Transcript Analyzing the Paper

The melanocortin-1 receptor gene mediates femalespecific mechanisms of analgesia in mice and humans.
(2003) Proc. Natl. Acad. Sci USA.
Mogil JS, Wilson SG, Chesler EJ, Rankin AL, Nemmani KV, Lariviere WR, Groce
MK, Wallace MR, Kaplan L, Staud R, Ness TJ, Glover TL, Stankova M, Mayorov A,
Hruby VJ, Grisel JE, Fillingim RB.
Dr. Jeffrey S. Mogil
McGill University, Montreal
Background of
Qualitative Sex
Differences in Analgesia
http://www.sci.uidaho.edu/biosci/labs/magnusson/research/
• Swim stress-induced analgesia is reversed by NMDA
Receptor antagonist (MK-801) in male mice only, suggesting
that females have a descending pathway not mediated by
NMDAR (4: 1993).
Background of
Qualitative Sex
Differences in Analgesia
http://www.sci.uidaho.edu/biosci/labs/magnusson/research/
• Swim stress-induced analgesia is reversed by NMDA
Receptor antagonist (MK-801) in male mice only, suggesting
that females have a descending pathway not mediated by
NMDAR (4: 1993).
• The same sex-specific effect is observed in analgesia
resulting from administration of  -opioid agonists (9: 1997).
Background of
Qualitative Sex
Differences in Analgesia
http://www.sci.uidaho.edu/biosci/labs/magnusson/research/
• Swim stress-induced analgesia is reversed by NMDA
Receptor antagonist (MK-801) in male mice only, suggesting
that females have a descending pathway not mediated by
NMDAR (4: 1993).
• The same sex-specific effect is observed in analgesia
resulting from administration of  -opioid agonists (9: 1997).
• A previous linkage study by the Mogil group implicates the
distal portion of chromosome 8 as the QTL for swim stressinduced analgesia in mice (7: 1997).
Background of
Qualitative Sex
Differences in Analgesia
http://www.sci.uidaho.edu/biosci/labs/magnusson/research/
• Swim stress-induced analgesia is reversed by NMDA
Receptor antagonist (MK-801) in male mice only, suggesting
that females have a descending pathway not mediated by
NMDAR (4: 1993).
• The same sex-specific effect is observed in analgesia
resulting from administration of  -opioid agonists (9: 1997).
• A previous linkage study by the Mogil group implicates the
distal portion of chromosome 8 as the QTL for swim stressinduced analgesia in mice (7: 1997).
• This paper looks at sex-specific  -opioid analgesia through
QTL mapping (2003).
Stress-Induced Pain Inhibition
Periaqueductal gray
(PAG) of midbrain
Brainstem
Dorsal horn of
spinal cord
Modified pain information
OW!
Nociceptor
Incoming pain
information
Actual or impending
tissue damage in
periphery
Abstract
Sex specificity of neural mechanisms modulating nociceptive information has been
demonstrated in rodents, and these qualitative sex differences appear to be relevant to
analgesia from κ-opioid receptor agonists, a drug class reported to be clinically effective
only in women. Via quantitative trait locus mapping followed by a candidate gene strategy
using both mutant mice and pharmacological tools, we now demonstrate that the
melanocortin-1 receptor (Mc1r) gene mediates κ-opioid analgesia in female mice only.
This finding suggested that individuals with variants of the human MC1R gene, associated
in our species with red hair and fair skin, might also display altered κ-opioid analgesia. We
found that women with two variant MC1R alleles displayed significantly greater analgesia
from the κ-opioid, pentazocine, than all other groups. This study demonstrates an
unexpected role for the MC1R gene, verifies that pain modulation in the two sexes
involves neurochemically distinct substrates, and represents an example of a direct
translation of a pharmacogenetic finding from mouse to human.
What was the research question of the Mogil group? Read
the abstract above and click to reveal the answer.
Abstract
Sex specificity of neural mechanisms modulating nociceptive information has been
demonstrated in rodents, and these qualitative sex differences appear to be relevant to
analgesia from κ-opioid receptor agonists, a drug class reported to be clinically effective
only in women. Via quantitative trait locus mapping followed by a candidate gene strategy
using both mutant mice and pharmacological tools, we now demonstrate that the
melanocortin-1 receptor (Mc1r) gene mediates κ-opioid analgesia in female mice only.
This finding suggested that individuals with variants of the human MC1R gene, associated
in our species with red hair and fair skin, might also display altered κ-opioid analgesia. We
found that women with two variant MC1R alleles displayed significantly greater analgesia
from the κ-opioid, pentazocine, than all other groups. This study demonstrates an
unexpected role for the MC1R gene, verifies that pain modulation in the two sexes
involves neurochemically distinct substrates, and represents an example of a direct
translation of a pharmacogenetic finding from mouse to human.
What was the research question of the Mogil group? Read
the abstract above and click to reveal the answer.
What gene mediates female-specific κ-opioid analgesia in mice? Is this
gene involved in sex differences in analgesia in humans, as well?
