SvanhildSvanhild

NornesNornesMorgan NewmanMorgan NewmanMichael Michael LardelliLardelli

--Uni. Of AdelaideUni. Of Adelaide

Using Using zebrafishzebrafish in human disease research:in human disease research:some advantages, disadvantages and ethical some advantages, disadvantages and ethical

considerations.considerations.

Giuseppe Giuseppe VerdileVerdileRalph MartinsRalph Martins

--The The McCuskerMcCusker

Foundation for Foundation for Alzheimer’s Alzheimer’s Disease ResearchDisease Research

••

ZebrafishZebrafish

biology including advantagesbiology including advantages••

DisadvantagesDisadvantages

••

(Ethical considerations)(Ethical considerations)••

Examples of unique utility from our workExamples of unique utility from our work

Zebrafish (Danio rerio) - Biology

•

Freshwater teleost from northern India•

~ 3cm adult length•

Lifespan = 1.5 –

2 years•

Most fertile period = 3 –

9 months•

Fertile temperature ~ 25 –

31 ºC•

Generation time = 2 –

4 months•

Genome size 1.7 Gb

in 25 Chromosomes

Embryos:•

~ 0.5mm embryo diameter•

200 per female per week•

Transparent•

External development•

Rapid, synchronous embryogenesis

•

Hatching at 2 –

3 days•

Dead embryos not resorbed•

Drugs absorbed from medium © The Exploratorium, www.exploratorium.edu

© University of Oregon, 1994-2008,

Eugene, Oregon.

Zebrafish (Danio rerio) - Biology

•

Freshwater teleost from northern India•

~ 3cm adult length•

Lifespan = 1.5 –

2 years•

Most fertile period = 3 –

9 months•

Fertile temperature ~ 25 –

31 ºC•

Generation time = 2 –

4 months•

Genome size 1.7 Gb

in 25 Chromosomes

Embryos:•

~ 0.5mm embryo diameter•

200 per female per week•

Transparent•

External development•

Rapid, synchronous embryogenesis

•

Hatching at 2 –

3 days•

Dead embryos not resorbed•

Drugs absorbed from medium © The Exploratorium, www.exploratorium.edu

Zebrafish (Danio rerio) – Some of the experimental techniques possible

•

Mutation screening – diploid and haploid by X/γ-radiation, chemical or transposon/virus-based insertional mutagensis

•

Cloning – by repeated haploidisation and diploidisation (creates isogenics) or nuclear transfer

•

Blockage of specific protein synthesis in embryos -

by injection of antisense morpholino oligonucleotides

•

Transgenics – Gal4/UAS, GFP, Sleeping beauty transposon vector etc. (Best to use zebrafish promoters including heat shock promoters).

•

In situ immunohistochemistry (variable depending upon antibody)•

In situ transcript hybridisation (excellent and can be scaled up due to high embryo numbers produced by zebrafish)

•

Time lapse microscopy etc. of living embryos•

Arraying of zebrafish/Chemical Genomics in microtitre trays for drug testing on development, behaviour, disease models etc.

•

Conditional ablation of specific cells – using E.coli nitroreductase and metronidazole

•

Storage of genetic lines as frozen sperm.•

TILLING - Targeting Induced Local Lesions in Genomes •

Directed mutagenesis of genes (?!) – using “designed zinc-finger nucleases”.

Zebrafish – Other advantages

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Relatively low cost

•

High density culture

•

Visually fascinating(very media friendly)!

© The Exploratorium, www.exploratorium.edu

© The Exploratorium, www.exploratorium.edu© The Exploratorium, www.exploratorium.edu

Chemical genomics

Screen for compounds that modify a disease model

Or

Screen for mutations that modify response to a compound

A zebrafish mutant strain lacking skin pigmentation, Casper, is useful in transplantation, tumorigenesis and metastasis studies etc.

(Created by Richard White, MD, PhD, a clinical fellow in the Stem Cell Program at Children's Hospital Boston, with others in the laboratory of Leonard Zon, MD. )

The embryo as a “test-cell”

Some advantages of testing molecular interactions in embryos rather than cultured cells :•

Normal gene expression background

•

Easy to perform complex manipulations of genetic state

•

Developmental phenotype indicates change in gene activity !

Zebrafish (Danio rerio) – Disadvantages

•

Non-mammalian physiology –

some drugs affecting zebrafish

do not affect mammals and vice-versa due to e.g. different rates of metabolism

•

Some anatomical differences from mammals

•

Non-placental –

testing drug interactions with placenta not possible

•

Great capacity for regeneration

Zebrafish (Danio rerio) – Ethical considerations

•

Pain –

Pain

perception by fish disputed.

