansions in ALS. We analyzed the nucleotide repeat lengths of 6 genes (ATXN8, ATXN10, PPP2R2B, NOP56, DMPK, and JPH3) that have previously been associated with neurologic or neuromuscular disorders, in several hundred sporadic patients with ALS and healthy control subjects. We report no association between ALS and repeat length in any of these genes, suggesting that variation in the noncoding repetitive regions in these genes does not contribute to ALS.

    View details for DOI 10.1016/j.neurobiolaging.2013.09.024

    View details for PubMedID 24269018

  • Evaluating noncoding nucleotide repeat expansions in amyotrophic lateral sclerosis NEUROBIOLOGY OF AGING Figley, M. D., Thomas, A., Gitler, A. D. 2014; 35 (4)

    View details for Web of Science ID 000330283300032

    View details for PubMedID 24269018

  • TDP-43 in ALS: stay on target…almost there. Neuron Jovicic, A., Gitler, A. D. 2014; 81 (3): 463-465


    ALS is associated with RNA processing impairments involving the RNA-binding protein TDP-43. Pioneering a novel RNA beacon to illuminate RNA trafficking in neurons, Alami et al. (2014) discover a cytoplasmic function for TDP-43, suggesting a new disease mechanism.

    View details for DOI 10.1016/j.neuron.2014.01.034

    View details for PubMedID 24507183

  • Therapeutic modulation of eIF2 alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models NATURE GENETICS Kim, H., Raphael, A. R., LaDow, E. S., McGurk, L., Weber, R. A., Trojanowski, J. Q., Lee, V. M., Finkbeiner, S., Gitler, A. D., Bonini, N. M. 2014; 46 (2): 152-?

    View details for DOI 10.1038/ng.2853

    View details for Web of Science ID 000331208300011

    View details for PubMedID 24336168

  • Exome sequencing to identify de novo mutations in sporadic ALS trios. Nature neuroscience Chesi, A., Staahl, B. T., Jovicic, A., Couthouis, J., Fasolino, M., Raphael, A. R., Yamazaki, T., Elias, L., Polak, M., Kelly, C., Williams, K. L., Fifita, J. A., Maragakis, N. J., Nicholson, G. A., King, O. D., Reed, R., Crabtree, G. R., Blair, I. P., Glass, J. D., Gitler, A. D. 2013; 16 (7): 851-855


    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, we assessed the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n = 141 exomes). We found that amino acid-altering de novo mutations were enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex (nBAF) component SS18L1 (also known as CREST). CREST mutations inhibited activity-dependent neurite outgrowth in primary neurons, and CREST associated with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.

    View details for DOI 10.1038/nn.3412

    View details for PubMedID 23708140

  • Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways. journal of neuroscience Staahl, B. T., Tang, J., Wu, W., Sun, A., Gitler, A. D., Yoo, A. S., Crabtree, G. R. 2013; 33 (25): 10348-10361


    During the development of the vertebrate nervous system, neural progenitors divide, generate progeny that exit mitosis, and then migrate to sites where they elaborate specific morphologies and synaptic connections. Mitotic exit in neurons is accompanied by an essential switch in ATP-dependent chromatin regulatory complexes from the neural progenitor Brg/Brm-associated factor (npBAF) to neuron-specific nBAF complexes that is in part driven by miR-9/9* and miR-124. Recapitulating this microRNA/chromatin switch in fibroblasts leads to their direct conversion to neurons. We have defined the kinetics of neuron-specific BAF complex assembly in the formation of induced neurons from mouse embryonic stem cells, human fibroblasts, and normal mouse neural differentiation and, using proteomic analysis, found that this switch also includes the removal of SS18 and its replacement by CREST at mitotic exit. We found that switching of chromatin remodeling mechanisms is highly correlated with a broad switch in the use of neurogenic transcription factors. Knock-down of SS18 in neural stem cells causes cell-cycle exit and failure to self-renew, whereas continued expression of SS18 in neurons blocks dendritic outgrowth, underlining the importance of subunit switching. Because dominant mutations in BAF subunits underlie widely different human neurologic diseases arising in different neuronal types, our studies suggest that the characteristics of these diseases must be interpreted in the context of the different BAF assemblies in neurons rather than a singular mammalian SWItch/sucrose nonfermentable (mSWI/SNF) complex.

    View details for DOI 10.1523/JNEUROSCI.1258-13.2013

    View details for PubMedID 23785148

  • Stress granules as crucibles of ALS pathogenesis. journal of cell biology Li, Y. R., King, O. D., Shorter, J., Gitler, A. D. 2013; 201 (3): 361-372


    Amyotrophic lateral sclerosis (ALS) is a fatal human neurodegenerative disease affecting primarily motor neurons. Two RNA-binding proteins, TDP-43 and FUS, aggregate in the degenerating motor neurons of ALS patients, and mutations in the genes encoding these proteins cause some forms of ALS. TDP-43 and FUS and several related RNA-binding proteins harbor aggregation-promoting prion-like domains that allow them to rapidly self-associate. This property is critical for the formation and dynamics of cellular ribonucleoprotein granules, the crucibles of RNA metabolism and homeostasis. Recent work connecting TDP-43 and FUS to stress granules has suggested how this cellular pathway, which involves protein aggregation as part of its normal function, might be coopted during disease pathogenesis.

    View details for DOI 10.1083/jcb.201302044

    View details for PubMedID 23629963

  • Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS NATURE Kim, H. J., Kim, N. C., Wang, Y., Scarborough, E. A., Moore, J., Diaz, Z., MacLea, K. S., Freibaum, B., Li, S., Molliex, A., Kanagaraj, A. P., Carter, R., Boylan, K. B., Wojtas, A. M., Rademakers, R., Pinkus, J. L., Greenberg, S. A., Trojanowski, J. Q., Traynor, B. J., Smith, B. N., Topp, S., Gkazi, A., Miller, J., Shaw, C. E., Kottlors, M., Kirschner, J., Pestronk, A., Li, Y. R., Ford, A. F., Gitler, A. D., Benatar, M., King, O. D., Kimonis, V. E., Ross, E. D., Weihl, C. C., Shorter, J., Taylor, J. P. 2013; 495 (7442): 467-?


    Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

    View details for DOI 10.1038/nature11922

    View details for Web of Science ID 000316682800037

    View details for PubMedID 23455423

  • Parallel PARKing: Parkinson's genes function in common pathway. Neuron Chuang, R. S., Gitler, A. D. 2013; 77 (3): 377-379


    Parkinson's disease (PD) is associated with diverse genetic and environmental susceptibilities. Functional connections between PD genes have remained elusive. In this issue of Neuron, MacLeod et al. (2013) link three PD susceptibility genes, LRRK2, PARK16, and VSP35, to a common cellular pathway and show how these deficits contribute to dysfunction.

    View details for DOI 10.1016/j.neuron.2013.01.014

    View details for PubMedID 23395366

  • Yeast genetic screen reveals novel therapeutic strategy for ALS Rare Diseases Figley, M., Gitler, AD 2013; 1 (1): e24420
  • Yeast genetic screen reveals novel therapeutic strategy for ALS. Rare diseases (Austin, Tex.) Figley, M. D., Gitler, A. D. 2013; 1


    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by a selective loss of motor neurons. There is no cure and few effective treatments. The RNA-binding protein TDP-43 contributes to the pathogenesis of ALS. TDP-43 is depleted from the nucleus and accumulates in cytoplasmic aggregates in the degenerating neurons and glia of most ALS patients. Furthermore, mutations in the TDP-43 gene cause rare familial and sporadic forms of the disease. Thus, therapeutic strategies targeting TDP-43 may be efficacious. We have used the yeast model system to identify the mechanisms by which TDP-43 aggregation contributes to ALS and to identify approaches to protect cells from the toxic effects of TDP-43 aggregation. Using an unbiased yeast genetic screen we discovered Dbr1 as a potent suppressor of TDP-43 toxicity. Yeast cells in which Dbr1 is deleted are resistant to TDP-43 toxicity. Dbr1 inhibition in mammalian cells is also sufficient to protect against TDP-43 cytotoxicity. Here, we review this recent discovery, highlighting future approaches aimed at extending these studies and pursuing Dbr1 as a novel therapeutic target for ALS.

