Search

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.

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.

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. (C) 2011 Elsevier Inc. All rights reserved.

We hypothesized that the NF1 protein Neurofibromin has functions in addition to its Ras-GAP activity and proposed to develop a yeast NF1 model to define new functions as well as novel regulators of known functions. In this 3 year project, we categorized genetic hits that we recovered from yeast screens. We divided hits into Ras-dependent and Ras-independent categories. We have identified proteins that physically interact with IRA1 and IRA2 (the yeast homologs of human Neurofibromin). We have also focused on a novel genetic interaction between IRA1 and IRA2 and yeast genes involved in peroxisome formation and function in yeast. These studies will illuminate the cellular pathways that Ira1 and Ira2 regulate and how neurofibromin deficiency contributes to disease., The original document contains color images.

We hypothesized that the NF1 protein Neurofibromin has functions in addition to its Ras-GAP activity and proposed to develop a yeast NF1 model to define new functions as well as novel regulators of known functions. In year 1 of this project, we categorized genetic hits that we recovered from yeast screens. We divided hits into Ras-dependent and Ras-independent categories. In year 2 of this project, we have identified proteins that physically interact with IRA1 and IRA2 (the yeast homologs of human Neurofibromin). These data will facilitate future years of funding for this project, as we integrate the genetic and physical interaction data in order to get a better picture of the cellular pathways that Ira1 and Ira2 regulate, and then to validate if these interactions are relevant in animal models of the disease., The original document contains color images.

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, yet the functional roles of the genes identified by these assays often remain enigmatic. By comparing the results of these two assays across various cellular responses, we found that they are consistently distinct. Moreover, genetic screens tend to identify response regulators, while 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 harnessed this approach to reveal cellular pathways related to alpha-synuclein, a small lipid-binding protein implicated in several neurodegenerative disorders including Parkinson disease. For this we screened an established yeast model for alphasynuclein toxicity to identify genes that when overexpressed alter cellular survival. Application of our algorithm to these data and data from mRNA profiling provided functional explanations for many of these genes and revealed novel relations between alpha-synuclein toxicity and basic cellular pathways., MGH/MIT Morris Udall Center of Excellence in PD Research

We hypothesized that the NF1 protein Neurofibromin has functions in addition to its Ras-GAP activity and proposed to develop a yeast NF1 model to define new functions as well as novel regulators of known functions. In year 1 of this project, we categorized genetic hits that we recovered from yeast screens. We divided hits into Ras-dependent and Ras-independent categories. To do this, we used two approaches. First, we assayed mutant yeast strains from our screens by idodine staining, providing an indirect readout for ras signaling levels. For strains that showed elevated ras signaling, we next used Ras-GTP pull-down assays to define if the genes functioned upstream or downstream of ras. The most significant finding during the first year of this project is that we defined which of our genes that interact with IRA1 or IRA2 are involved in ras signaling and which are not. Furthermore, of the genes involved in ras signaling, we now have evidence placing these either upstream or downstream of ras. These data will facilitate future years of funding for this project, as we define if the genetic modifiers interact physically with Ira1 and/or Ira2, as well as generating transgenic flies to validate our results in Drosophila., The original document contains color images.

My doctoral dissertation involves studying the growth and development of the vasculature and centers around two projects involving novel endothelial cell signaling mechanisms. I begin by analyzing the role of the Nf1 gene in cardiovascular development. The cardiac phenotype of homozygous null Nf1 embryos is complex and the structures in the affected hearts are derived from multiple cell types. Using Cre/loxP technology, I ablated Nf1 function in myocardium, cardiac neural crest, and endothelium. Endothelial-specific Nf1 inactivation recapitulated the complete cardiac phenotype seen in null embryos. I found elevated ras/MAPK signaling and ectopic activation of NFATc1. A subset of the endothelial-specific Nf1 knockout mice survived embryogenesis and developed a leukemia resembling the type children with NF1 are predisposed to. I continued to investigate endothelial cell signaling pathways and explored the hypothesis: mechanisms that guide axons in the nervous system may have been co-opted to function independently in endothelial cells to pattern the vasculature. I identified a novel Plexin gene that we called plexinD1. In the nervous system, Plexins are receptors for the Semaphorin family of axon guidance molecules. The sequence of plexinD1 was highly similar to other Plexin family members, however its expression pattern was restricted to endothelium. To explore the in vivo role of plexinD1, I generated a mouse knockout. plexinD1−/− mice survived embryogenesis and were born, but all became cyanotic and died within 24 hours of birth and all had congenital heart defects. The phenotype was remarkably similar to sema3C−/− and to endothelial-restricted Neuropilin-1 knockouts, suggesting a possible connection in a common signaling pathway. Consistent with this, I was able to immunoprecipitate a complex containing PlexinD1 and Npn1. Moreover, in an in vitro cell-based system, this interaction enhanced Sema3C binding to Npn1. I identified a zebrafish plexinD1 ortholog. Intrig