Lynne Sneddon

et al. of Roslin

Institute found neurons responding appropriately to pain-inducing stimuli in fish. They also injected bee venom or acetic acid into lips of rainbow trout and observed behaviours they interpreted as experience of pain (rubbing lips onto stones, increased ventilation rate, rocking) which were greater than in controls. Morphine apparently acted as an analgesic.

James Rose of University of Wyoming was very critical of this study. Criticised Sneddon

et al.’s understanding of definition of “pain”

versus nociception, formulation of experiment (volume of toxin injected) and inconsistency in interpretation of results. He believes fish brain lacks structure for awareness of pain.

Zebrafish (Danio rerio) – Ethical considerations

•

Consciousness / brain size

Adult 0.1g (goldfish) Rat, 2 gHuman 1,300 g

Embryo 10,000 neurons Ant, 10,000 –

100,000 neuronsDrosophila, 100,000Honey Bee, 850,000Rat, 2x107

Human, 1x1011

Hill and Walsh, 2005,Nature 437: 64-7

Grandel et al., 2006, Dev Biol 295: 263-77

Hill and Walsh, 2005,Nature 437: 64-7

Alzheimer's DiseaseAlzheimer's Disease

► Most prevalent form of dementia Australia 2004: 100,000 patients

► Complex molecular changes (not well understood)

► <5% of cases are familial (FAD) with an early onset and autosomal dominant inheritance

► >95% of cases are sporadic with a late onsetof > 65 years

Neuropathology of ADNeuropathology of AD►

Amyloid plaques Aggregates of the amyloid beta (Aβ) peptide

►

Neurofibrillary tangles (NFT)Aggregates of phosphorylated Tau

►

Extensive loss of neurons

NFTAmyloid plaque

http://www.alzheimer.med.br/goetz.htm

Genes Associated with ADGenes Associated with AD

►

Amyloid Precursor Protein (APP) Cleaved to release Aβ peptide

►

Presenilins

(PSEN) PSEN1 and PSEN2 essential for APP cleavage

►

Microtubule-Associated Protein Tau

(MAPT)NFTs contain aggregates of hyperphosphorylated Tau

►

APOEAllele 4 is a genetic risk factor for sporadic AD

Three types of protein changeThree types of protein change due to gene mutationdue to gene mutation

normal

changed amino acid(“missense”)

internal deletion(e.g. splicing error)

Truncation(e.g. non-sense mutation or splicing error)

Common (>160 in PSEN1)

Rare (2 in PSEN1)

Common (but NONE

everfound in PSEN1)

http://www.alzforum.org/res/com/mut/pre/diagram2006.asp

160 amino acid changes in PSEN1 (red dots)

http://www.alzforum.org/res/com/mut/pre/diagram2006.asp

Two internal deletions in PSEN1We tried to recreate these in zebrafish

embryos

Failed attempt to create internal deletion still has VERY strong

PSEN1 loss phenotype!

Negative control (normal)

Phenotypic effects of injection of a morpholino designed to create an internal deletion in PSEN1

At 48 hours post fertilization:•

“Hydrocephalus”•

Pigmentation loss

After much testing we found that our attempts to create After much testing we found that our attempts to create internal deletions had, instead, created small amounts of internal deletions had, instead, created small amounts of truncationstruncations

normal

changed amino acid(“missense”)

internal deletion(e.g. splicing error)

Truncation(e.g. non-sense mutation or splicing error)

Common (160+ in PSEN1)

Rare (2 in PSEN1)

Common (none

in PSEN1)

normal

changed amino acid(“missense”)

internal deletion(e.g. splicing error)

Truncation(e.g. non-sense mutation or splicing error)

Common (160+ in PSEN1)

Rare (2 in PSEN1)

Common (none

in PSEN1)

These mutations were once thought to have no function. We now know that they are so potent that they would kill any developing embryo –

so cannot be found in human families or

in animal mutation screens.

http://www.alzforum.org/res/com/mut/pre/diagram2006.asp

Truncation in yellow region of PSEN1 protein creates very potent “dominant negative” forms

that interfere with normal PSEN1 protein

These observations could not have been made using cultured cells or mice

Could truncated PSEN1 protein contribute to Could truncated PSEN1 protein contribute to sporadic Alzheimersporadic Alzheimer’’s disease? s disease? We know thatWe know that::

•

PSEN1 is the

major site for mutations causing FAD.

•

Most FAD mutations in PSEN1 appear to dominantly reduce

protein activity. i.e. Reduced PSEN1 activity

causes Alzheimer’s disease.

•

Mechanisms are known that can cause protein truncation at low levels in ageing cells. Is decreased PSEN1 activity a “molecular common link”

between inherited and sporadic

Alzheimer’s disease?

Thanks for support from:

NHMRCCancer Council SACMGD