    View details for DOI 10.4161/rdis.24420

    View details for PubMedID 25002991

  • A Template for New Drugs Against Alzheimer's Disease Cell Aguzzi, A., Gitler, A. D. 2013; 154 (6): 1182–1184
  • Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models NATURE GENETICS Armakola, M., Higgins, M. J., Figley, M. D., Barmada, S. J., Scarborough, E. A., Diaz, Z., Fang, X., Shorter, J., Krogan, N. J., Finkbeiner, S., Farese, R. V., Gitler, A. D. 2012; 44 (12): 1302-1309


    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in the gene encoding TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most individuals with ALS. Thus, strategies aimed at targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here, we report results from two genome-wide loss-of-function TDP-43 toxicity suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was deletion of DBR1, which encodes an RNA lariat debranching enzyme. We show that, in the absence of Dbr1 enzymatic activity, intronic lariats accumulate in the cytoplasm and likely act as decoys to sequester TDP-43, preventing it from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rat neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43-mediated cytotoxicity and suggest that decreasing Dbr1 activity could be a potential therapeutic approach for ALS.

    View details for DOI 10.1038/ng.2434

    View details for Web of Science ID 000311713200006

    View details for PubMedID 23104007

  • TDP-43 and FUS/TLS yield a target-rich haul in ALS NATURE NEUROSCIENCE Gitler, A. D. 2012; 15 (11): 1467-1469

    View details for Web of Science ID 000310424900001

    View details for PubMedID 23103989

  • Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Weisberg, S. J., Lyakhovetsky, R., Werdiger, A., Gitler, A. D., Soen, Y., Kaganovich, D. 2012; 109 (39): 15811-15816


    Neurodegenerative diseases constitute a class of illnesses marked by pathological protein aggregation in the brains of affected individuals. Although these disorders are invariably characterized by the degeneration of highly specific subpopulations of neurons, protein aggregation occurs in all cells, which indicates that toxicity arises only in particular cell biological contexts. Aggregation-associated disorders are unified by a common cell biological feature: the deposition of the culprit proteins in inclusion bodies. The precise function of these inclusions remains unclear. The starting point for uncovering the origins of disease pathology must therefore be a thorough understanding of the general cell biological function of inclusions and their potential role in modulating the consequences of aggregation. Here, we show that in human cells certain aggregate inclusions are active compartments. We find that toxic aggregates localize to one of these compartments, the juxtanuclear quality control compartment (JUNQ), and interfere with its quality control function. The accumulation of SOD1G93A aggregates sequesters Hsp70, preventing the delivery of misfolded proteins to the proteasome. Preventing the accumulation of SOD1G93A in the JUNQ by enhancing its sequestration in an insoluble inclusion reduces the harmful effects of aggregation on cell viability.

    View details for DOI 10.1073/pnas.1205829109

    View details for Web of Science ID 000309604500060

    View details for PubMedID 22967507

  • Modeling Human Disease SCIENCE Gitler, A. D., Lehmann, R. 2012; 337 (6092): 269-269

    View details for DOI 10.1126/science.1227179

    View details for Web of Science ID 000306542600001

    View details for PubMedID 22822114

  • ALS-Associated Ataxin 2 PolyQ Expansions Enhance Stress-Induced Caspase 3 Activation and Increase TDP-43 Pathological Modifications JOURNAL OF NEUROSCIENCE Hart, M. P., Gitler, A. D. 2012; 32 (27): 9133-9142


    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by the loss of motor neurons. The degenerating motor neurons of ALS patients are characterized by the accumulation of cytoplasmic inclusions containing phosphorylated and truncated forms of the RNA-binding protein TDP-43. Ataxin 2 intermediate-length polyglutamine (polyQ) expansions were recently identified as a risk factor for ALS; however, the mechanism by which they contribute to disease is unknown. Here, we show that intermediate-length ataxin 2 polyQ expansions enhance stress-induced TDP-43 C-terminal cleavage and phosphorylation in human cells. We also connect intermediate-length ataxin 2 polyQ expansions to the stress-dependent activation of multiple caspases, including caspase 3. Caspase activation is upstream of TDP-43 cleavage and phosphorylation since caspase inhibitors block these pathological modifications. Analysis of the accumulation of activated caspase 3 in motor neurons revealed a striking association with ALS cases harboring ataxin 2 polyQ expansions. These findings indicate that activated caspase 3 defines a new pathological feature of ALS with intermediate-length ataxin 2 polyQ expansions. These results provide mechanistic insight into how ataxin 2 intermediate-length polyQ expansions could contribute to ALS--by enhancing stress-induced TDP-43 pathological modifications via caspase activation. Because longer ataxin 2 polyQ expansions are associated with a different disease, spinocerebellar ataxia 2, these findings help explain how different polyQ expansions in the same protein can have distinct cellular consequences, ultimately resulting in different clinical features. Finally, since caspase inhibitors are effective at reducing TDP-43 pathological modifications, this pathway could be pursued as a therapeutic target in ALS.

    View details for DOI 10.1523/JNEUROSCI.0996-12.2012

    View details for Web of Science ID 000306193900004

    View details for PubMedID 22764223

  • Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis HUMAN MOLECULAR GENETICS Couthouis, J., Hart, M. P., Erion, R., King, O. D., Diaz, Z., Nakaya, T., Ibrahim, F., Kim, H., Mojsilovic-Petrovic, J., Panossian, S., Kim, C. E., Frackelton, E. C., Solski, J. A., Williams, K. L., Clay-Falcone, D., Elman, L., McCluskey, L., Greene, R., Hakonarson, H., Kalb, R. G., Lee, V. M., Trojanowski, J. Q., Nicholson, G. A., Blair, I. P., Bonini, N. M., Van Deerlin, V. M., Mourelatos, Z., Shorter, J., Gitler, A. D. 2012; 21 (13): 2899-2911


    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

    View details for DOI 10.1093/hmg/dds116

    View details for Web of Science ID 000305457700006

    View details for PubMedID 22454397

    View details for PubMedCentralID PMC3373238

  • The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease BRAIN RESEARCH King, O. D., Gitler, A. D., Shorter, J. 2012; 1462: 61-80


    Prions are self-templating protein conformers that are naturally transmitted between individuals and promote phenotypic change. In yeast, prion-encoded phenotypes can be beneficial, neutral or deleterious depending upon genetic background and environmental conditions. A distinctive and portable 'prion domain' enriched in asparagine, glutamine, tyrosine and glycine residues unifies the majority of yeast prion proteins. Deletion of this domain precludes prionogenesis and appending this domain to reporter proteins can confer prionogenicity. An algorithm designed to detect prion domains has successfully identified 19 domains that can confer prion behavior. Scouring the human genome with this algorithm enriches a select group of RNA-binding proteins harboring a canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of 210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these are in the top 60 prion candidates in the entire genome. Startlingly, these RNA-binding prion candidates are inexorably emerging, one by one, in the pathology and genetics of devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), Alzheimer's disease and Huntington's disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing prion candidates, form cytoplasmic inclusions in the degenerating motor neurons of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently, perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing prion candidate, has been connected with ALS and FTLD-U. We strongly suspect that we have now merely reached the tip of the iceberg. We predict that additional RNA-binding prion candidates identified by our algorithm will soon surface as genetic modifiers or causes of diverse neurodegenerative conditions. Indeed, simple prion-like transfer mechanisms involving the prion domains of RNA-binding proteins could underlie the classical non-cell-autonomous emanation of neurodegenerative pathology from originating epicenters to neighboring portions of the nervous system. This article is part of a Special Issue entitled RNA-Binding Proteins.

