Using zebrafish in human disease research: some advantages, disadvantages and ethical considerations.

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1 Using zebrafish in human disease research: some advantages, disadvantages and ethical considerations. Svanhild Nornes Morgan Newman Michael Lardelli - Uni. Of Adelaide Giuseppe Verdile Ralph Martins - The McCusker Foundation for Alzheimer s Disease Research

2 Zebrafish biology including advantages Disadvantages (Ethical considerations) Examples of unique utility from our work

3 Zebrafish (Danio rerio) - Biology Freshwater teleost from northern India ~ 3cm adult length Lifespan = years Most fertile period = 3 9 months Fertile temperature ~ 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 University of Oregon, , Eugene, Oregon. The Exploratorium,

4 Zebrafish (Danio rerio) - Biology Freshwater teleost from northern India ~ 3cm adult length Lifespan = years Most fertile period = 3 9 months Fertile temperature ~ 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,

5 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.

6 Zebrafish Other advantages Relatively low cost High density culture Visually fascinating (very media friendly)! The Exploratorium, The Exploratorium, The Exploratorium,

7 Chemical genomics Screen for compounds that modify a disease model Or Screen for mutations that modify response to a compound

8 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. )

9 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!

10 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

11 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.

12 Zebrafish (Danio rerio) Ethical considerations Consciousness / brain size Adult 0.1g (goldfish) Embryo 10,000 neurons Rat, 2 g Human 1,300 g Ant, 10, ,000 neurons Drosophila, 100,000 Honey Bee, 850,000 Rat, 2x10 7 Human, 1x10 11

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

14 Grandel et al., 2006, Dev Biol 295: Hill and Walsh, 2005, Nature 437: 64-7

15 Alzheimer'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 onset of > 65 years

16 Neuropathology of AD Amyloid plaques Aggregates of the amyloid beta (Aβ) peptide Neurofibrillary tangles (NFT) Aggregates of phosphorylated Tau NFT Amyloid plaque Extensive loss of neurons

17 Genes 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 APOE Allele 4 is a genetic risk factor for sporadic AD

18 Three types of protein change due to gene mutation normal changed amino acid ( missense ) Common (>160 in PSEN1) internal deletion (e.g. splicing error) Rare (2 in PSEN1) Truncation (e.g. non-sense mutation or splicing error) Common (but NONE ever found in PSEN1)

19 160 amino acid changes in PSEN1 (red dots)

20 Two internal deletions in PSEN1 We tried to recreate these in zebrafish embryos

21 Phenotypic effects of injection of a morpholino designed to create an internal deletion in PSEN1 Negative control (normal) Failed attempt to create internal deletion still has VERY strong PSEN1 loss phenotype! At 48 hours post fertilization: Hydrocephalus Pigmentation loss

22 After much testing we found that our attempts to create internal deletions had, instead, created small amounts of truncations normal changed amino acid ( missense ) Common (160+ in PSEN1) internal deletion (e.g. splicing error) Rare (2 in PSEN1) Truncation (e.g. non-sense mutation or splicing error) Common (none in PSEN1)

23 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. normal changed amino acid ( missense ) Common (160+ in PSEN1) internal deletion (e.g. splicing error) Rare (2 in PSEN1) Truncation (e.g. non-sense mutation or splicing error) Common (none in PSEN1)

24 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

25 Could truncated PSEN1 protein contribute to sporadic Alzheimer s s disease? We 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?

26 Thanks for support from: NHMRC Cancer Council SA CMGD