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Nervous System Function in Drosophila Randall D. Shortridge, Ph.D. Biological Sciences, SUNY-Buffalo 645-2363 ext 137 Cooke Hall, Room 336 [email protected] Project Collaborators Randall D. Shortridge, Ph.D., Biological Sciences, SUNY-Buffalo; Molecular biology, transgenics, immunology, biochemistry, bioinformatics. Satpal Singh, Ph.D., Pharmacology and Toxicology, SUNY-Buffalo; Electrophysiology, pharmacology, physiology, genetics. Why study the nervous system? movie Neuronal Disease • Diverse causes and common occurrence • Severely impacts the quality of life • Understanding causes can lead to treatments or cure Why use fruit flies to study the nervous system? 1. Relevance Studies can reveal complex processes in humans. 2. Amenable to a variety of research approaches Genetics, behavioral analysis, transgenics, molecular biology, biochemistry, electrophisiology, immunology. From: Genome Research, 2001, 11(6), 1114-25. 75% of Human disease genes listed in the Online Mendilian Inheritance in Man (OMM) database have counterparts in Drosophila melanogaster. “In essence, we are nothing but a big fly,” Dr. Charles Zuker, Professor of Biology at UCSD – reported by ABC News Maggie Fox, March 23, 2003 Brundlefly in The Fly, 1986, starring Jeff Goldblum, Geena Davis Drosophila Genetics 1. Drosophila is the best genetically-characterized Metazoan. 2. Easy to induce mutations and isolate mutant lines. 3. Easy to transfer genes back into fly (transgenics). 4. Thousands of mutants available in stock centers. 5. Sophisticated mutant lines available for use in research studies (eg. enhancer-traps). 6. Allows a correlation of gene to function (functional genetics). Forward Genetics; Functional Genetics Genetic-Molecular approach (Drosophila) allows a direct correlation of specific genes to their in vivo function. Organism (mutant phenotype) Tissue Gene product Functional lesion Genetics and the making of FrankenFly Bride of Frankenstein, Universal Studios, 1933 Cover of Science, March 24, 1995 Eye on wing Eye on leg Eye on antenna Halder et al., (1995) Science, 267:1788 The Nobel Prize in Physiology or Medicine 1995 "for their discoveries concerning the genetic control of early embryonic development" Edward B. Lewis Christiane NüssleinVolhard Eric F. Wieschaus California Institute of Technology Pasadena, CA, USA Max-Planck-Institut für Entwicklungsbiologie Tübingen, Federal Republic of Germany Princeton University Princeton, NJ, USA b. 1918 b. 1942 b. 1947 Basic Strategy 1. Isolate Drosophila mutations that disrupt function of the nervous system (TS paralysis). 2. Identify the genes involved. 3. Determine the mechanism of gene expression in the nervous system. The power of this approach is that it allows a direct correlation of specific gene products to their in vivo function. Equipment used in Temperature-Sensitive (TS) Paralytic Assays Aquarium with heater Flies Worker Stopwatch Writing pad Pencil Temperature-Sensitive (TS) Paralytic Mutant Assays movie TS mutants are identified by paralysis at 38oC. Convention for naming TS paralytic mutants Drosophila TS mutants are named after individuals associated with the 1991-1994 Buffalo Bills football team (eg. Norwood, Levy, Andre, Kelly, Thurmond). Scott Norwood’s game-ending FG miss in Superbowl XXV Kelly was intercepted four times in Superbowl XXVI Approximately 30 total TS alleles isolated 5 being actively studied at the present time. Some TS paralytic mutants are bang-sensitive movie Bang-sensitive assay: Vortex flies for 10 seconds then time paralytics for recovery. Recovery times vary among mutants from a few seconds to 20 minutes. TS paralytic mutants and altered heart rates Larvae Normal movie Reduced movie Paralytic mutants under active study Ion Current Heart Rates Bang Sensitivity levy Yes (COX VIa subunit) Ca2+ K+ 140% Yes kelly Yes (novel) Ca2+ only 80% No norwood Yes (novel) K+ only 100% No andre 1 of 2 (novel) Ca2+ only 120% Yes thurmond 1 of 11 (novel) Ca2+ only 122% No Mutation Gene Identified? Genetic Analysis of the levy1 Mutation DNA sequence analysis of levy1 identifies a mutation in the CG17280 gene CG17280: 743 nucleotides, 2 exons, 1 intron, 109 amino acids, 47% identical to the human COX subunit VIa precursor WT A.A cDNA levy cDNA A.A 32 44 …S G G Y K V W K R L S F F ...TCTGGTGGCTACAAGGTGTGGAAGCGCCTGTCCTTCTTC ...TCTG--TGGCTACAAGGTGTGGAAGCGCCTGTCCTTCTTC ....S V A T R C G S A C P S S 32 44 Exon-exon boundary WT A.A cDNA levy cDNA A.A 45 56 V A V P A V G L C M L N... GTGGCCGTGCCCGCCGTGGGACTGTGCATGCTGAAC... GTGGCCGTGCCCGCCGTGGGACTGTGCATGCTGAAC... W P C P P W D C A C Stop 45 54 The levy1 mutation is predicted to cause a highly truncated form of the CG17280 (COX VIa) protein STOP WT Exon 1 Exon 2 Intron levy1 mutation alters splicing at intron-exon junction, resulting in a frame-shift defect. STOP levy1 Exon 1 The predicted transcript is detected by DNA sequencing after RT-PCR in levy1 mutant. Rescue of levy mutant defect by gene therapy Transgenic Drosophila: 1. Insert gene into transposable element. 