    View details for DOI 10.1016/j.brainres.2012.01.016

    View details for Web of Science ID 000306444700007

    View details for PubMedID 22445064

  • The Role of the Parkinson's Disease Gene PARK9 in Essential Cellular Pathways and the Manganese Homeostasis Network in Yeast PLOS ONE Chesi, A., Kilaru, A., Fang, X., Cooper, A. A., Gitler, A. D. 2012; 7 (3)


    YPK9 (Yeast PARK9; also known as YOR291W) is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase. However, its substrate specificity is unknown. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset parkinsonism. We previously described a strong genetic interaction between Ypk9 and another Parkinson's disease (PD) protein α-synuclein in multiple model systems, and a role for Ypk9 in manganese detoxification in yeast. In humans, environmental exposure to toxic levels of manganese causes a syndrome similar to PD and is thus an environmental risk factor for the disease. How manganese contributes to neurodegeneration is poorly understood. Here we describe multiple genome-wide screens in yeast aimed at defining the cellular function of Ypk9 and the mechanisms by which it protects cells from manganese toxicity. In physiological conditions, we found that Ypk9 genetically interacts with essential genes involved in cellular trafficking and the cell cycle. Deletion of Ypk9 sensitizes yeast cells to exposure to excess manganese. Using a library of non-essential gene deletions, we screened for additional genes involved in tolerance to excess manganese exposure, discovering several novel pathways involved in manganese homeostasis. We defined the dependence of the deletion strain phenotypes in the presence of manganese on Ypk9, and found that Ypk9 deletion modifies the manganese tolerance of only a subset of strains. These results confirm a role for Ypk9 in manganese homeostasis and illuminates cellular pathways and biological processes in which Ypk9 likely functions.

    View details for DOI 10.1371/journal.pone.0034178

    View details for Web of Science ID 000304046900051

    View details for PubMedID 22457822

  • The modulation of Amyotrophic Lateral Sclerosis risk by Ataxin-2 intermediate polyglutamine expansions is a specific effect NEUROBIOLOGY OF DISEASE Gispert, S., Kurz, A., Waibel, S., Bauer, P., Liepelt, I., Geisen, C., Gitler, A. D., Becker, T., Weber, M., Berg, D., Andersen, P. M., Krueger, R., Riess, O., Ludolph, A. C., Auburger, G. 2012; 45 (1): 356-361


    Full expansions of the polyglutamine domain (polyQ≥34) within the polysome-associated protein ataxin-2 (ATXN2) are the cause of a multi-system neurodegenerative disorder, which usually presents as a Spino-Cerebellar Ataxia and is therefore known as SCA2, but may rarely manifest as Levodopa-responsive Parkinson syndrome or as motor neuron disease. Intermediate expansions (27≤polyQ≤33) were reported to modify the risk of Amyotrophic Lateral Sclerosis (ALS). We have now tested the reproducibility and the specificity of this observation. In 559 independent ALS patients from Central Europe, the association of ATXN2 expansions (30≤polyQ≤35) with ALS was highly significant. The study of 1490 patients with Parkinson's disease (PD) showed an enrichment of ATXN2 alleles 27/28 in a subgroup with familial cases, but the overall risk of sporadic PD was unchanged. No association was found between polyQ expansions in Ataxin-3 (ATXN3) and ALS risk. These data indicate a specific interaction between ATXN2 expansions and the causes of ALS, possibly through altered RNA-processing as a common pathogenic factor.

    View details for DOI 10.1016/j.nbd.2011.08.021

    View details for Web of Science ID 000297883500040

    View details for PubMedID 21889984

  • Distinct TDP-43 pathology in ALS patients with ataxin 2 intermediate-length polyQ expansions Acta Neuropathol Hart MP, Brettschneider J, Lee VM, Trojanowski JQ, Gitler AD 2012; 124 (2): 221-230
  • A yeast functional screen predicts new candidate ALS disease genes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Couthouis, J., Hart, M. P., Shorter, J., DeJesus-Hernandez, M., Erion, R., Oristano, R., Liu, A. X., Ramos, D., Jethava, N., Hosangadi, D., Epstein, J., Chiang, A., Diaz, Z., Nakaya, T., Ibrahim, F., Kim, H., Solski, J. A., Williams, K. L., Mojsilovic-Petrovic, J., Ingre, C., Boylan, K., Graff-Radford, N. R., Dickson, D. W., Clay-Falcone, D., Elman, L., McCluskey, L., Greene, R., Kalb, R. G., Lee, V. M., Trojanowski, J. Q., Ludolph, A., Robberecht, W., Andersen, P. M., Nicholson, G. A., Blair, I. P., King, O. D., Bonini, N. M., Van Deerlin, V., Rademakers, R., Mourelatos, Z., Gitler, A. D. 2011; 108 (52): 20881-20890


    Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.

    View details for DOI 10.1073/pnas.1109434108

    View details for Web of Science ID 000298479900012

    View details for PubMedID 22065782

  • Local RNA Translation at the Synapse and in Disease JOURNAL OF NEUROSCIENCE Liu-Yesucevitz, L., Bassell, G. J., Gitler, A. D., Hart, A. C., Klann, E., Richter, J. D., Warren, S. T., Wolozin, B. 2011; 31 (45): 16086-16093


    Local regulation of protein synthesis in neurons has emerged as a leading research focus because of its importance in synaptic plasticity and neurological diseases. The complexity of neuronal subcellular domains and their distance from the soma demand local spatial and temporal control of protein synthesis. Synthesis of many synaptic proteins, such as GluR and PSD-95, is under local control. mRNA binding proteins (RBPs), such as FMRP, function as key regulators of local RNA translation, and the mTORC1 pathway acts as a primary signaling cascade for regulation of these proteins. Much of the regulation occurs through structures termed RNA granules, which are based on reversible aggregation of the RBPs, some of which have aggregation prone domains with sequence features similar to yeast prion proteins. Mutations in many of these RBPs are associated with neurological diseases, including FMRP in fragile X syndrome; TDP-43, FUS (fused in sarcoma), angiogenin, and ataxin-2 in amyotrophic lateral sclerosis; ataxin-2 in spinocerebellar ataxia; and SMN (survival of motor neuron protein) in spinal muscular atrophy.

    View details for DOI 10.1523/JNEUROSCI.4105-11.2011

    View details for Web of Science ID 000296799700007

    View details for PubMedID 22072660

  • Neuroscience. Another reason to exercise. Science Gitler, A. D. 2011; 334 (6056): 606-607

    View details for DOI 10.1126/science.1214714

    View details for PubMedID 22053033

  • Model Organisms Reveal Insight into Human Neurodegenerative Disease: Ataxin-2 Intermediate-Length Polyglutamine Expansions Are a Risk Factor for ALS JOURNAL OF MOLECULAR NEUROSCIENCE Bonini, N. M., Gitler, A. D. 2011; 45 (3): 676-683


    Model organisms include yeast Saccromyces cerevisae and fly Drosophila melanogaster. These systems have powerful genetic approaches, as well as highly conserved pathways, both for normal function and disease. Here, we review and highlight how we applied these systems to provide mechanistic insight into the toxicity of TDP-43. TDP-43 accumulates in pathological aggregates in ALS and about half of FTD. Yeast and fly studies revealed an interaction with the counterparts of human Ataxin-2, a gene whose polyglutamine repeat expansion is associated with spinocerebellar ataxia type 2. This finding raised the hypothesis that repeat expansions in ataxin-2 may associate with diseases characterized by TDP-43 pathology such as ALS. DNA analysis of patients revealed that intermediate-length polyglutamine expansions in ataxin-2 are a risk factor for ALS, such that repeat lengths are greater than normal, but lower than that associated with spinocerebellar ataxia type 2 (SCA2), and are more frequent in ALS patients than in matched controls. Moreover, repeat expansions associated with ALS are interrupted CAA-CAG sequences as opposed to the pure CAG repeat expansions typically associated with SCA2. These studies provide an example of how model systems, when extended to human cells and human patient tissue, can reveal new mechanistic insight into disease.

