Of Mouse and Man: A description of translational research and training at the University of Colorado

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Of Mouse and ManA description of translational
research and training at the University of
ColoradoAllan C. Collins, Ph.D.
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Institute for Behavioral Genetics (IBG)
A division of the Graduate School at the
University of Colorado, Boulder. Faculty and
students come from three campuses, several
departments, and schools.
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• IBGers study genetic and environmental influences
  on the abuse of drugs, with special emphasis on
  tobacco (nicotine) and alcohol.

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• The research route
• Humans
• Mouse (my lab)
• Humans (Dr. Marissa Ehringer)
• Mouse (Dr. Jerry Stitzel)
• Humans in Tanzania (the old guy with white hair)
• i.e., we do translational research.

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Genes, Environment, and Tobacco Use

• A multi-step process that frequently begins
  during adolescence

Initiation (Impulsivity)
Initial response
Continued use
Abuse
Dependence
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Can we learn anything about genetic regulation of
tobacco abuse from studies with humans?
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Genetic strategies that are used in humans

• Twin studies
• Family studies
• Proband studies
• Adoption studies (great at evaluating role for
  ENVIRONMENT).

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Questions that early human genetic studies
partially answered

• Is tobacco abuse heritable? (Yes, h2 0.5-0.8)
• Do environmental factors influence tobacco abuse?
  (Environment is a major factor that influences
  initiation genes influence persistence.)
• Is tobacco abuse influenced by one or many genes?
  (Most likely many, each with a small effect.)
• Are there genes that increase and/or decrease
  tobacco abuse potential? (Yes. Understanding
  genes that decrease vulnerability to addiction
  may be more important than understanding genes
  that promote addiction.)

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Can we learn anything about the genetics of
smoking from a mouse?
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Mouse Researchers Use Two Complementary Genetic
Strategies

• Forward genetics (similar to human research)
• Identify variability in phenotypes
• Attempt to identify genes that cause variability
• Detect associations between genes and phenotypes
• NOTE ASSOCIATION IS NOT CAUSATION!
• Reverse genetics (not possible in humans)
• Genetically modify genes
• Test for effects of gene manipulation on
  phenotypes
• Can evaluate cause-effect relationships, as long
  as youre very careful in interpreting results!

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Is it possible to develop a comprehensive animal
(mouse) model of smoking or any other form of
substance abuse? NO!(Mice wont strike a
match.)
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Mouse models have been developed for several
simple components of nicotine addiction.

• Acute (first dose) sensitivities.
• Tolerance
• Rapid tolerance (first dose)
• Chronic tolerance
• Reinforcement
• Oral preference and CPP
• Mice dont self-administer nicotine i.v.
• Withdrawal.

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The Sensitivity Model

• High sensitivity to positive actions increases
  vulnerability to addiction.
• Low sensitivity to toxic actions increases
  vulnerability to addiction.
• Low sensitivity could be innate (genetically
  determined)
• Low sensitivity could be acquired (drug tolerance
  and/or environmental mediation).
• Could be due to altered metabolism or CNS
  sensitivity.

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Strain differences in sensitivity to a first dose
were seen in 1977,
in a 1989 study with 19 strains,
and in subsequent studies that have included over
50 strains.
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• Inbred mouse strains differ dramatically in
  sensitivity to an acute dose of nicotine.

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First dose sensitivity to nicotine

• Weve measured gt 10 different responses.
• Principal components analyses indicate that
  sensitivities to nicotine fall into two general
  domains (one typified by effects on locomotor
  activity and body temperature i.e., depressant
  effects, and the second typified by
  nicotine-induced seizures i.e., stimulant
  effects).

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Do inbred mouse strains differ in the rate of
nicotine metabolism?
Small strain differences in metabolism could not
account for most of the strain differences in
first-dose sensitivity.
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The pharmacologists mantra

• D R ? DR ? Response
• Biochemical questions that we have addressed
• Where are nicotinic receptors expressed in the
  brain?
• What are the subunit compositions of native
  nicotinic receptors?
• Are nicotinic receptors changed by chronic
  nicotine treatment? YES! AND THEY ARE HUNGRY!
• Behavioral questions that we have addressed
• Do nicotinic receptors modulate nicotine-related
  behaviors?
• Given that there are many effects of nicotine, do
  different receptor subtypes modulate different
  behaviors?
• Do nicotinic receptors modulate some
  alcohol-related behaviors?
• Do nicotinic receptors modulate normal
  behaviors?

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In Situ Hybridization for nAChR Subunits
a2
a3
a4
a5
a6
a7
b2
b3
b4
Sections approximately 2.8 mm Bregma.
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Nicotinic Acetylcholine Receptor Top View
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Binding of 125IA85380 To Mouse Brain
a4/
b2/
(about -1.5 mm Bregma)
a4/- b2/-
a4/-
b2/-
a4-/-
b2-/-
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Genetic Factors Regulate Levels of Receptor
Expression
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Differences in sensitivity are associated with
differences in levels of receptor expression
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Mouse strains differ in sensitivity to the
antinociceptive effects of nicotine
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STUDIES WITH INBRED STRAINS LED TO THE POSTULATE
THAT ANIMALS WITH MORE NICOTINIC RECEPTORS HAVE
GREATER SENSITIVITY TO NICOTINE.D R ? DR ?
Response
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Location of a Mouse a4 Polymorphism
Extracellular
Intracellular
GAASLTESKPTGSPASLKTRPSQLPVSDQTSPCKCTCKEPSPVSPITVLK
AGGTKAPPQHLP GAASLTESKPTGSPASLKTRPSQLPVSDQASPCKCT
CKEPSPVSPITVLKAGGTKAPPQHLP
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A/T Differences in 86Rb flux
19 Inbred Strains
Dobelis et al. (2002) Mol. Pharmacol. 62 334-42.
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The Chrna4 T529A polymorphism alters the ratio of
high to low sensitivity a4b2 nAChRs
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Does the a4 Polymorphism Seem to Influence
Anything Interesting?

