Bio

Academic Appointments


Honors & Awards


  • Young Scientist Lectureship Award, International Society for Neurochemistry (2013)
  • Eppendorf and Science Prize for Neurobiology, Finalist, Eppendorf and Science Magazine (2011)
  • Instituto Paulo Gontijo International Medicine PG Award, Instituto Paulo Gontijo (2011)
  • Addgene Innovation Award, Addgene (2011)
  • Scientist to Watch, The Scientist (2010)
  • NIH Directorís 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


Protein folding is critically important for all life, from microbes to man. A bafflingly diverse set of cellular mechanisms has evolved to coordinate this process. Not unexpectedly, problems in protein folding are the root cause of many of the most devastating diseases, which represent a major challenge to public health worldwide, especially as our population continues to age. Referred to collectively as protein-misfolding disorders, these truly disastrous neurodegenerative diseases include Alzheimer disease, Parkinson disease and ALS (Lou Gehrigís disease). Understanding at a mechanistic level the cellular consequences of protein misfolding will help to suggest potential strategies for therapeutic intervention. We use the bakerís yeast, Saccharomyces cerevisiae, as a model system to study the cell biology underpinning protein-misfolding diseases. We do not limit ourselves to one model system or experimental approach. We start with yeast, perform genetic screens, and then move to other model systems (e.g. mammalian tissue culture, mouse, fly, zebrafish) and even work with human patient samples (tissue sections, patient-derived cells, including iPS cells, and next generation sequencing approaches to look for mutations in novel genes).

Parkinson's disease and alpha-synuclein

We have focused on the Parkinson disease (PD) linked protein, alpha-synuclein. By performing high-throughput genome-wide screens in yeast, we identified a set of genes, many with clear human homologs, which are able to antagonize cellular toxicity associated with the accumulation of misfolded alpha-synuclein. Remarkably, some genes are also able to rescue neuron loss in animal models of PD (Cooper et al., Science 2006). We recently found that one of the genes from our alpha-synuclein toxicity modifier screen is the yeast homolog of the human PARK9 gene and that yeast PARK9 functions to protect cells from manganese toxicity, an environmental risk factor for PD and PD-like syndromes (Gitler et al., Nature Genetics 2009).

New yeast models of neurodegenerative diseases

We recently developed a yeast model to study the ALS disease proteins TDP-43 and FUS (Johnson et al., PNAS 2008; Sun et al., 2011). We have used yeast and in vitro biochemistry (in collaboration with Jim Shorter at PENN) to analyze the effects of ALS-linked TDP-43 and FUS mutations on aggregation and toxicity (Johnson et al., JBC 2009; Sun et al., 2011). We are now using these models to perform high-throughput genetic screens to elucidate the molecular pathways affected by TDP-43 and FUS aggregation.

Ataxin-2 and ALS

Interestingly, one of the hits from our yeast TDP-43 genetic modifier screen 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), and used biochemistry to show the proteins physically associate in an RNA-dependent manner.To extend our findings to human disease, we analyzed the ataxin 2 gene in a large number of ALS patients and healthy controls and found intermediate-length polyQ expansions in ataxin 2 significantly associated with increased risk for ALS (Elden et al., Nature 2010). We continue to characterize the role of ataxin 2 in ALS as well as other neurodegenerative disease situations (Hart et al., 2012; Hart and Gitler, 2012).

New ALS Disease Genes

We have begun a novel functional screen in yeast to identify new human ALS disease genes. From this seemingly simple yeast screen, we were able to predict a set of ALS candidate disease genes and, remarkably, have already identified mutations in two of them in human ALS patients (Couthouis et al., 2011; Couthouis et al., 2012). We continue to sequence more genes in a large cohort of sporadic and familial ALS patients using standard as well as next generation sequencing approaches.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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)

    Abstract

    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

  • 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

    Abstract

    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

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

    Abstract

    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 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

    Abstract

    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-?

    Abstract

    Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2? phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2? phosphorylation in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

    View details for DOI 10.1038/ng.2853

    View details for Web of Science ID 000331208300011

    View details for PubMedID 24336168

  • 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

    Abstract

    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

    Abstract

    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-?

    Abstract

    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

    Abstract

    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 Web of Science ID 000317030800001

    View details for PubMedID 23395366

  • A Template for New Drugs Against Alzheimer's Disease Cell Aguzzi, A., Gitler, A. D. 2013; 154 (6): 1182?1184
  • Yeast genetic screen reveals novel therapeutic strategy for ALS Rare Diseases Figley, M., Gitler, AD 2013; 1 (1): e24420
  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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)

    Abstract

    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 ?-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

    Abstract

    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
  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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)

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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)

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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 (http://www.addgene.org/yeast_gateway).

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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 SYMPOSIA ON QUANTITATIVE BIOLOGY Gitler, A. D., Brown, C. B., Kochilas, L., Li, J., Epstein, J. A. 2002; 67: 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

    Abstract

    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

    Abstract

    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

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