    View details for DOI 10.1007/s12031-011-9548-9

    View details for Web of Science ID 000296518900045

    View details for PubMedID 21660502

  • RNA-binding proteins with prion-like domains in ALS and FTLD-U PRION Gitler, A. D., Shorter, J. 2011; 5 (3): 179-187


    Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) is a debilitating, and universally fatal, neurodegenerative disease that devastates upper and lower motor neurons. The causes of ALS are poorly understood. A central role for RNA-binding proteins and RNA metabolism in ALS has recently emerged. The RNA-binding proteins, TDP-43 and FUS, are principal components of cytoplasmic inclusions found in motor neurons of ALS patients and mutations in TDP-43 and FUS are linked to familial and sporadic ALS. Pathology and genetics also connect TDP-43 and FUS with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). It was unknown whether mechanisms of FUS aggregation and toxicity were similar or different to those of TDP-43. To address this issue, we have employed yeast models and pure protein biochemistry to define mechanisms underlying TDP-43 and FUS aggregation and toxicity, and to identify genetic modifiers relevant for human disease. We have identified prion-like domains in FUS and TDP-43 and provide evidence that these domains are required for aggregation. Our studies have defined key similarities as well as important differences between the two proteins. Collectively, however, our findings lead us to suggest that FUS and TDP-43, though similar RNA-binding proteins, likely aggregate and confer disease phenotypes via distinct mechanisms.

    View details for DOI 10.4161/pri.5.3.17230

    View details for Web of Science ID 000298921800011

    View details for PubMedID 21847013

  • High-throughput Yeast Plasmid Overexpression Screen JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Fleming, M. S., Gitler, A. D. 2011

    View details for DOI 10.3791/2836

    View details for Web of Science ID 000209215000027

  • A yeast model for polyalanine-expansion aggregation and toxicity MOLECULAR BIOLOGY OF THE CELL Konopka, C. A., Locke, M. N., Gallagher, P. S., Ngan Pham, N., Hart, M. P., Walker, C. J., Gitler, A. D., Gardner, R. G. 2011; 22 (12): 1971-1984


    Nine human disorders result from the toxic accumulation and aggregation of proteins with expansions in their endogenous polyalanine (polyA) tracts. Given the prevalence of polyA tracts in eukaryotic proteomes, we wanted to understand the generality of polyA-expansion cytotoxicity by using yeast as a model organism. In our initial case, we expanded the polyA tract within the native yeast poly(Adenine)-binding protein Pab1 from 8A to 13A, 15A, 17A, and 20A. These expansions resulted in increasing formation of Pab1 inclusions, insolubility, and cytotoxicity that correlated with the length of the polyA expansion. Pab1 binds mRNA as part of its normal function, and disrupting RNA binding or altering cytoplasmic mRNA levels suppressed the cytotoxicity of 17A-expanded Pab1, indicating a requisite role for mRNA in Pab1 polyA-expansion toxicity. Surprisingly, neither manipulation suppressed the cytotoxicity of 20A-expanded Pab1. Thus longer expansions may have a different mechanism for toxicity. We think that this difference underscores the potential need to examine the cytotoxic mechanisms of both long and short expansions in models of expansion disorders.

    View details for DOI 10.1091/mbc.E11-01-0037

    View details for Web of Science ID 000291548400003

    View details for PubMedID 21508314

  • Evaluating the prevalence of polyglutamine repeat expansions in amyotrophic lateral sclerosis NEUROLOGY Lee, T., Li, Y. R., Chesi, A., Hart, M. P., Ramos, D., Jethava, N., Hosangadi, D., Epstein, J., Hodges, B., Bonini, N. M., Gitler, A. D. 2011; 76 (24): 2062-2065


    Given the recent finding of an association between intermediate-length polyglutamine (polyQ) expansions in ataxin 2 and amyotrophic lateral sclerosis (ALS), we sought to determine whether expansions in other polyQ disease genes were associated with ALS.We assessed the polyQ lengths of ataxin 1, ataxin 3, ataxin 6, ataxin 7, TBP, atrophin 1, and huntingtin in several hundred patients with sporadic ALS and healthy controls.Other than ataxin 2, we did not identify a significant association with the other polyQ genes and ALS.These data indicate that the effects of ataxin 2 polyQ expansions on ALS risk are likely to be rooted in the biology of ataxin 2 or ataxin 2-specific interactions, rather than the presence of an expanded polyQ repeat per se. These findings have important consequences for understanding the role of ataxin 2 in ALS pathogenesis and provide a framework for future mechanistic studies.

    View details for Web of Science ID 000291588800009

    View details for PubMedID 21562248

  • Ataxin-2 intermediate-length polyglutamine expansions in European ALS patients HUMAN MOLECULAR GENETICS Lee, T., Li, Y. R., Ingre, C., Weber, M., Grehl, T., Gredal, O., De Carvalho, M., Meyer, T., Tysnes, O., Auburger, G., Gispert, S., Bonini, N. M., Andersen, P. M., Gitler, A. D. 2011; 20 (9): 1697-1700


    Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease primarily affecting motor neurons. We recently identified intermediate-length polyglutamine (polyQ) expansions (27-33 Qs) in ataxin 2 as a genetic risk factor for sporadic ALS in North American ALS patients. To extend these findings, we assessed the ataxin 2 polyQ repeat length in 1294 European ALS patients and 679 matched healthy controls. We observed a significant association between polyQ expansions and ALS (>30 Qs; P= 6.2 × 10(-3)). Thus, intermediate-length ataxin 2 polyQ repeat expansions are associated with increased risk for ALS also in the European cohort. The specific polyQ length cutoff, however, appears to vary between different populations, with longer repeat lengths showing a clear association. Our findings support the hypothesis that ataxin 2 plays an important role in predisposing to ALS and that polyQ expansions in ataxin 2 are a significant risk factor for the disease.

    View details for DOI 10.1093/hmg/ddr045

    View details for Web of Science ID 000289311400003

    View details for PubMedID 21292779

  • Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease Protein FUS/TLS PLOS BIOLOGY Sun, Z., Diaz, Z., Fang, X., Hart, M. P., Chesi, A., Shorter, J., Gitler, A. D. 2011; 9 (4)


    TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.

    View details for DOI 10.1371/journal.pbio.1000614

    View details for Web of Science ID 000289938900009

    View details for PubMedID 21541367

  • PolyQ Repeat Expansions in ATXN2 Associated with ALS Are CAA Interrupted Repeats PLOS ONE Yu, Z., Zhu, Y., Chen-Plotkin, A. S., Clay-Falcone, D., McCluskey, L., Elman, L., Kalb, R. G., Trojanowski, J. Q., Lee, V. M., Van Deerlin, V. M., Gitler, A. D., Bonini, N. M. 2011; 6 (3)


    Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27-33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥ 34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1-3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2.

    View details for DOI 10.1371/journal.pone.0017951

    View details for Web of Science ID 000289054600022

    View details for PubMedID 21479228

  • TDP-43 toxicity in yeast METHODS Armakola, M., Hart, M. P., Gitler, A. D. 2011; 53 (3): 238-245


    The budding yeast Saccharomyces cerevisiae is an emerging tool for investigating the molecular pathways that underpin several human neurodegenerative disorders associated with protein misfolding. Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disease primarily affecting motor neurons. The protein TDP-43 has recently been demonstrated to play an important role in the disease, however, the mechanisms by which TDP-43 contributes to pathogenesis are unclear. To explore the mechanistic details that result in aberrant accumulation of TDP-43 and to discover potential strategies for therapeutic intervention, we employed a yeast TDP-43 proteinopathy model system. These studies allowed us to determine the regions of TDP-43 required for aggregation and toxicity and to define the effects of ALS-linked mutant forms of TDP-43. We have also been able to harness the power of yeast genetics to identify potent modifiers of TDP-43 toxicity using high-throughput yeast genetic screens. Here, we describe the methods and approaches that we have used in order to gain insight into TDP-43 biology and its role in disease. These approaches are readily adaptable to other neurodegenerative disease proteins.

