Transcript shortridge

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
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Reduced
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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