Nervous System Function in Drosophila

Randall D. Shortridge, Ph.D.

645-2363 ext 137Cooke Hall, Room 336

rds@buffalo.edu

Biological Sciences, SUNY-Buffalo

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?

Neuronal Disease • Diverse causes and common occurrence• Severely impacts the quality of life • Understanding causes can lead to treatments or cure

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Why use fruit flies to study the nervous system?

1. Relevance

2. Amenable to a variety of research approachesGenetics, behavioral analysis, transgenics, molecular biology, biochemistry, electrophisiology, immunology.

Studies can reveal complex processes in humans.

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).

Genetic-Molecular approach (Drosophila) allows a direct correlation of specific genes to their in vivo function.

Organism (mutant phenotype)

Gene product

Functional lesion

Tissue

Forward Genetics; Functional Genetics

Bride of Frankenstein, Universal Studios, 1933

Cover of Science, March 24, 1995

Genetics and the making of FrankenFly

Halder et al., (1995) Science, 267:1788

Eye on antenna

Eye on wing

Eye on leg

Edward B. Lewis Christiane Nüsslein-Volhard

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

The Nobel Prize in Physiology or Medicine 1995

"for their discoveries concerning the genetic control of early embryonic development"

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.

Basic Strategy

Equipment used in Temperature-Sensitive (TS) Paralytic Assays

Aquarium with heater

PencilWriting pad

Flies

Stopwatch

Worker

Temperature-Sensitive (TS) Paralytic Mutant Assays

TS mutants are identified by paralysis at 38oC. movie

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).

Approximately 30 total TS alleles isolated

5 being actively studied at the present time.

Scott Norwood’s game-ending FG miss in Superbowl XXV Kelly was intercepted four times in Superbowl XXVI

Some TS paralytic mutants are bang-sensitive

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.

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TS paralytic mutants and altered heart rates

Normal

Reduced

Larvae

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Mutation Gene Identified? Ion

CurrentHeart Rates

Bang Sensitivity

levyYes (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

Paralytic mutants under active study

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

32 44WT A.A …S G G Y K V W K R L S F FcDNA ...TCTGGTGGCTACAAGGTGTGGAAGCGCCTGTCCTTCTTClevy cDNA ...TCTG--TGGCTACAAGGTGTGGAAGCGCCTGTCCTTCTTC A.A ....S V A T R C G S A C P S S 32 44

45 56WT A.A V A V P A V G L C M L N...cDNA GTGGCCGTGCCCGCCGTGGGACTGTGCATGCTGAAC...levy cDNA GTGGCCGTGCCCGCCGTGGGACTGTGCATGCTGAAC...A.A W P C P P W D C A C Stop 45 54

Exon-exon boundary

The levy1 mutation is predicted to cause a highly truncated form of the CG17280 (COX VIa) protein

WT

levy1

Exon 1

Exon 1

Exon 2

Intron

STOP

STOP

levy1 mutation alters splicing at intron-exon junction, resulting in a frame-shift defect.

The predicted transcript is detected by DNA sequencing after RT-PCR in levy1 mutant.

Rescue of levy mutant defect by gene therapy

1. Insert gene into transposable element.

2. Inject modified DNA into early embryo.

3. Isolate fly carrying DNA insert.

Transgenic Drosophila:

levy embryolevy mutant expressing CG17280

(transformant)

levy transformants are not TS paralytic

DNA constructinject

CG17280 (levy)

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

2.50

2.00

1.50

1.00

0.5

0

CO

X A

ctiv

ity (n

mol

/min

/g)

WT

(CS)

levy

TFc2

TFc1

levy

/ +

TF1

TF2

ATP is reduced in levy1 after paralysis at 38oC

120

100

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TP le

vel (

% C

S)

WT

(CS)

levy

TFc

TF

After 38oC, 5 min (paralytic)

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leve

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CS)

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levy

TFc

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21oC 2 days old adults

ATP is reduced in levy1 mutants by age

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ATP

leve

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CS)

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levy

TFc

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21oC 2 days old adults

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TP le

vel (

% C

S)

WT

(CS)

levy

TFc

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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

levy mutationFig from: R. Poyton, Nature Genetics 1998, 20:316

Cytochrome C Oxidase (COX) Deficiency

Fatal Infantile MyopathyHypotoniaWeaknessRespiratory distressRenal tubular defect: glycosuria, amino aciduria, etcMitochondrial myopathy with selective absence of subunits VII a/b of COX

Benign Infantile MyopathyHypotoniaWeaknessRespiratory insufficiencySpontaneous recovery by age 2-3Mitochondrial myopathy with selective absence of subunits VII a/b and II of COX

Leigh's Syndrome (Subacute Necrotizing Encephalomyelopathy)HypotoniaEpisodic vomiting and feeding problemsLoss of motor and verbal skillsHearing and visual lossSpasticityMuscle biopsy normal except for COX deficiency with absence of all subunits

Leigh’s DiseaseSynonyms:

• Leigh Necrotizing Encephalopathy• Leigh’s Syndrome • Necrotizing Encephalomyelopathy of Leigh’s • SNE • Subacute Necrotizing Encephalopathy

Closely Related Disorders:• Autism• Alzheimer's

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

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 wild-type.

5. Identify mutant in andre (2 candidate genes) or in 25 other TS-paralytic 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