    View details for DOI 10.1016/j.ymeth.2010.11.006

    View details for Web of Science ID 000288523200010

    View details for PubMedID 21115123

  • High-throughput yeast plasmid overexpression screen. Journal of visualized experiments : JoVE Fleming, M. S., Gitler, A. D. 2011


    The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology (1-5). In the last few years, several genomewide libraries of yeast strains and plasmids have been generated (6-10). These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches (11-16). Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

    View details for DOI 10.3791/2836

    View details for PubMedID 21841759

  • Prion-like disorders: blurring the divide between transmissibility and infectivity JOURNAL OF CELL SCIENCE Cushman, M., Johnson, B. S., King, O. D., Gitler, A. D., Shorter, J. 2010; 123 (8): 1191-1201


    Prions are proteins that access self-templating amyloid forms, which confer phenotypic changes that can spread from individual to individual within or between species. These infectious phenotypes can be beneficial, as with yeast prions, or deleterious, as with mammalian prions that transmit spongiform encephalopathies. However, the ability to form self-templating amyloid is not unique to prion proteins. Diverse polypeptides that tend to populate intrinsically unfolded states also form self-templating amyloid conformers that are associated with devastating neurodegenerative disorders. Moreover, two RNA-binding proteins, FUS and TDP-43, which form cytoplasmic aggregates in amyotrophic lateral sclerosis, harbor a 'prion domain' similar to those found in several yeast prion proteins. Can these proteins and the neurodegenerative diseases to which they are linked become 'infectious' too? Here, we highlight advances that define the transmissibility of amyloid forms connected with Alzheimer's disease, Parkinson's disease and Huntington's disease. Collectively, these findings suggest that amyloid conformers can spread from cell to cell within the brains of afflicted individuals, thereby spreading the specific neurodegenerative phenotypes distinctive to the protein being converted to amyloid. Importantly, this transmissibility mandates a re-evaluation of emerging neuronal graft and stem-cell therapies. In this Commentary, we suggest how these treatments might be optimized to overcome the transmissible conformers that confer neurodegeneration.

    View details for DOI 10.1242/jcs.051672

    View details for Web of Science ID 000276568200002

    View details for PubMedID 20356930

  • GTPase Activity Plays a Key Role in the Pathobiology of LRRK2 PLOS GENETICS Xiong, Y., Coombes, C. E., Kilaru, A., Li, X., Gitler, A. D., Bowers, W. J., Dawson, V. L., Dawson, T. M., Moore, D. J. 2010; 6 (4)


    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with late-onset, autosomal-dominant, familial Parkinson's disease (PD) and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s) underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker's yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human alpha-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.

    View details for DOI 10.1371/journal.pgen.1000902

    View details for Web of Science ID 000277354200019

    View details for PubMedID 20386743

  • TDP-43 Is Intrinsically Aggregation-prone, and Amyotrophic Lateral Sclerosis-linked Mutations Accelerate Aggregation and Increase Toxicity JOURNAL OF BIOLOGICAL CHEMISTRY Johnson, B. S., Snead, D., Lee, J. J., McCaffery, J. M., Shorter, J., Gitler, A. D. 2009; 284 (30): 20329-20339


    Non-amyloid, ubiquitinated cytoplasmic inclusions containing TDP-43 and its C-terminal fragments are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Importantly, TDP-43 mutations are linked to sporadic and non-SOD1 familial ALS. However, TDP-43 is not the only protein in disease-associated inclusions, and whether TDP-43 misfolds or is merely sequestered by other aggregated components is unclear. Here, we report that, in the absence of other components, TDP-43 spontaneously forms aggregates bearing remarkable ultrastructural similarities to TDP-43 deposits in degenerating neurons of ALS and FTLD-U patients [corrected] . The C-terminal domain of TDP-43 is critical for spontaneous aggregation. Several ALS-linked TDP-43 mutations within this domain (Q331K, M337V, Q343R, N345K, R361S, and N390D) increase the number of TDP-43 aggregates and promote toxicity in vivo. Importantly, mutations that promote toxicity in vivo accelerate aggregation of pure TDP-43 in vitro. Thus, TDP-43 is intrinsically aggregation-prone, and its propensity for toxic misfolding trajectories is accentuated by specific ALS-linked mutations.

    View details for DOI 10.1074/jbc.M109.010264

    View details for Web of Science ID 000268097400059

    View details for PubMedID 19465477

  • Disease models and mechanisms in the classroom DISEASE MODELS & MECHANISMS Gitler, A. D. 2009; 2 (3-4): 103-106


    At the University of Pennsylvania (PENN), we devote an entire graduate-level course to the study of human disease models: Seminar on Current Genetic Research: Modeling Human Disease in Diverse Genetic Systems.

    View details for DOI 10.1242/dmm.002600

    View details for Web of Science ID 000268254800003

    View details for PubMedID 19259378

  • Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity NATURE GENETICS Yeger-Lotem, E., Riva, L., Su, L. J., Gitler, A. D., Cashikar, A. G., King, O. D., Auluck, P. K., Geddie, M. L., Valastyan, J. S., Karger, D. R., Lindquist, S., Fraenkel, E. 2009; 41 (3): 316-323


    Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens. By comparing the results of these two assays across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We used this approach to reveal cellular pathways responding to the toxicity of alpha-synuclein, a protein implicated in several neurodegenerative disorders including Parkinson's disease. For this we screened an established yeast model to identify genes that when overexpressed alter alpha-synuclein toxicity. Bridging these data and data from mRNA profiling provided functional explanations for many of these genes and identified previously unknown relations between alpha-synuclein toxicity and basic cellular pathways.

    View details for DOI 10.1038/ng.337

    View details for Web of Science ID 000263640200012

    View details for PubMedID 19234470

  • alpha-Synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity NATURE GENETICS Gitler, A. D., Chesi, A., Geddie, M. L., Strathearn, K. E., Hamamichi, S., Hill, K. J., Caldwell, K. A., Caldwell, G. A., Cooper, A. A., Rochet, J., Lindquist, S. 2009; 41 (3): 308-315


    Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. alpha-Synuclein (alpha-syn) has a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between alpha-syn and the yeast ortholog of the PD-linked gene ATP13A2 (also known as PARK9). Dopaminergic neuron loss caused by alpha-syn overexpression in animal and neuronal PD models is rescued by coexpression of PARK9. Further, knockdown of the ATP13A2 ortholog in Caenorhabditis elegans enhances alpha-syn misfolding. These data provide a direct functional connection between alpha-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. We discovered that yeast PARK9 helps to protect cells from manganese toxicity, revealing a connection between PD genetics (alpha-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of alpha-syn-induced neuron loss in animals, establishing a diverse, highly conserved interaction network for alpha-syn.

    View details for DOI 10.1038/ng.300

    View details for Web of Science ID 000263640200011

    View details for PubMedID 19182805

  • Evidence That alpha-Synuclein Does Not Inhibit Phospholipase D BIOCHEMISTRY Rappley, I., Gitler, A. D., Selvy, P. E., LaVoie, M. J., Levy, B. D., Brown, H. A., Lindquist, S., Selkoe, D. J. 2009; 48 (5): 1077-1083


    Alpha-synuclein (alphaSyn) is a small cytosolic protein of unknown function, which is highly enriched in the brain. It is genetically linked to Parkinson's disease (PD) in that missense mutations or multiplication of the gene encoding alphaSyn causes early onset familial PD. Furthermore, the neuropathological hallmarks of both sporadic and familial PD, Lewy bodies and Lewy neurites, contain insoluble aggregates of alphaSyn. Several studies have reported evidence that alphaSyn can inhibit phospholipase D (PLD), which hydrolyzes phosphatidylcholine to form phosphatidic acid and choline. Although various hypotheses exist regarding the roles of alphaSyn in health and disease, no other specific biochemical function for this protein has been reported to date. Because PLD inhibition could represent an important function of alphaSyn, we sought to extend existing reports on this interaction. Using purified proteins, we tested the ability of alphaSyn to inhibit PLD activity in cell-free assays. We also examined several cell lines and transfection conditions to assess whether alphaSyn inhibits endogenous or overexpressed PLD in cultured mammalian cells. In yeast, we extended our previous report of an interaction between alphaSyn and PLD-dependent phenotypes, for which PLD activity is absolutely necessary. Despite testing a range of experimental conditions, including those previously published, we observed no significant inhibition of PLD by alphaSyn in any of these systems. We propose that the previously reported effects of alphaSyn on PLD activity could be due to increased endoplasmic reticulum-related stress associated with alphaSyn overexpression in cells, but are not likely due to a specific and direct interaction between alphaSyn and PLD.

