MEDMIN16 – Part 3
Animal models for neurological diseases
Erik Storkebaum, 07/11/2019, 13.30-15.15
Learning outcomes:
- Explain which genetic strategies can be used to generate an animal model, as well as pros
and cons of each strategy.
- Know which parameters to consider for selecting a genetic strategy and apply this knowledge
to a given inherited neurological disease.
- Have a general insight on how to phenotype animal models for neurological diseases
- Know which organisms are frequently used to model neurological diseases, as well as their
pros and cons
- Be able to give and overview of genetic methods used to generate animal models.
Characteristics of a good disease model
A perfect disease model recapitulates the molecular mechanisms underlying the human disease
(=molecular pathogenesis) = primary criterion
→ (i) it recapitulates the key disease feature
(ii) it responds to (drug) treatments in the same way as human patients = ‘surrogate’ criteria
You don’t know the underlying cause, so in the end you hope to know the underlying molecular
mechanisms.
The more the disease is recapitulated in the animal model, the more you are recapitulating the
molecular pathogenesis.
Sporadic versus familial forms of disease
Gene that gives rise to PD: 15-20 genes that can be the genetic cause for PD.
Inherited forms of disease are good to create in animal models.
Develop animal model for sporadic form: genetically manipulate the mouse, to induce exactly the
same mutation. This is more like an inherited form, it is not easy to make an animal model for a
sporadic form.
Disease:
Sporadic/idiopathic: no family history
Familial/inherited: caused by a mutation in a single gene
For many diseases, a fraction of patients has a sporadic form, and a fraction has a familial form (often
the minority), e.g. AD, PD, ALS.
Some diseases are all genetic/familial, e.g. Charcot-Marie-Tooth disease, Huntington’s disease.
Inheritance pattern of monogenic disease
First question: what is the inheritance pattern?
- Dominant inheritance
- Recessive inheritance
Dominant: when a mutation in one of two gene copies (heterozygosis) is sufficient to cause disease.
Recessive: when both gene copies have to be mutant to cause disease (homozygosis).
Remark: X-linked versus autosomal inheritance
Types of mutation
Missense mutation: a mutation in the coding region of a gene that results in the exchange of one
amino acid by another one.
1
,Nonsense mutation: a mutation in the coding region of a gene that results in the creation of a stop
codon → production of a truncated protein or nonsense-mediated mRNA decay.
Frameshift mutation: when nucleotides are inserted or deleted in the coding region of a gene,
resulting in a shift in the reading frame.
In-frame deletion: small vs large deletion.
In-frame insertion: → one or more additional amino acids.
Splice site mutation: results in intron retention or altered inclusion/exclusion of an exon.
Duplication: of one or more genes.
Nucleotide repeat expansion.
Effect of mutation on protein function
Haploinsufficiency: one copy of the good gene (heterozygous) is not enough to be healthy.
Dominant-negative function: 25% of healthy functional genes, is not enough to be healthy.
Loss-of-function:
Full loss-of-function (amorph)
Partial loss-of-function (hypomorph)
Special cases:
- Haploinsufficiency
- dominant-negative function (antimorph)
Gain-of-function
Gain of wild type function (hypermorph)
Gain of toxic function (neomorph)
In general, a disease-causing mutation can result in loss of protein function either by reducing or
abolishing protein expression, or, when the protein is still expressed, by disrupting the normal,
physiological function of the protein.
Haploinsufficiency: when disease is caused by loss of 50% of gene function.
Dominant negative mechanism: when the mutation results in loss of function, not only of the mutant
protein, but also of a fraction of the wild type protein, produced by the wild type allele in a
heterozygous patient. This can occur e.g. when proteins function as homodimers or multimers.
Gain of toxic function: when the mutation results in the acquisition of a novel ‘function’ (often a novel
protein-protein interaction) that the wild type protein does not possess, and which is toxic to the
affected cell types.
Note: in some cases, a combination of disease mechanisms is possible. E.g. a mutation can result in
both a (partial) loss of protein function and a gain of toxic function.
Mentimeter
What are the possible effects of … on protein function?
2
,Genetic strategies for generating an animal disease model
Knock-out model: Only when you think the disease is caused by a full loss of function.
RNAi = RNA interference
BAC = Bacterial artificial chromosomes
Knock-in (KI) model: introduction of disease-causing mutations in the orthologous gene.
Knock-out (KO) model: inactivation of the orthologous disease gene.
o Full body knock-out
o ‘conditional’ knock-out (tissue-specific and temporal control).
Knock-down (KD) model: using transgenic RNAi, resulting in partial loss of function.
Overexpression (OE) model: overexpression of the disease gene, typically with disease
mutations.
o cDNA transgene
o genomic transgene
o duplication (e.g. BAC transgene)
Knock-out: protein is no longer expressed → results in full loss of protein function.
Knock-down: expression of the protein is (severely) reduced, but typically some residual protein is
expressed → results in partial loss of protein function.