2. Inject modified DNA into early embryo. 3. Isolate fly carrying DNA insert. CG17280 (levy) inject DNA construct levy embryo levy mutant expressing CG17280 (transformant) levy transformants are not TS paralytic Levy mutation shows neurodegeneration “Swiss cheese” effect in brain Drastic reduction in life span Levels of COX activity is reduced by the levy1 mutation, but restored in transformants 2.00 1.50 1.00 TFc2 TFc1 TF2 TF1 levy / + 0 levy 0.5 WT (CS) COX Activity (nmol/min/mg) 2.50 ATP is reduced in levy1 after paralysis at 38oC 120 100 40 TFc TF levy 0 2 days old adults TFc 20 TF 20 60 levy 40 80 WT (CS) 60 0 21oC ATP level (% CS) 80 WT (CS) ATP level (% CS) 120 100 After 38oC, 5 min (paralytic) ATP is reduced in levy1 mutants by age TFc 2 days old adults TFc 20 0 TF levy 20 40 TF 40 60 levy 60 80 WT (CS) ATP level (% CS) 80 0 21oC 120 100 WT (CS) ATP level (% CS) 120 100 30 days old adults Age-dependent Bang Sensitivity of levy1 (21oC) Mitochondrial Respiration Machinery (Oxidative Phosphorylation) levy is a part of Complex IV (Cyt C Oxidase) Ox-Phos Figure from Alberts et al., MB of the Cell, 4e. Gene contributions to assembling the mitochondrial COX complex Fig from: R. Poyton, Nature Genetics 1998, 20:316 levy mutation Cytochrome C Oxidase (COX) Deficiency Fatal Infantile Myopathy Benign Infantile Myopathy Hypotonia Weakness Respiratory distress Renal tubular defect: glycosuria, amino aciduria, etc Mitochondrial myopathy with selective absence of subunits VII a/b of COX Hypotonia Weakness Respiratory insufficiency Spontaneous recovery by age 2-3 Mitochondrial myopathy with selective absence of subunits VII a/b and II of COX Leigh's Syndrome (Subacute Necrotizing Encephalomyelopathy) Hypotonia Episodic vomiting and feeding problems Loss of motor and verbal skills Hearing and visual loss Spasticity Muscle biopsy normal except for COX deficiency with absence of all subunits Leigh’s Disease Synonyms: • Leigh Necrotizing Encephalopathy • Leigh’s Syndrome • Necrotizing Encephalomyelopathy of Leigh’s • SNE • Subacute Necrotizing Encephalopathy Symptoms: • Classical Leigh’s has onset after 3 months of age, but adult onset possible. • 50% survival to 3 years; less than 20% by mid teens • Progressive neurodegeneration • Loss of motor skills is first noticeable trait • Lack of muscle tone (hypotonia); clumsiness; tremors; absence of reflexes; muscle spasms; seizures; visual disturbance; heart enlargement; partial paralysis, mental retardation. • Increased CO2 in blood; lactic acidosis Causes: • COX deficiency • Lesion in non-COX Ox-Phos proteins Closely Related Disorders: • Autism • Alzheimer's levy Mutant and Leigh’s Disease Parallels 1. Cytochrome C oxidase (COX) deficiency. 2. Muscular dysfunction (TS paralysis) 3. Brain (neuronal) degeneration. 4. Reduced life span. 5. Motor defects (paralysis; bang sensitivity; climbing behavior?; jumping ability?) 6. Visual dysfunction (?) 7. Ion channel regulatory defects (?) Examples of Available Ph.D. or M.S Research Projects 1. What gene encodes the IKF (potassium) channel? Use RNAi to knockout candidate genes (8). 2. Will levy’s brother substitute for levy to rescue paralysis in the mutant? Construct hybrid genes and transfer back into mutant lines. 3. What are the expression profiles of levy and levy’s brother? Are they tissue-specific like in humans? RT-PCR, Northerns, epitope tags, transformants expressing reporter constructs. 4. Identify mutation in the thurmond gene. PCR amplify 10 candidate genes from mutant and compare sequence to wildtype. 5. Identify mutant in andre (2 candidate genes) or in 25 other TSparalytic mutant lines. (same as above). A multidisciplinary approach is used in analyzing the nervous system 1. Electrophysiology 2. Pharmacology 3. Genetics 4. Immunology 5. Molecular Biology 6. Transgenics 7. Physiology 8. Biochemistry 9. Bioinformatics The Far Side In the Fly House of Horrors