Model organisms Flashcards
Why do we use animal models in neuroscience research?
> A means to address a scientific question
In vivo vs in vitro: better understanding of the causes, mechanisms, pathways
- from molecule to mind
What is Charles Darwin’s ‘Tree of life’ (1837)?
> Illustrates the phylogenetic relationship
- how knowledge gained in a specific animal relates to others and humans
> Darwin’s idea: animals might be related to each other
What is the phylogenetic relationship?
Relationship between species
What does the phylogenetic tree of animals show?
Phylogenetic relationships can be remote
- e.g. humans are separated from insects by almost 500 million years
- yet, there is still a trace
- > we can learn about humans from insects
What is Darwin’s concept of homology, and homologous structures (1876)?
“The relative position or connection in homologous parts; they may differ to almost any extent in form and size, and yet remain connected together in the same invariable order.”
What are homologous genes/proteins?
Sequence identity between orthologous genes/proteins from different species
-> confirmation of Darwin’s hypothesis of homology
What is structural homology?
Different species have similar structures and structure positions
What are the 6 things we can learn from animal models?
Knowledge and understanding about the function of
1. A gene and its encoded proteins (including various protein isoforms)
- Gene-protein interaction
- Signalling pathway and how it works
- Formation/specification of cell types, tissues and organs
- Circuits and networks in the nervous system
- Genes and circuits in relation to disease
To gain knowledge and understanding from animal models, what do functional studies involve?
- Mutating, inactivating or over expressing a gene/protein
- Finding interacting/binding partners
- Screening for enhancers/suppressors of ‘disease gene/protein’
- Epistasis tests and manipulation of a signalling pathway
- who comes first and next - Targeted activation/inactivation of neural circuits
- Regulation and function of behaviour
What does epistasis refer to?
“Any relationship of nonadditive interaction between two or more genes in their combined effects on a phenotype”
To what extent (in vivo) functional studies are possible in animals and humans?
> Ethics committee approval and Home Office (UK) consent are mandatory requirements for work with vertebrates
> Only cell culture or non-invasive studies with written consent are possible regarding humans
What underlies insect and mammalian brain development?
Conserved genetic program that underlies the formation of an insect and mammalian brain
- even though they are different in size and composition
- some genes in a fly and a mouse show structural similarity, are expressed in similar ways, and seem to have similar functions
- > theme, architectural plan with similarities and dissimilarities
What is Saccharomyces cerevisiae?
Baker’s and brewer’s yeast
What is a yeast?
A eukaryotic cell
- all mammals are made of them
How has baker’s/brewer’s yeast been used scientifically?
To discover genes and their function in the regulation of the cell cycle/division - which is conserved across the animal kingdom
How is the YFP protein used in functional studies?
To highlight the cell body and nucleus
How did Couthouis et al. (2011) use Saccharomyces cerevisiae - yeast?
- TDP-43 and FUS proteins form aggregates and inhibit growth of yeast cultures
-> aggregate formation is associated to the viability of yeast
=> TDP-43 and FUS proteins build toxic aggregates - Researchers used yeast as an expression system to express several RNA binding proteins
- 3 readouts to screen for genes and proteins to form aggregates: cytoplasm diffuse, nucleus diffuse, cytoplasm aggregates - Cytotoxic effect of TAF15:
- increasing amount of the RNA binding protein TAF15 cause aggregate formation
=> TAF15: novel aggregate formation protein
- formation of pellets in Eppendorg tube
-> protein accumulates and precipitates - Human homologue of RNA binding proteinTAF15 forms aggregates
- found in post-mortem ALS patient tissue
=> Screen in yeast lead to the discovery of a novel disease-related gene in humans
What is amyotrophic lateral sclerosis (ALS)?
Progressive nervous system disease causing degeneration of upper and lower motor neurons (through brain, spinal cord and muscles)
- most common form of motor neuron disease
What is Caenorhabditis elegans and what are its characteristics?
The worm:
- simple anatomy
- 959 somatic nuclei
- 302 neurons
- small
- 250 progeny per generation
- easily cultivated
- eats bacteria
- grown on agar plates seeded bacteria
- rapid development (3-day life cycle)
What is a pedigree?
Tree of life starting at the top from one single cell and all derived cells
What did John Sulston receive a Nobel prize for?
Discovery of cell lineages and the clonal origin of cell types and tissues using C. elegans (worms)
What in the C. elegans (worm) makes it possible to study cell fate decisions and how they’re determined?
Invariant lineages that give rise to the animal
What characterises the C. elegans’ generation cycle?
> Short generation cycle
Worms generate lots of progeny
> Larval stages can be interrupted when there’s an overcrowding in the culture or wether there is a shortage of food
> At larval stages L1 to L2, the worm can go into dauer stage
What in the C. elegans makes it an ideal model organism to study the genetic basis of aging?
Worms have a short life cycle and don’t get very old
What are the 2 genetic mutations that regulate the lifespan in C. elegans, found by Kenyon and colleagues (1993)?
daf-2 and daf-16
> daf-2 can inhibit or suppress daf-16
> high levels of daf-16 increase worm’s lifespan
> daf-2 mutation reduces daf-2 activity
-> increased daf-16 -> increased lifespan
What did Gems and Partridge (2013) reveal about the daf-2 genetic mutation?
> daf-2 is an insulin receptor, universally conserved receptor molecule, involved in nutrition and aging
> reduced daf-2 and daf-16 do not increase lifespan
-> daf-2 single mutant must have positive effect on lifespan via the activity of daf-16
> There are homologous genes in Drosophila (fly) and mouse
=> evolutionary conserved pathway involved in longevity and aging
What is Drosophila melanogaster and what are its characteristics?
