Animal Models

Question 1

Intro

Answer 1

Drosophilia:

• C. elegans are the only organism for which the entire connectome (complete set of synaptic connections) has been deciphered —> invaluable for guided developmental and neural circuit research
• C. elegans has a transparent body which is great for developmental and imaging studies
• *Rodents and nonhuman primates**
• mouse specifically are the only mammals for which the production of transgenic and knockout animals is routine —> facilitates genetic manipulation and allows genetically identified neuronal populations to be recorded and manipulated
• historically, rats were used more for behavioural paradigms while mice were use for genetic manipulation

Question 2

Forward Genetics

Answer 2

= traces an observed phenotype to a gene

—> screens use random mutagenesis to identify genes that control complex biological processes

• a series of unknown genes play a role in the process of interest —> can use chemical mutagens, radiation, or transposon insertion to mutagenize a population of animals —> each animal carries a different set of random mutations of a single gene or few genes —> screen for mutations that disrupt the biological process of interest based on the phenotypes exhibited by the offspring
• particularly powerful in tackling problems for which the cellular and molecular pathways are poor understood
  
  • identifies genes involved in a specific process without any bias or knowledge as to what is expected

Question 3

Reverse Genetics

Answer 3

= follows a gene to its associated phenotype

—> disrupting a pre-designated gene

• with genome sequences completed for most model organisms, researchers can use databases to identify genes that may perform certain functions based on expression patterns and predicted protein sequences
• essentially hypothesis driven
• to test function of candidate gene, you must create a loss-of-function mutation and examine the phenotypes of the animals (deleting the gene) —> is this gene indeed necessary for what we think it is
• most popular technique for gene deletion is homologous recombination
  
  • piece of endogenous DNA is replaced by a piece of in vitro engineered DNA that have identical end sequences
  • knockout = type of homologous recombination
  • many variations of knockout now exist, making the technique more versatile - knock-in
    
    • e.g. single nucleotides can be made to test the contribution of specific AA residues to protein function
    • knock-in mouse can express a marker gene

Question 4

Conditional knockout mice more detailed

Answer 4

• first developed using the Cre/LoxP system
• Cre recombinase is an enzyme that catalyzes recombination between two sequence-specific DNA elements called loxP sites
• when two loxP sites are in the same orientation, a recombination event will delete the intervening sequence

—> hundreds of transgenic Cre lines with different patterns of Cre expression have been generated —> gene deletion only occurs after the Cre transgene is first expressed

Question 5

Animal models of neurodegenerative diseases (overview)

Answer 5

—> animal models of adult-onset neurodegenerative diseases have enhanced the understanding of the molecular pathogenesis of AD, PD etc.

• genetic forms of the human disorders do not perfectly phenocopy diseases, but in many cases are still excellent surrogates —> can still be used as models of the disorders to provide insight into molecular mechanisms to help identify disease-modifying therapies
• arguably, no animal model fully phenocopies the human disease
• BUT some models develop a more complete neurodegenerative cascade, but it still remains uncertain whether the entire sequence of pathophysiological events that occur in a disease are captured
• animal models have been used to develop, test, and refine therapies BUT animal models have poor predictive power for drug efficacy in human neurodegenerative diseases
  
  • translation failure not always due to shortcomings of the animal model but due to the models being limited in terms of maturation and complexity
    
    • e.g. lack of complex neuronal circuits, full glia complexity, no immunologic components etc.
  —> still need engineered animal models that recapitulate the complexity of an intact nervous system

Question 6

Common Challenges in modelling neurodegenerative disorders in rodents

Answer 6

• potentially limited relevance of models based on expression of rare genetic variants —> may be inappropriate when molecular and cellular events leading to the neurodegeneration differ from human disease
• short life-span of rodents —> may lead to an incomplete development of pathological hallmarks and/or neurodegeneration —> major limitations in the modelling of age-related diseases
• inherent differences in the development and function of rodent and human brains —> need to be cautious when interpreting behavioural deficits in animal models when the human disease is characterized by cognitive, emotional and language deficits
• there are genomic differences between rodent and human e.g. amino acid changes has a different impact in various species —> not always the same
• using inbred animals → does not reflect genetic diversity of a population

Question 7

Animal Models of AD and related disorders

Answer 7

—> modelled either based on amyloid pathology or tau pathology

Amyloid Pathology Models Problems

• even though Aß aggregates can be produced and overexpressed, these models do not show behavioral abnormalities or show only subtle abnormalities
• models mimic some aspects of preclinical asymptomatic AD but lack the tau pathology, robust neurodeeneration, and neurotransmitter abnormalities that are associated with the symptomatic phases of human AD —> animal models which accumulate Aß are not AD models