Hypothesis
What was their hypothesis about the identity of the gene?
Hypothesis
What was their hypothesis about the identity of the gene?
They believed it would be located somewhere in the distal portion
of chromosome 8. Other than that, they had no idea what gene it
would be!
“I think nonhypothesis-driven research is probably more
important than hypothesis-driven research. . . . You find genes
that weren't high probability candidates beforehand. . . . We can
find completely novel things, very surprising things." JSM
Stress-Induced Analgesia Differs
Between
Sexes
opioid
receptors
glutamate
& aspartate
NMDAR
Modified pain information
Male Mouse: pain inhibition
mediated by NMDAR
Stress-Induced Analgesia Differs
Between
Sexes
opioid
receptors
estrogen
receptors
glutamate
& aspartate
NMDAR
??? receptor
Modified pain information
Male Mouse: pain inhibition
mediated by NMDAR
Female Mouse: an additional
pathway mediated by an
unknown receptor
Figure 1: Research Question
Looking at Figure 1, what
question was the Mogil group
trying to answer when they
designed this study?
Figure 1: Research Question
Looking at Figure 1, what
question was the Mogil group
trying to answer when they
designed this study?
What region of the genome
contains genes that mediate
female-specific stress-induced
analgesia?
What method was used to
answer this question?
Figure 1: Research Question
Looking at Figure 1, what
question was the Mogil group
trying to answer when they
designed this study?
What region of the genome
contains genes that mediate
female-specific stress-induced
analgesia?
What method was used to
answer this question?
Quantitative trait locus (QTL)
mapping
Figure 1: Quantitative Trait Locus
“The technique is blind.
(QTL) Mapping The disadvantage is it
takes a bloody long
time.” JSM
• Microsatellites used as markers for linkage
mapping of a certain trait
– Microsatellite: dinucleotide repeats of variable lengths
– At the same microsatellite locus on the genome, the
number of repeats varies between individuals (or, in
this case, between strains)
Figure 1: Quantitative Trait Locus
“The technique is blind.
(QTL) Mapping The disadvantage is it
takes a bloody long
time.” JSM
• Microsatellites used as markers for linkage
mapping of a certain trait
– Microsatellite: dinucleotide repeats of variable lengths
– At the same microsatellite locus on the genome, the
number of repeats varies between individuals (or, in
this case, between strains)
• For each microsatellite locus
– PCR using primers for DNA on either side of
microsatellite marker
– PCR produces will vary in size depending on the
number of repeats
Figure 1: Creation of B6D2F2 mice
B6
C57BL/6J:
MK-801 sensitive
X
D2
DBA/J2:
MK-801-insensitive strain
Figure 1: Creation of B6D2F2 mice
B6
X
C57BL/6J:
MK-801 sensitive
All
heterozygous
B6D2F1
D2
DBA/J2:
MK-801-insensitive strain
X
B6D2F1
F1 B6D2
Figure 1: Creation of B6D2F2 mice
B6
X
C57BL/6J:
MK-801 sensitive
All
heterozygous
B6D2F1
D2
DBA/J2:
MK-801-insensitive strain
X
F1 B6D2
B6D2F1
F2
1:2:1
B6D2F2
B6
D2
B6 B6B6 B6D2
D2 B6D2 D2D2
Due to independent assortment and crossing over, each F2 mouse could be
B6/B6, B6/D2, or D2/D2 at each of the microsatellite markers genotyped.
Mice were injected with U50,488 ( opiod agonist) to activate the
descending analgesia pathways and
nociceptive sensitivity was measured
using a hot-water tail-withdrawal
assay.
likelihood of QTL
Figure 1: Results and Conclusion
A statistical linkage analysis was
performed for the microsatellite
genotypes and the analgesia
phenotypes.
Microsatellite Markers
What can be concluded from these data?
at D8Mit56
Genotype at D8Mit56
Mice were injected with U50,488 ( opiod agonist) to activate the
descending analgesia pathways and
nociceptive sensitivity was measured
using a hot-water tail-withdrawal
assay.
likelihood of QTL
Figure 1: Results and Conclusion
A statistical linkage analysis was
performed for the microsatellite
genotypes and the analgesia
phenotypes.
Microsatellite Markers
What can be concluded from these data?
at D8Mit56
κ-opiod analgesia is linked to the
distal portion of chromosome 8
(near 67cM) in females. There is no
linkage of κ-opioid analgesia in this
region for males.
Genotype at D8Mit56
Candidate Gene: Melanocortin-1
Receptor (Mc1r)
The Mogil group decided to test Mc1r from among the candidate
genes near the QTL because one of the lab members happened upon
a paper that demonstrated MC1R expression in the areas of the brain
associated with analgesia.
Candidate Gene: Melanocortin-1
Receptor (Mc1r)
The Mogil group decided to test Mc1r from among the candidate
genes near the QTL because one of the lab members happened upon
a paper that demonstrated MC1R expression in the areas of the brain
associated with analgesia.
MC1R is commonly known for its role in the control of melanin synthesis.