Fruit fly
- 180 Mb sequenced genome comprising 4 chromosomes encoding approx. 13,600 genes
- 65% structural identity to human genes related to neurological disorders
- Complex brain, behaviour, learning, and memory
- Sophisticated genetic toolbox for molecular, cellular and behavioural studies in vivo
- Major discovery in 1912 in the lab of Thomas Hunt Morgan, Columbia University
What makes Drosophila melanogaster a powerful genetic model organism?
> Short life cycle
> Small size -> multiple laboratory cultures of genetically modified animals (flies)
What in Drosophila melanogaster are powerful genetic tools for cell and circuit manipulations?
> Transactivation system
- e.g. GAL4 / UAS system
> Molecular cloning: you can generate transgenic flies that have an enhancer that drives GAL4
- e.g. enhancer specific to DA neurons: activation of DA neurons -> enhancer is transcriptionally active = recognises the abstract activating sequences of a second set
What is GAL4?
Transcriptional factor from yeast, that can specifically bind to UAS - upstream activating sequence
How is Drosophila used in the study of mental health disorders?
Even at complex level involved in regulation of voluntary movements, there are similar circuits in a mammal and an insect (e.g. Drosophila)
What is the principle of forward genetics?
Find the genetic basis of a phenotype
- mutate genome
- observe mutant phenotype
- Identify protein and gene
What is the principle of reverse genetics?
- Known gene
- Alter its function
- Determine the phenotype resulting from that genetic sequence
How did Hirth (2010) cause induced Parkinsonism in Drosophila?
Modification of Drosophila’s mitochondrial activity - defective in Parkinsonian patients
What is the behavioural phenotype caused by disease-related genes/proteins in Hirth’s study (2010)?
Parkinsonian-like phenotype in Drosophila
- knock-out flies (induced Parkinsonism) take significantly longer to induce movement
What characterises the mitochondrial dysfunction in Drosophila?
It has a much more pronounced effect when inactivated in dopamine neurons vs. other neurons
-> essential clues to the study of Parkinson’s disease
What is Danio rerio and what characterises it?
Zebrafish: it’s a vertebrate
- much closer to us than insects or worms
What are the 6 functional in vivo studies possible in a vertebrate?
- Naturalistic behaviour
- High-speed behaviour tracking
- Computational models
- Functional imaging
- Circuit manipulation
- Genetics
What are the advantages of the zebrafish?
> Transparent embryo
- fast embryonic development
- fast development
> Genetically identical offspring
What is the autism-related gene found by Hoffman and colleagues (2016) and how did they find it?
Mutation of cntnap2a, related to:
- autism in humans
- hyperactivity
- Induced mutation of this gene in zebrafish
- Behaviour observation
- mutant fish is more active at nighttime vs. wild type
What did Biran and Levkowitz (2016) discover in a screen to identify autism gene-related defects, and how?
Phenotypic suppressors of autism gene-related defects
- ASD-linked genes
- cntnap2 gene locus
- cntnap2-deficient zebrafish
- Rest-activity behavioural test
- Estrogenic phenotypic suppressors of hyperactivity
vs. wild type zebrafish - quantitative behavioural profiling
In a functional in vivo study to identify a disease gene-related defects, is the mutant zebrafish made to have the disease?
No
- we study a phenotype expressing an identified disease-related gene
- e.g. the zebrafish isn’t made to be autistic
What is Mus musculus and what are its characteristics?
Mouse
- mammal with social behaviour
- 90% of genes homologous to human
What is the impact of Parkinson’s disease on the brain?
> Loss of nigrostriatal pathway
> Degenerative loss of dopaminergic neurons in the substantia nigra pars compacta (SNc)
What are the symptoms of Parkinson’s disease?
- Shuffling
- Tremor
- Rigidity
- Bradykinesia
What are the input nuclei of the functional (motor) mammalian basal ganglia?
Dorsal striatum: caudate nucleus and putamen
What are the intrinsic nuclei of the functional (motor) mammalian basal ganglia?
- External segment of globus pallidus (GPe)
- Subthalamic nucleus (STN)
What are the output nuclei of the functional (motor) mammalian basal ganglia?
- Substantia nigra pars reticulata (SNr)
- Internal segment of the globus pallidus (GPi)
What is the neuromodulator of the functional (motor) mammalian basal ganglia?
Substantia nigra pars compacta (SNc)
What happens at the functional mammalian basal ganglia in Parkinson’s disease?
Lack of striatal dopamine innervation (from substantia nigra pars compacta)
- dopamine no longer reaches the striatum
How did Kravitz and colleagues (2010, 2014) mediate complex behaviour in a mouse model?
Targeted manipulation of neural circuitry
- Modified adinovirus
- protein ChR2 over-activates neuron when activated by a pulse
- > remote control with light to activate neuron - Researchers injected the virus in mouse’s basal ganglia
- Virus injected with modification, so the gene is altered to be expressed only in D1 (direct) pathway or only in D2 (indirect pathway)
What did Kravitz and colleagues (2010, 2014) show with target manipulation activation and artificially induced Parkinsonian animal model?
> Dopamine antagonist 6-OHDA has depleted DA
= artificially induced Parkinsonian model
-> nigrostriatal pathway is gone with the depleted DA in striatum
> D1 laser on makes the mouse move
-> D1 receptor on striatum is closely related to movement initiation and maintenance
> D2 laser on inhibits movement in the mouse
-> D2 activity inhibits movement
=> D1 and D2 have a crucial role in voluntary movement
=> Activation of D1 can overcome a parkinsonian phenotype
What gives great power to animal models in the study of mental health disorders?
The evolutionary conservation of genes, pathways and their dysfunctions