Which parameters to consider for selection of a genetic strategy?
Most important step: 4 – effect of disease mutation on protein function
1. Inheritance pattern:
dominant versus recessive
autosomal versus X-linked
2. Location of the mutation:
in coding region
in regulatory region (e.g. promoter, intron)
3. Nature of the mutation:
missense mutation
nonsense mutation
frameshift mutation
deletion
insertion
splice site mutation
duplication
nucleotide repeat expansion
4. Effect of disease mutation on protein function (often not known!):
loss-of-function:
o full loss-of-function (amorph)
o partial loss-off-function (hypomorph)
o special cases:
Haploinsufficiency
Dominant-negative function (antimorph)
Gain-of-function:
o Gain of wild type function (hypermorph)
o Gain-of-toxic-function (neomorph)
Which genetic strategy when?
You have to be able to do the reasoning in the table on the exam! The WG assignment is an example
of an exam question.
3
, Pros & cons of different genetic strategies
Knock-in: very safe choice
Genetic strategy Pros Cons
knock-in - identical genetic situation as in patients - technically challenging and time consuming
- no need to know the effect of the disease - risk of no phenotype e.g. adult-onset
mutation on protein function upfront neurodegenerative diseases; human/primate-
specific function of disease protein
- whole body mutant (but can be made
conditional)
knock-out - you will learn about the physiological function of - only good model if disease is caused by full
the disease gene loss of gene function (note: haplo-
- may be technically easier to generate than KI, but insufficiency)
still challenging - risk of no phenotype or embryonic lethality
- can be rendered cell-type/tissue specific
knock-down - not full loss of function, thus suited to model - level of knock-down?
partial loss of function - potential off-target effects
- technically easier to generate than KI/KO
- can be cell-type/tissue specific or ubiquitous
overexpression - relatively easy to generate - transgene expression levels? → need to
- human disease protein can be expressed generate control animal expressing the wild
- can be cell-type/tissue specific or ubiquitous type disease protein at similar levels
- only suited to model gain of function or
dominant negative disease mechanisms
How to phenotype animal models?
Disease features you wish to recapitulate in the animal model:
Clinical features:
o Examples: motor deficits, memory dysfunction, behavioral deficits
o Evaluate by suited behavioral tests
Neuropathological hallmarks:
o Examples: neuronal degeneration and loss, ‘inclusion’ formation e.g. amyloid beta
plaques (AD), Lewy bodies (PD), TDP-43-containing aggregates (ALS)
o Evaluated by histological analyses
4
Animal models for neurological diseases
Erik Storkebaum, 07/11/2019, 13.30-15.15
Learning outcomes:
- Explain which genetic strategies can be used to generate an animal model, as well as pros
and cons of each strategy.
- Know which parameters to consider for selecting a genetic strategy and apply this knowledge
to a given inherited neurological disease.
- Have a general insight on how to phenotype animal models for neurological diseases
- Know which organisms are frequently used to model neurological diseases, as well as their
pros and cons
- Be able to give and overview of genetic methods used to generate animal models.
Characteristics of a good disease model
A perfect disease model recapitulates the molecular mechanisms underlying the human disease
(=molecular pathogenesis) = primary criterion
→ (i) it recapitulates the key disease feature
(ii) it responds to (drug) treatments in the same way as human patients = ‘surrogate’ criteria
You don’t know the underlying cause, so in the end you hope to know the underlying molecular
mechanisms.
The more the disease is recapitulated in the animal model, the more you are recapitulating the
molecular pathogenesis.
Sporadic versus familial forms of disease
Gene that gives rise to PD: 15-20 genes that can be the genetic cause for PD.
Inherited forms of disease are good to create in animal models.
Develop animal model for sporadic form: genetically manipulate the mouse, to induce exactly the
same mutation. This is more like an inherited form, it is not easy to make an animal model for a
sporadic form.
Disease:
Sporadic/idiopathic: no family history
Familial/inherited: caused by a mutation in a single gene
For many diseases, a fraction of patients has a sporadic form, and a fraction has a familial form (often
the minority), e.g. AD, PD, ALS.
Some diseases are all genetic/familial, e.g. Charcot-Marie-Tooth disease, Huntington’s disease.
Inheritance pattern of monogenic disease
First question: what is the inheritance pattern?
- Dominant inheritance
- Recessive inheritance
Dominant: when a mutation in one of two gene copies (heterozygosis) is sufficient to cause disease.
Recessive: when both gene copies have to be mutant to cause disease (homozygosis).
Remark: X-linked versus autosomal inheritance
Types of mutation
Missense mutation: a mutation in the coding region of a gene that results in the exchange of one
amino acid by another one.
1
,Nonsense mutation: a mutation in the coding region of a gene that results in the creation of a stop
codon → production of a truncated protein or nonsense-mediated mRNA decay.
Frameshift mutation: when nucleotides are inserted or deleted in the coding region of a gene,
resulting in a shift in the reading frame.
