Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

College of Charleston

The infantile onset form of Neuronal Ceroid Lipofuscinoses (INCL) is the earliest and most severe form of NCL, with neurological symptoms that reflect massive neurodegeneration in the CNS and retina. INCL is due to recessively inherited mutations at the CLN1 locus. This locus encodes the evolutionarily conserved enzyme palmitoyl-protein thioesterase 1 (PPT1), indicating an essential role for protein palmitoylation in normal neuronal function.

An over-expression system for characterizing Ppt1 function in Drosophila.

Infantile neuronal ceroid lipofuscinosis (INCL) is a pediatric neurodegenerative disease caused by mutations in the human CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase 1 (PPT1), suggesting an important role for the regulation of palmitoylation in normal neuronal function. To further elucidate Ppt1 function, we performed a gain-of-function modifier screen in Drosophila using a collection of enhancer-promoter transgenic lines to suppress or enhance the degeneration produced by overexpression of Ppt1 in the adult visual system. Modifier genes identified in our screen connect Ppt1 function to synaptic vesicle cycling, endo-lysosomal trafficking, synaptic development, and activity-dependent remodeling of the synapse. Furthermore, several homologs of the modifying genes are known to be regulated by palmitoylation in other systems and may be in vivo substrates for Ppt1. Our results complement recent work on mouse Ppt1(-/-) cells that shows a reduction in synaptic vesicle pools in primary neuronal cultures and defects in endosomal trafficking in human fibroblasts. The pathways and processes implicated by our modifier loci shed light on the normal cellular function of Ppt1. A greater understanding of Ppt1 function in these cellular processes will provide valuable insight into the molecular etiology of the neuronal dysfunction underlying the disease.

Genetic modifiers of Drosophila palmitoyl-protein thioesterase 1-induced degeneration.

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization,RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627 and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2)and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.

The Drosophila protein palmitoylome: characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases.

We hold these truths to be self-evident, that all flies and men are created equal: Recent progress on human disease models.

Infantile-onset Neuronal Ceroid Lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate post-translational modification from an unknown set of substrate proteins. To better understand the function of Ppt1 in neurons, we performed an unbiased dominant loss-of-function genetic modifier screen in Drosophila using a previously characterized Ppt1 gain-of-function system. The enhancers and suppressors identified in our screen make novel connections between Ppt1 and genes involved in cellular trafficking and the modulation of synaptic growth. We further support the relevance of our screen by demonstrating that Garland cells from Ppt1 loss-of-function mutants have defects in endocytic trafficking. Endocytic tracer uptake and ultrastructural analysis of these non-neuronal cells points to Ppt1 playing a role in modulating the early stages of vesicle formation. This work lays the groundwork for further experimental exploration of these processes to better understand their contributions to the INCL disease process.

Identifying cellular pathways modulated by Drosophila palmitoyl-protein thioesterase 1 function.

The Internet is enhancing and challenging traditional approaches to teaching undergraduate neuroscience. In addition to the new FUN-supported development of a Society for Neuroscience Portal for higher education, there is a wealth of available teaching resources currently housed on the web. This article discusses the current state of digital libraries and introduces a series of exemplary web-based classroom resources.

Teaching undergraduate neuroscience in the digital age.

Collaborative and international scientific efforts continue to be of increasing importance in the development of successful educational and research programs. The goal of our study abroad program, Neuroscience Seminar in Germany, is to bring this fact to light for undergraduates and make them aware of the global opportunities that exist in the neurosciences and related biological sciences. Here we discuss our experience of conducting a four-week summer study abroad course in collaboration with two universities associated with the German Graduate Schools of Neuroscience: Munich Center for Neurosciences - Ludwig-Maximilians-Universität (MCN-LMU) and Charité - Universitätsmedizin, Berlin (a joint institution of the Freie Universität and the Humboldt-Universität). This course combined the historical foundations of neuroscience in Germany with current research programs at these two prominent German research universities. Two weeks were spent at each location and faculty members from the participating universities provided seminars, laboratory exercises, demonstrations and tours. Students were presented with background reading and lecture material prior to the seminars and activities. Additionally, they were responsible for leading seminar-style class discussions through brief presentations and submitting written critical analyses of primary research papers associated with the laboratory exercises. These assignments provided a means to assess learning outcomes, coupled with course evaluations. Overall, this experience may serve as a template for those interested in study abroad course development and research opportunities in the neurosciences.