    View details for DOI 10.1021/bi801871h

    View details for Web of Science ID 000263047900029

    View details for PubMedID 19146388

  • Discovery and characterization of three novel synuclein genes in zebrafish DEVELOPMENTAL DYNAMICS Sun, Z., Gitler, A. D. 2008; 237 (9): 2490-2495


    The neuronal protein alpha-synuclein has been linked to the pathogenesis of synucleinopathies, a collection of neurodegenerative disorders, including Parkinson's disease. alpha-Synuclein belongs to a family of synuclein genes that includes beta- and gamma-synuclein. However, despite being associated with several fatal human neurodegenerative diseases, little is known about the normal function of synucleins. Here we report the cloning and characterization of three synucleins from zebrafish, sncga, sncgb, and sncb. The sequences of these zebrafish synucleins are very similar to those of the human proteins. We used whole-mount in situ hybridization to analyze their spatial and temporal expression patterns during development. sncgb was expressed exclusively in the notochord, while sncga and sncb were expressed strongly in the nervous system. Our identification of synuclein genes in zebrafish and the characterization of their expression patterns will facilitate future experiments aimed at assessing their functions in normal physiology as well as their role in pathophysiology.

    View details for DOI 10.1002/dvdy.21569

    View details for Web of Science ID 000259289800021

    View details for PubMedID 18521955

  • A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDR-43 aggregation and cellular toxicity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. D. 2008; 105 (17): 6439-6444


    Protein misfolding is intimately associated with devastating human neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. Although disparate in their pathophysiology, many of these disorders share a common theme, manifested in the accumulation of insoluble protein aggregates in the brain. Recently, the major disease protein found in the pathological inclusions of two of these diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), was identified as the 43-kDa TAR-DNA-binding protein (TDP-43), providing a molecular link between them. TDP-43 is a ubiquitously expressed nuclear protein that undergoes a pathological conversion to an aggregated cytoplasmic localization in affected regions of the nervous system. Whether TDP-43 itself can convey toxicity and whether its abnormal aggregation is a cause or consequence of pathogenesis remain unknown. We report a yeast model to define mechanisms governing TDP-43 subcellular localization and aggregation. Remarkably, this simple model recapitulates several salient features of human TDP-43 proteinopathies, including conversion from nuclear localization to cytoplasmic aggregation. We establish a connection between this aggregation and toxicity. The pathological features of TDP-43 are distinct from those of yeast models of other protein-misfolding diseases, such as polyglutamine. This suggests that the yeast model reveals specific aspects of the underlying biology of the disease protein rather than general cellular stresses associated with accumulating misfolded proteins. This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies.

    View details for DOI 10.1073/pnas.0802082105

    View details for Web of Science ID 000255534100041

    View details for PubMedID 18434538

  • The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gitler, A. D., Bevis, B. J., Shorter, J., Strathearn, K. E., Hamamichi, S., Su, L. J., Caldwell, K. A., Caldwell, G. A., Rochet, J., McCaffery, J. M., Barlowe, C., Lindquist, S. 2008; 105 (1): 145-150


    alpha-Synuclein (alpha-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast alpha-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER-->Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER-->Golgi trafficking. The homologous Rab1 rescues alpha-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of alpha-syn pathobiology. In a cell-free system with purified transport factors alpha-syn inhibited ER-->Golgi trafficking in an alpha-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of alpha-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with alpha-syn and with diverse vesicle markers, suggesting that alpha-syn can impair multiple trafficking steps. Other Rabs did not ameliorate alpha-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, alpha-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.

    View details for DOI 10.1073/pnas.0710685105

    View details for Web of Science ID 000252435300030

    View details for PubMedID 18162536

  • Beer and bread to brains and beyond: Can yeast cells teach us about neurodegenerative disease? NEUROSIGNALS Gitler, A. D. 2008; 16 (1): 52-62


    For millennia, humans have harnessed the astonishing power of yeast, producing such culinary masterpieces as bread, beer and wine. Therefore, in this new millennium, is it very farfetched to ask if we can also use yeast to unlock some of the modern day mysteries of human disease? Remarkably, these seemingly simple cells possess most of the same basic cellular machinery as the neurons in the brain. We and others have been using the baker's yeast, Saccharomyces cerevisiae, as a model system to study the mechanisms of devastating neurodegenerative diseases such as Parkinson's, Huntington's, Alzheimer's and amyotrophic lateral sclerosis. While very different in their pathophysiology, they are collectively referred to as protein-misfolding disorders because of the presence of misfolded and aggregated forms of various proteins in the brains of affected individuals. Using yeast genetics and the latest high-throughput screening technologies, we have identified some of the potential causes underpinning these disorders and discovered conserved genes that have proven effective in preventing neuron loss in animal models. Thus, these genes represent new potential drug targets. In this review, I highlight recent work investigating mechanisms of cellular toxicity in a yeast Parkinson's disease model and discuss how similar approaches are being applied to additional neurodegenerative diseases.

    View details for DOI 10.1159/000109759

    View details for Web of Science ID 000252706300007

    View details for PubMedID 18097160

  • A suite of Gateway (R) cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae YEAST Alberti, S., Gitler, A. D., Lindquist, S. 2007; 24 (10): 913-919


    In the post-genomic era, academic and biotechnological research is increasingly shifting its attention from single proteins to the analysis of complex protein networks. This change in experimental design requires the use of simple and experimentally tractable organisms, such as the unicellular eukaryote Saccharomyces cerevisiae, and a range of new high-throughput techniques. The Gateway system has emerged as a powerful high-throughput cloning method that allows for the in vitro recombination of DNA with high speed, accuracy and reliability. Two Gateway-based libraries of overexpression plasmids containing the entire complement of yeast open reading frames (ORFs) have recently been completed. In order to make use of these powerful resources, we adapted the widely used pRS series of yeast shuttle vectors for use in Gateway-based cloning. The resulting suite of 288 yeast Gateway vectors is based upon the two commonly used GPD and GAL1 promoter expression systems that enable expression of ORFs, either constitutively or under galactose-inducible conditions. In addition, proteins of interest can be fused to a choice of frequently used N- or C-terminal tags, such as EGFP, ECFP, EYFP, Cerulean, monomeric DsRed, HA or TAP. We have made this yeast Gateway vector kit available to the research community via the non-profit Addgene Plasmid Repository (

    View details for DOI 10.1002/yea.1502

    View details for Web of Science ID 000250352000007

    View details for PubMedID 17583893

  • Prime time for alpha-synuclein JOURNAL OF NEUROSCIENCE Gitler, A. D., Shorter, J. 2007; 27 (10): 2433-2434
  • Kermit 2/XGIPC, an IGF1 receptor interacting protein, is required for IGF signaling in Xenopus eye development DEVELOPMENT Wu, J., O'Donnell, M., Gitler, A. D., Klein, P. S. 2006; 133 (18): 3651-3660


    GIPC is a PDZ-domain-containing protein identified in vertebrate and invertebrate organisms through its interaction with a variety of binding partners including many membrane proteins. Despite the multiple reports identifying GIPC, its endogenous function and the physiological significance of these interactions are much less studied. We have previously identified the Xenopus GIPC homolog kermit as a frizzled 3 interacting protein that is required for frizzled 3 induction of neural crest in ectodermal explants. We identified a second Xenopus GIPC homolog, named kermit 2 (also recently described as an IGF receptor interacting protein and named XGIPC). Despite its high amino acid similarity with kermit, kermit 2/XGIPC has a distinct function in Xenopus embryos. Loss-of-function analysis indicates that kermit 2/XGIPC is specifically required for Xenopus eye development. Kermit 2/XGIPC functions downstream of IGF in eye formation and is required for maintaining IGF-induced AKT activation. A constitutively active PI3 kinase partially rescues the Kermit 2/XGIPC loss-of-function phenotype. Our results provide the first in vivo loss of function analysis of GIPC in embryonic development and also indicate that kermit 2/XGIPC is a novel component of the IGF pathway, potentially functioning through modulation of the IGF1 receptor.

    View details for DOI 10.1242/dev.02547

    View details for Web of Science ID 000240046500016

    View details for PubMedID 16914488

  • alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models SCIENCE Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell, G. A., Marsischky, G., Kolodner, R. D., LaBaer, J., Rochet, J., Bonini, N. M., Lindquist, S. 2006; 313 (5785): 324-328


    Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.