Tau Pathology Models

• in comparison to the Aß models, the animals exhibit overt neurdegenerative changes
• BUT there is concern whether the tauopathy in these models are relevant to the tauopathy in AD
• a mutant tau transgene expressed in the spinal cord leads to motor phenotype, while expression in forebrain, hippocampus, or entorhinal cortex results in tau pathology and neurodegeneration in those regions —> mutations in tau can drive tau aggregation which produce neurodegeneration
• BUT it is still unclear why only specific neuronal population degenerated in AD although tau is widely expressed throughout the human CNS

Question 8

Animal Models of PD

Answer 8

• characterized by progressive loss of DA neurons in the substantia nigra and misfolded alpha-synuclein in Lewy bodies and neurites
• many different causes of PD have been found, both genetic and environmental

Pharmacological models of PD

• historically used to advance successful therapies for PD —> lead to L-dopa treatment
• effective models for symptomatic therapies for the motor symptoms of PD, but other therapeutic approaches have failed to demonstrate any utility in the identification of disease-modifying therapies

Genetically based models of alpha-synuclein pathology

• models of alpha-synuclein-induced degeneration have allowed the expression of varying degrees of neurodegeneration where animals develop many clinical biochemical feature of PD
• BUT degeneration occurs in the absence of any measurable loss of DA neurons
• BUT PRO: exhibit nonDA deficits such as anxiety, GI issues, and non-DA related motor dysfunction
• absence of DA neuron loss is viewed by some as a major shortcomings of these models

Question 9

Improving Translatability

Answer 9

• one of the primary goals of animal model development is to identify key points in the process of neurodegeneration that represent therapeutic targets
• clinical trials for AD and PD based on therapeutics that showed success in animal models have largely failed —>may be attributed to numerous factors in both preclinical and clinical studies e.g. imperfect animal models, overly optimist interpretation of preclinical data, clinical trials conducted in absence of informative biomarkers
• one major issue: lack of alignment between clinical and preclinical studies—> e.g. it is important to consider timing of treatment initiation and whether it alters the onset or progression of the disease —> if onset is altered rather than progression, then this will likely have no effect in patients that are already affected
• in AD: therapies are only tested EITHER in an Aß model or tau model, but not both; animal models often only at asymptomatic stage leading to therapies failing in humans with symptomatic AD
• Generally: therapies need to be tested in multiple models and we should not rely on results from just one —> likely to fail in clinical trial
• importance of effect size and significance in preclinical studies —> too often small significant effect sizes in preclinical trials are used to justify clinical trials —> BUT unlikely to find evidence of these small effects
• claims about what a model shows should remain conservative —> due to imprecise correlations between models and the extent to which they phenocopy human disease

Question 10

Shortcomings of rodent models for psychiatric disorders especially SCZ

Answer 10

• rodent models replicate only CERTAIN neurophysiological, neuroanatomical and/or behavioral features of genetic mutations implicated in SCZ or ASD —> none fully replicates the complexity of these disorders —> each model provides only a specific facet that then needs to be integrated into a greater whole that reflects the heterogeneity of the disorder
• animal models cannot be used to assess the necessary depth —> how can one research thought processes, perception and abstract learning in animals when they cannot convey these fully through language —> can only assess core features of disorders indirectly with a focus on simpler behavioural and physiological features—> translation to complex human symptomatology not always clear
• induction of disease states in rodents may require pharmacological or other insults which to not accurately replicate causes of psychiatric disorders in humans
• even transgenic approaches may be inaccurate —> a single gene knockout is unlikely to capture the complexity of genetic causes of psychiatric disorders —> genomic landscapes of genes may differ in rodents and humans
• humans and rodents have different lifespans —> may not be appropriately congruent with respect to timeline of disease development
• pharmacology of potential drug treatments may differ in species —> creating false positives and negatives in preclinical studies

Question 11

Behavioral models of depression - adult stress model examples

Answer 11

• learned helplessness
  
  • exposure to uncontrollable stressful like events makes people feel a loss of control and can lead to depressive behavouirs
  • rodents develop deficits in escape, cognitive, and rewarded behvaiours following repeated unavoidable and uncontrollabe shocks
  • helpless behaviour is measured based on performance of active escape test
  • Pros: can be used to measure escape performance of mice with different mutations —> target genes of depression may affect the vulnerability to develop a depressive-like state
  • cons: requires very strong stressors —> ethic concerns; not long lasting
• chronic mild stress
  
  • likely the most valid animal model of depression
  • aims to model chronic depressive-like state that develops over time in response to stress
  • animals are exposed to series of mild and unpredictable stressors during at least two weeks
  • results in long lasting changes of behavioral, neurochemical, neuroimmune, and neuroendocrinological variables —> reversed by chronic but not acute antidepressant treatment
  • can be used to screen and test potential antidepressant compounds and to develop new treatment strategies
  • Pros: most valid model of depression
  • cons: labor intensive, demanding of space, long duration