Neuroscience study abroad: developing a short-term summer course.

Although powerful bioinformatics tools are available for free on the web and are used by neuroscience professionals on a daily basis, neuroscience students are largely ignorant of them. This Neuroinformatics module weaves together several bioinformatics tools to make a comprehensive unit. This unit encompasses quantifying a phenotype through a Quantitative Trait Locus (QTL) analysis, which links phenotype to loci on chromosomes that likely had an impact on the phenotype. Students then are able to sift through a list of genes in the region(s) of the chromosome identified by the QTL analysis and find a candidate gene that has relatively high expression in the brain region of interest. Once such a candidate gene is identified, students can find out more information about the gene, including the cells/layers in which it is expressed, the sequence of the gene, and an article about the gene. All of the resources employed are available at no cost via the internet. Didactic elements of this instructional module include genetics, neuroanatomy, Quantitative Trait Locus analysis, molecular techniques in neuroscience, and statistics-including multiple regression, ANOVA, and a bootstrap technique. This module was presented at the Faculty for Undergraduate Neuroscience (FUN) 2011 Workshop at Pomona College and can be accessed at http://mdcune.psych.ucla.edu/modules/bioinformatics.

Teaching neuroinformatics with an emphasis on quantitative locus analysis.

Wolfram syndrome (WFS) is a progressive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. WFS1 and WFS2 are caused by recessive mutations in the genes Wolfram Syndrome 1 (WFS1) and CDGSH iron sulfur domain 2 (CISD2), respectively. To explore the function of CISD2, we performed genetic studies in flies with altered expression of its Drosophila orthologue, cisd2. Surprisingly, flies with strong ubiquitous RNAi-mediated knockdown of cisd2 had no obvious signs of altered life span, stress resistance, locomotor behavior or several other phenotypes. We subsequently found in a targeted genetic screen, however, that altered function of cisd2 modified the effects of overexpressing the fly orthologues of two lysosomal storage disease genes, palmitoyl-protein thioesterase 1 (PPT1 in humans, Ppt1 in flies) and ceroid-lipofuscinosis, neuronal 3 (CLN3 in humans, cln3 in flies), on eye morphology in flies. We also found that cln3 modified the effects of overexpressing Ppt1 in the eye and that overexpression of cln3 interacted with a loss of function mutation in cisd2 to disrupt locomotor ability in flies. Follow-up multi-species bioinformatic analyses suggested that a gene network centered on CISD2, PPT1 and CLN3 might impact disease through altered carbohydrate metabolism, protein folding and endopeptidase activity. Human genetic studies indicated that copy number variants (duplications and deletions) including CLN3, and possibly another gene in the CISD2/PPT1/CLN3 network, are over-represented in individuals with developmental delay. Our studies indicate that cisd2, Ppt1 and cln3 function in concert in flies, suggesting that CISD2, PPT1 and CLN3 might also function coordinately in humans. Further, our studies raise the possibility that WFS2 and some lysosomal storage disorders might be influenced by common mechanisms and that the underlying genes might have previously unappreciated effects on developmental delay.

Genetic studies in Drosophila and humans support a model for the concerted function of CISD2, PPT1 and CLN3 in disease.

Fruit flies of the genus Drosophila have been an attractive and effective genetic model organism since Thomas Hunt Morgan and colleagues made seminal discoveries with them a century ago. Work with Drosophila has enabled dramatic advances in cell and developmental biology, neurobiology and behavior, molecular biology, evolutionary and population genetics, and other fields. With more tissue types and observable behaviors than in other short-generation model organisms, and with vast genome data available for many species within the genus, the fly's tractable complexity will continue to enable exciting opportunities to explore mechanisms of complex developmental programs, behaviors, and broader evolutionary questions. This primer describes the organism's natural history, the features of sequenced genomes within the genus, the wide range of available genetic tools and online resources, the types of biological questions Drosophila can help address, and historical milestones.

Christopher Korey

Department of Biology

College of Charleston

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Christopher Korey

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College of Charleston

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Department of Biology