In-frame deletion: small vs large deletion.
In-frame insertion: → one or more additional amino acids.
Splice site mutation: results in intron retention or altered inclusion/exclusion of an exon.
Duplication: of one or more genes.
Nucleotide repeat expansion.
Effect of mutation on protein function
Haploinsufficiency: one copy of the good gene (heterozygous) is not enough to be healthy.
Dominant-negative function: 25% of healthy functional genes, is not enough to be healthy.
Loss-of-function:
Full loss-of-function (amorph)
Partial loss-of-function (hypomorph)
Special cases:
- Haploinsufficiency
- dominant-negative function (antimorph)
Gain-of-function
Gain of wild type function (hypermorph)
Gain of toxic function (neomorph)
In general, a disease-causing mutation can result in loss of protein function either by reducing or
abolishing protein expression, or, when the protein is still expressed, by disrupting the normal,
physiological function of the protein.
Haploinsufficiency: when disease is caused by loss of 50% of gene function.
Dominant negative mechanism: when the mutation results in loss of function, not only of the mutant
protein, but also of a fraction of the wild type protein, produced by the wild type allele in a
heterozygous patient. This can occur e.g. when proteins function as homodimers or multimers.
Gain of toxic function: when the mutation results in the acquisition of a novel ‘function’ (often a novel
protein-protein interaction) that the wild type protein does not possess, and which is toxic to the
affected cell types.
Note: in some cases, a combination of disease mechanisms is possible. E.g. a mutation can result in
both a (partial) loss of protein function and a gain of toxic function.
Mentimeter
What are the possible effects of … on protein function?
2
,Genetic strategies for generating an animal disease model
Knock-out model: Only when you think the disease is caused by a full loss of function.
RNAi = RNA interference
BAC = Bacterial artificial chromosomes
Knock-in (KI) model: introduction of disease-causing mutations in the orthologous gene.
Knock-out (KO) model: inactivation of the orthologous disease gene.
o Full body knock-out
o ‘conditional’ knock-out (tissue-specific and temporal control).
Knock-down (KD) model: using transgenic RNAi, resulting in partial loss of function.
Overexpression (OE) model: overexpression of the disease gene, typically with disease
mutations.
o cDNA transgene
o genomic transgene
o duplication (e.g. BAC transgene)
Knock-out: protein is no longer expressed → results in full loss of protein function.
Knock-down: expression of the protein is (severely) reduced, but typically some residual protein is
expressed → results in partial loss of protein function.
Which parameters to consider for selection of a genetic strategy?
Most important step: 4 – effect of disease mutation on protein function
1. Inheritance pattern:
dominant versus recessive
autosomal versus X-linked
2. Location of the mutation:
in coding region
in regulatory region (e.g. promoter, intron)
3. Nature of the mutation:
missense mutation
nonsense mutation
frameshift mutation
deletion
insertion
splice site mutation
duplication
nucleotide repeat expansion
4. Effect of disease mutation on protein function (often not known!):
loss-of-function:
o full loss-of-function (amorph)
o partial loss-off-function (hypomorph)
o special cases:
Haploinsufficiency
Dominant-negative function (antimorph)
Gain-of-function:
o Gain of wild type function (hypermorph)
o Gain-of-toxic-function (neomorph)
Which genetic strategy when?
You have to be able to do the reasoning in the table on the exam! The WG assignment is an example
of an exam question.
3
, Pros & cons of different genetic strategies
Knock-in: very safe choice
Genetic strategy Pros Cons
knock-in - identical genetic situation as in patients - technically challenging and time consuming
- no need to know the effect of the disease - risk of no phenotype e.g. adult-onset
mutation on protein function upfront neurodegenerative diseases; human/primate-
specific function of disease protein
- whole body mutant (but can be made
conditional)
knock-out - you will learn about the physiological function of - only good model if disease is caused by full
the disease gene loss of gene function (note: haplo-
- may be technically easier to generate than KI, but insufficiency)
still challenging - risk of no phenotype or embryonic lethality
- can be rendered cell-type/tissue specific
knock-down - not full loss of function, thus suited to model - level of knock-down?
partial loss of function - potential off-target effects
- technically easier to generate than KI/KO
- can be cell-type/tissue specific or ubiquitous
overexpression - relatively easy to generate - transgene expression levels? → need to
- human disease protein can be expressed generate control animal expressing the wild
- can be cell-type/tissue specific or ubiquitous type disease protein at similar levels
- only suited to model gain of function or
dominant negative disease mechanisms
How to phenotype animal models?
Disease features you wish to recapitulate in the animal model:
Clinical features:
o Examples: motor deficits, memory dysfunction, behavioral deficits
o Evaluate by suited behavioral tests
Neuropathological hallmarks:
o Examples: neuronal degeneration and loss, ‘inclusion’ formation e.g. amyloid beta
plaques (AD), Lewy bodies (PD), TDP-43-containing aggregates (ALS)
o Evaluated by histological analyses
4