    View details for DOI 10.1126/science.1129462

    View details for Web of Science ID 000239154300036

    View details for PubMedID 16794039

  • Insertion of Cre into the Pax3 locus creates a new allele of Splotch and identifies unexpected Pax3 derivatives DEVELOPMENTAL BIOLOGY Engleka, K. A., Gitler, A. D., Zhang, M. Z., Zhou, D. D., High, F. A., Epstein, J. A. 2005; 280 (2): 396-406


    Pax3 is a transcription factor expressed in the dorsal neural tube and somite of the developing embryo. It plays critical roles in pre-migratory neural crest cells and in myogenic precursors of skeletal muscle. Pax3-deficient Splotch embryos display neural tube and neural crest defects and lack hypaxial muscles. We have created a new allele of Splotch by replacing the first coding exon with a gene encoding Cre recombinase. This functions as a null allele and no Pax3 protein is detected in homozygous embryos. Heterozygous Pax3(Cre/+) mice display a white belly spot, as do Splotch heterozygotes. Homozygous Pax3(Cre/Cre) embryos are embryonic lethal. We have used Pax3(Cre/+) mice to fate-map Pax3 derivatives in the developing mouse. As expected, neural crest and some somitic derivatives are identified. However, we also detect previously unappreciated derivatives of Pax3-expressing precursors in the colonic epithelium of the hindgut and within the urogenital system.

    View details for DOI 10.1016/j.ydbio.2005.02.002

    View details for Web of Science ID 000228377600011

    View details for PubMedID 15882581

  • Yeast cells as a discovery platform for neurodegenerative disease 9th Annual International Conference on Research in Computational Molecular Biology (RECOMB 2005) Lindquist, S., Fraenkel, E., Outeiro, T., Gitler, A., Su, J., Cashikar, A., Jagadish, S. SPRINGER-VERLAG BERLIN. 2005: 102–102
  • Semaphorin-plexin signaling guides patterning of the developing vasculature DEVELOPMENTAL CELL Torres-Vazquez, J., Gitler, A. D., Fraser, S. D., Berk, J. D., Pham, V. N., Fishman, M. C., Childs, S., Epstein, J. A., Weinstein, B. M. 2004; 7 (1): 117-123


    Major vessels of the vertebrate circulatory system display evolutionarily conserved and reproducible anatomy, but the cues guiding this stereotypic patterning remain obscure. In the nervous system, axonal pathways are shaped by repulsive cues provided by ligands of the semaphorin family that are sensed by migrating neuronal growth cones through plexin receptors. We show that proper blood vessel pathfinding requires the endothelial receptor PlexinD1 and semaphorin signals, and we identify mutations in plexinD1 in the zebrafish vascular patterning mutant out of bounds. These results reveal the fundamental conservation of repulsive patterning mechanisms between axonal migration in the central nervous system and vascular endothelium during angiogenesis.

    View details for Web of Science ID 000222696300015

    View details for PubMedID 15239959

  • PlexinD1 and semaphorin signaling are required in endothelial cells for cadiovascular development DEVELOPMENTAL CELL Gitler, A. D., Lu, M. M., Epstein, J. A. 2004; 7 (1): 107-116


    The identification of new signaling pathways critical for cardiac morphogenesis will contribute to our understanding of congenital heart disease (CHD), which remains a leading cause of mortality in newborn children worldwide. Signals mediated by semaphorin ligands and plexin receptors contribute to the intricate patterning of axons in the central nervous system. Here, we describe a related signaling pathway involving secreted class 3 semaphorins, neuropilins, and a plexin receptor, PlexinD1, expressed by endothelial cells. Interruption of this pathway in mice results in CHD and vascular patterning defects. The type of CHD caused by inactivation of PlexinD1 has previously been attributed to abnormalities of neural crest. Here, we show that this form of CHD can be caused by cell-autonomous endothelial defects. Thus, molecular programs that mediate axon guidance in the central nervous system also function in endothelial cells to orchestrate critical aspects of cardiac morphogenesis.

    View details for Web of Science ID 000222696300014

    View details for PubMedID 15239958

  • Tie2-cre-induced inactivation of a conditional mutant Nf1 allele in mouse results in a myeloproliferative disorder that models juvenile myelomonocytic leukemia PEDIATRIC RESEARCH Gitler, A. D., Kong, Y., Choi, J. K., Zhu, Y., Pear, W. S., Epstein, J. A. 2004; 55 (4): 581-584


    Neurofibromatosis type one (NF1) is a common genetic disorder affecting 1:4000 births and is characterized by benign and malignant tumors. Children with NF1 are predisposed to juvenile myelomonocytic leukemia. The Nf1 gene encodes neurofibromin, which can function as a Ras GTPase-activating protein. Neurofibromin deficiency in mice leads to mid-gestation lethality due to cardiovascular defects. We have previously shown that conditional inactivation of Nf1 using Tie2-Cre recapitulates the heart defects seen in Nf1(-/-) embryos. Tie2-Cre transgenic mice express Cre recombinase in all endothelial cells. Here, we show that Tie2-Cre-mediated deletion of Nf1 also leads to excision of Nf1 in the hematopoietic lineage. Surviving mice exhibit a myeloproliferative disorder similar to juvenile myelomonocytic leukemia seen in NF1 patients. These mice provide a useful model to study neurofibromin deficiency in hematopoiesis. Furthermore, defects in Tie2-Cre-expressing progenitors that result in heart and blood defects suggest that related heart and blood disorders in NF1 and other syndromes represent disorders of the hemangioblast.

    View details for DOI 10.1203/01.PDR.0000113462.98851.2E

    View details for Web of Science ID 000220478700009

    View details for PubMedID 14739366

  • Molecular markers of cardiac endocardial cushion development DEVELOPMENTAL DYNAMICS Gitler, A. D., Lu, M. M., Jiang, Y. Q., Epstein, J. A., Gruber, P. J. 2003; 228 (4): 643-650


    Endocardial cushions are precursors of mature heart valves. They form within the looped heart tube as discrete swellings and develop into thin, pliable leaflets that prevent regurgitation of blood. The embryonic origins of cardiac valves include endothelial, myocardial, and neural crest cells. Recently, an increasing number of animal models derived from mutational screens, gene inactivation, and transgenic studies have identified specific molecules required for normal development of the cardiac valves, and critical molecular pathways are beginning to emerge. To further this process, we have sought to assemble a diverse set of molecular markers encompassing all stages of cardiac valve development. Here, we provide a detailed comparative gene expression analysis of thirteen endocardial cushion markers. We identify endocardial cushion expression of the transcription factor Fog1, and we demonstrate active Wnt/beta-catenin signaling in developing endocardial cushions suggesting pathways that have not been previously appreciated to participate in cardiac valve formation.

    View details for DOI 10.1002/dvdy.10418

    View details for Web of Science ID 000186954100009

    View details for PubMedID 14648841

  • Cloning and characterization of zebrafish tbx1 GENE EXPRESSION PATTERNS Kochilas, L. K., Potluri, V., Gitler, A., Balasubramanian, K., Chin, A. J. 2003; 3 (5): 645-651


    Tbx1 is one of the genes within the DiGeorge Critical Region (DGCR) and has been recently identified as the critical gene for the cardiovascular anomalies in the DiGeorge mouse models. We have cloned, sequenced and analyzed the zebrafish (Danio rerio) tbx1 cDNA. It encodes a protein of 460 amino acids that shares 64% identity and 67% similarity with the human TBX1 orthologue at the amino acid level. Although maternal expression was detected by RT-PCR, only zygotic expression could be detected by whole-mount in situ hybridization. Expression of zebrafish tbx1 by whole-mount in situ hybridization was first detected at 40% epiboly, 5.0 hours post fertilization (hpf) in the dorsal blastoderm margin. Through the stage of embryonic shield formation, tbx1 expression is restricted to the hypoblast, in the region of cells fated to become head and lateral plate mesoderm and pharyngeal endoderm. At 18 hpf, when the heart tube is beginning to assemble, three domains of tbx1 expression can be seen: cardiac precursors, pharyngeal arch precursors and otic vesicle. These three domains will remain the sites of tbx1 expression to varying degrees through at least 72 hpf. By 51 hpf, tbx1 expression can be seen in the cardiac outflow tract, the ventricle and the atrium, although by 72 hpf cardiac expression is strongest in the cardiac outflow tract. This newly identified tbx1 expression pattern in cardiac regions other than the cardiac outflow tract offers a new insight into the role of the tbx1 transcription factor in cardiac development.