• Many acute responses to nicotine
• Some acute responses to alcohol
• May influence the development of tolerance to
  nicotine
• May influence chronic nicotine-induced
  upregulation of receptors.

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THE a4 POLYMORPHISM ALSO SEEMS TO INFLUENCE
NICOTINE-INDUCED SEIZURES.
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The a4 A/T Poly Seems to Influence (i.e., is
ASSOCIATED with)

• nAChR receptor function
• EtOH enhancement of receptor function
• EtOH effects on receptor desensitization
• Sensitivity to several behavioral effects of
  nicotine
• Sensitivity to several behavioral effects of
  alcohol
• The development of tolerance and cross tolerance
  between nicotine and alcohol
• Severity of alcohol withdrawal
• Nicotine preference
• More

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Reverse Genetics Provides Converging Evidence

• Studies with null mutants
• a4 mutants (John Drago, Melbourne)
• b2 mutants (Marina Picciotto, Yale)
• Others (Beaudet, Baylor Heinemann, Salk)
• Studies with gain of function mutants
• Gain of function a4 mutants (Lester, Cal Tech).

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Gain of function mutation of a4 resulted in
changes in several addiction-related behaviors.
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Gain of function of a4 mutants are more sensitive
to nicotines analgesic actions.
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Nicotine preference is modulated by the A/T a4
polymorphism in wild-type but not b2 KO mice.

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Summary of Mouse Studies

• Inbred mouse strains differ in sensitivity to
  nicotine.
• Inbred strains differ in receptor expression.
• Polymorphisms exist in mouse nAChR genes that
  cause altered receptor function.
• Variations in receptor levels and function are
  ASSOCIATED with variation in sensitivity.
• Null and gain of function mutant mice show
  expected changes in behaviors helping to bridge
  the ASSOCIATION vs. CAUSATION gap.

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The highest affinity nAChRsare found on the lips!
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Back to Humans

• Modern studies are actually looking for
  associations between polymorphisms associated
  with genes.
• Candidate genes (e.g., DA receptors)
• Whole genome associations
• These studies have yielded provocative results.

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Polys in Chrna5 Smoking
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The cluster is associated with early use of
both booze and butts.
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And the sensitivity issue comes to the fore

• Addiction (Accepted April 9, 2008)
• Association of an SNP in neuronal acetylcholine
  receptor subunit alpha 5 (CHRNA5) with smoking
  status and with "pleasurable buzz" during early
  experimentation with smoking
• Short title Association of CHRNA5 SNP with
  early smoking
• Richard Sherva, Kirk Wilhelmsen, Cynthia S.
  Pomerleau, Scott A. Chasse, John P. Rice, Sandy
  M. Snedecor, Laura J. Bierut, Rosalind J. Neuman,
  and Ovide F. Pomerleau.

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Other nAChR subunit genes and sensitivity
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THE MOUSE STUDIES TOLD US THAT a4b2 RECEPTORS
PLAY CRITICAL ROLES IN ADDICTION STUFF!WERE WE
WRONG?
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Biochemical Methods for Measuring nAChR Function

• Synaptosome Assays
• Ion Flux
• Neurotransmitter Release
• Dopamine
• GABA
• Acetylcholine.

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DOPAMINERGIC TERMINAL
AXON
DA
DAT
VMAT
DA DA
DA
D2
K
DA
Ca
nAChR
DA DA
CHOLINERGIC TERMINAL
D1
D1
Na
VSCC
ACh
POSTSYNAPTIC NEURON
Ca
ACh
AXON
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Release Apparatus
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Nicotine-Stimulated Dopamine Release
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?4 Subunit Deletion Abolishes the ?-CTX
MII-resistant 3H Dopamine Release
?-Ctx MII-sensitive DA release
?-Ctx MII-resistant DA release
Total DA release
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?2 Subunit Deletion Abolishes All Components of
Striatal 3H Dopamine Release
?-Ctx MII-resistant DA release
?-Ctx MII-sensitive DA release
Total DA release
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?5 Subunit Deletion Decreases the ?-CTX
MII-resistant 3H Dopamine Release by 70
?-Ctx MII-resistant DA release
?-Ctx MII-sensitive DA release
Total DA release
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?3 Subunit Deletion Abolishes the ?-CTX
MII-sensitive 3H Dopamine Release
?-Ctx MII-resistant DA release
?-Ctx MII-sensitive DA release
Total DA release
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In Situ Hybridization for nAChR Subunits
a2
a3
a4
a5
a6
a7
b2
b3
b4
Sections approximately 2.8 mm Bregma.
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nAChR Subtypes in Dopaminergic Nerve Terminals

• a6b2b3 (a-Ctx MII-sensitive)
• a4a6b2b3 (a-Ctx MII-sensitive)
• a4b2 (a-Ctx MII-resistant) also expressed in
  GABA neurons
• a4a5b2 (a-Ctx MII-resistant) also expressed in
  GABA neurons.

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THE HUMAN STUDIES ARE DETECTING ASSOCIATIONSCan
we cross the correlation-causation bridge in
human studies?Probably not.How about mouse
studies?Maybe (I think so).
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And now,A WORD FROM OUR SPONSORS- or -Whats
the old guy gonna do next?A new kind of
research in a novel population.
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Ganako School in 2007
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Im teaching science at Mwenge University in
Tanzania
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THANKS

• Mike Marks, Sharon Grady
• Jeanne Wehner
• My grad students
• My postdocs
• My collaborators.