    View details for DOI 10.1016/S1567-133X(03)00108-X

    View details for Web of Science ID 000185582300014

    View details for PubMedID 12972000

  • Cardiac hypertrophy and histone deacetylase-dependent transcriptional repression mediated by the atypical homeodomain protein Hop JOURNAL OF CLINICAL INVESTIGATION Kook, H., Lepore, J. J., Gitler, A. D., Lu, M. M., Yung, W. W., Mackay, J., Zhou, R., Ferrari, V., Gruber, P., Epstein, J. A. 2003; 112 (6): 863-871


    Activation of multiple pathways is associated with cardiac hypertrophy and heart failure. Repression of antihypertrophic pathways has rarely been demonstrated to cause cardiac hypertrophy in vivo. Hop is an unusual homeodomain protein that is expressed by embryonic and postnatal cardiac myocytes. Unlike other homeodomain proteins, Hop does not bind DNA. Rather, it modulates cardiac growth and proliferation by inhibiting the transcriptional activity of serum response factor (SRF) in cardiomyocytes. Here we show that Hop can inhibit SRF-dependent transcriptional activation by recruiting histone deacetylase (HDAC) activity and can form a complex that includes HDAC2. Transgenic mice that overexpress Hop develop severe cardiac hypertrophy, cardiac fibrosis, and premature death. A mutant form of Hop, which does not recruit HDAC activity, does not induce hypertrophy. Treatment of Hop transgenic mice with trichostatin A, an HDAC inhibitor, prevents hypertrophy. In addition, trichostatin A also attenuates hypertrophy induced by infusion of isoproterenol. Thus, chromatin remodeling and repression of otherwise active transcriptional processes can result in hypertrophy and heart failure, and this process can be blocked with chemical HDAC inhibitors.

    View details for DOI 10.1172/JCI200319137

    View details for Web of Science ID 000185376400010

    View details for PubMedID 12975471

  • Regulating heart development: the role of Nf1. Cell cycle Gitler, A. D., Epstein, J. A. 2003; 2 (2): 96-98


    Neurofibromatosis type 1 (NF1) is one of the most common human genetic disorders and is associated with significant morbidity and mortality. The gene responsible for this disorder, NF1, encodes neurofibromin, which can function to down-regulate ras activity. Mutations that inactivate NF7 result in elevated levels of ras signaling and increased cell proliferation in some tissues. NF7 functions as a tumor suppressor gene; patients inherit one mutated copy and are believed to acquire a "second hit" in tissues that go on to form benign or malignant tumors. NF7 is expressed widely, yet certain tissues are more susceptible to growth dysregulation in NF1 patients. Cardiovascular defects also contribute to NF1, though the cause remains unclear. In a recent study, we used tissue-specific gene inactivation in mice to study the role of neurofibromin in heart development. A further understanding of neurofibromin function will help to elucidate the pathophysiology of NF1 and will also lead to a better understanding of cell cycle regulation and ras pathways in specific cell types. Finally, we comment on how similar genetic strategies can be used in mice to study the role of additional signaling pathways involved in heart development.

    View details for PubMedID 12695655

  • Nf1 has an essential role in endothelial cells NATURE GENETICS Gitler, A. D., Zhu, Y., Ismat, F. A., Lu, M. M., Yamauchi, Y., Parada, L. F., Epstein, J. A. 2003; 33 (1): 75-79


    Neurofibromatosis type 1 (NF1) or von Recklinghausen neurofibromatosis is a genetic disorder that occurs in 1 of 4000 births and is characterized by benign and malignant tumors. Cardiovascular defects also contribute to NF1, though the pathogenesis is still unclear. Deficiency in neurofibromin (encoded by Nf1) in mice results in mid-embryonic lethality owing to cardiac abnormalities previously thought to be secondary to cardiac neural-crest defects. Using tissue-specific gene inactivation, we show that endothelial-specific inactivation of Nf1 recapitulates key aspects of the complete null phenotype, including multiple cardiovascular abnormalities involving the endocardial cushions and myocardium. This phenotype is associated with an elevated level of ras signaling in Nf1(-/-) endothelial cells and greater nuclear localization of the transcription factor Nfatc1. Inactivation of Nf1 in the neural crest does not cause cardiac defects but results in tumors of neural-crest origin resembling those seen in humans with NF1. These results establish a new and essential role for Nf1 in endothelial cells and confirm the requirement for neurofibromin in the neural crest.

    View details for DOI 10.1038/ng1059

    View details for Web of Science ID 000180136100021

    View details for PubMedID 12469121

  • Regulating Heart Development The Role of Nf1 CELL CYCLE Gitler, A. D., Epstein, J. A. 2003; 2 (2): 96-98

    View details for DOI 10.4161/cc.2.2.299

    View details for Web of Science ID 000209690600010

  • Hop is an unusual homeobox gene that modulates cardiac development CELL Chen, F., Kook, H., Milewski, R., Gitler, A. D., Lu, M. M., Li, J., Nazarian, R., Schnepp, R., Jen, K., Biben, C., Runke, G., MacKay, J. P., Novotny, J., Schwartz, R. J., Harvey, R. P., Mullins, M. C., Epstein, J. A. 2002; 110 (6): 713-723


    Hop is a small, divergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development.

    View details for Web of Science ID 000178182800007

    View details for PubMedID 12297045

  • Neural crest migration and mouse models of congenital heart disease Cold Spring Harbor Symposium on Quantitative Biology Gitler, A. D., Brown, C. B., Kochilas, L., Li, J., Epstein, J. A. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 2002: 57–62

    View details for Web of Science ID 000183780700009

    View details for PubMedID 12858524

  • Cloning and expression analysis of murine lupin, a member of a novel gene family that is conserved through evolution and associated with Lupus inclusions DEVELOPMENT GENES AND EVOLUTION Lu, M. M., Chen, F., Gitler, A., Li, J., Jin, F. Z., Ma, X. K., Epstein, J. A. 2000; 210 (10): 512-517


    We describe here the first full-length sequence of a member of a novel gene family encoding a protein in the mouse that we call Lupin. Lupin is homologous to a human protein previously called p36, which was purified from alpha-interferon-treated cells that formed lupus inclusions. Lupus inclusions are dense intracellular deposits found in endothelial cells and lymphocytes of patients with systemic lupus erythematosis and AIDS. Proteins closely related to Lupin exist in evolutionarily divergent species including Caenorhabditis elegans, Drosophila and zebrafish. At least one other lupin-related gene is expressed in the mouse and in man. Lupin is expressed in mouse embryos and adults, notably in liver, spleen, central nervous system, multiple epithelia and all types of muscle. In skeletal muscle, expression analysis suggests that Lupin associates with the contractile apparatus.

    View details for Web of Science ID 000089605200005

    View details for PubMedID 11180800

  • Apakaochtodenes A and B: Two tetrahalogenated monoterpenes from the red marine alga Portieria hornemannii JOURNAL OF NATURAL PRODUCTS Gunatilaka, A. A., Paul, V. J., Park, P. U., Puglisi, M. P., Gitler, A. D., Eggleston, D. S., Haltiwanger, R. C., Kingston, D. G. 1999; 62 (10): 1376-1378


    The structure of apakaochtodene A, the minor isomer of two tetrahalogenated ochtodene monoterpenes, isolated from the red marine alga Portieria hornemannii (Lyngbye) Silva has been identified as 6(S)-bromo-1,4(S),8(R)-trichloro-2(Z)-ochtodene (1) by NMR spectral and X-ray crystallographic analysis. Its geometrical isomer, apakaochtodene B (2), which could not be separated from 1 and thus characterized as a 95:5 mixture of 2:1 had (1)H and (13)C NMR spectral characteristics similar to previously known ochtodene (3) and the related tetrahalogenated monoterpene 4.

    View details for Web of Science ID 000083371400006

    View details for PubMedID 10543896