α-MSH (α-melanocyte-stimulating hormone)
MC1R
cAMP
phaeomelanin
(red/yellow)
eumelanin
(black)
Testing the Candidate Gene
After picking MC1R as their candidate gene, the Mogil group set out to determine
whether its gene product is actually required for activity of the female-specific
analgesia pathway.
Testing the Candidate Gene
After picking MC1R as their candidate gene, the Mogil group set out to determine
whether its gene product is actually required for activity of the female-specific
analgesia pathway.
Formal proof that they picked the correct gene would require the group to
engineer their own knock-out mouse and then rescue melanocortin-1 receptor
function. “A dead boring task. . . . A waste of time and taxpayer money.” JSM
Testing the Candidate Gene
After picking MC1R as their candidate gene, the Mogil group set out to determine
whether its gene product is actually required for activity of the female-specific
analgesia pathway.
Formal proof that they picked the correct gene would require the group to
engineer their own knock-out mouse and then rescue melanocortin-1 receptor
function. “A dead boring task. . . . A waste of time and taxpayer money.” JSM
The Mogil group decided to depart from tradition and designed a group of
independent experiments that all set out to answer the same question:
Does MC1R mediate the female-specific stress-induced analgesia pathway?
Testing the Candidate Gene
After picking MC1R as their candidate gene, the Mogil group set out to determine
whether its gene product is actually required for activity of the female-specific
analgesia pathway.
Formal proof that they picked the correct gene would require the group to
engineer their own knock-out mouse and then rescue melanocortin-1 receptor
function. “A dead boring task. . . . A waste of time and taxpayer money.” JSM
The Mogil group decided to depart from tradition and designed a group of
independent experiments that all set out to answer the same question:
Does MC1R mediate the female-specific stress-induced analgesia pathway?
Their data can be grouped into three main lines of evidence:
Mutant Data (Table 1 and Figure 2)
Pharmacological Data (Figure 3)
Human Data (Table 2 and Figure 4)
Figure 2: Research Question
What method did they use to
answer their question?
Figure 2: Research Question
What method did they use to
answer their question?
Comparison of B6 and Mc1r
mutant mouse strains.
In which sex do you hypothesize that the
mutation will have no effect on analgesia?
Figure 2: Research Question
What method did they use to
answer their question?
Comparison of B6 and Mc1r
mutant mouse strains.
In which sex do you hypothesize that the
mutation will have no effect on analgesia?
Male e/e and B6 mice should
have an identical analgesia
phenotypes because their
descending analgesia
pathways should not be
mediated by MC1R.
Figure 2: Mc1r Null Mutant
B6
e/e “recessive
yellow” mouse
The Mogil group used the pre-existing e/e “recessive yellow” mouse,
which is a Mc1r mutant in B6 background. The Mc1r allele is the only
known mutation in the e/e mouse.
Figure 2: Mutant Data
opioid receptors
estrogen
receptors
NMDAR
?
Figure 2: Mutant Data
opioid receptors
NMDAR
antagonist (MK-801)
NMDAR
estrogen
receptors
?
Figure 2: Mutant Data
Stimulate stress
response
(U50,488)
opioid receptors
NMDAR
antagonist (MK-801)
NMDAR
estrogen
receptors
?
What can be concluded from these data?
Figure 2: Mutant Data
Stimulate stress
response
(U50,488)
opioid receptors
NMDAR
antagonist (MK-801)
NMDAR
estrogen
receptors
?
What can be concluded from these data?
e/e females display typically male
MK-801 sensitivity. Since the only
known difference between e/e and
B6 strains is the MC1R genotype, it
seems that MC1R is mediating
female-specific analgesia.
Figure 3: Pharmacological Data
Outbred Crl:CD-1 “mutt” mice
ANT peptide = MC1R
antagonist
Figure 3: Pharmacological Data
Outbred Crl:CD-1 “mutt” mice
ANT peptide = MC1R
antagonist
Males:
ANT has no effect
Figure 3: Pharmacological Data
Outbred Crl:CD-1 “mutt” mice
ANT peptide = MC1R
antagonist
Males:
ANT has no effect
MK-801 sensitive
Figure 3: Pharmacological Data
Outbred Crl:CD-1 “mutt” mice
ANT peptide = MC1R
antagonist
Males:
ANT has no effect
MK-801 sensitive
Females:
antagonizing MC1R results in
MK-801 sensitivity
Figure 3: Pharmacological Data
Outbred Crl:CD-1 “mutt” mice
ANT peptide = MC1R
antagonist
Males:
ANT has no effect
MK-801 sensitive
Females:
antagonizing MC1R results in
MK-801 sensitivity
Why: ANT blocked femalespecific pathway, causing
mouse to “switch systems”
and have male-type
NMDAergic analgesia profile
Melanocortin-1 Receptor in Humans
"The fun thing about this finding was it could have been any gene. It
wasn't any gene; it was the redhead gene." JSM
Melanocortin-1 Receptor in Humans
"The fun thing about this finding was it could have been any gene. It
wasn't any gene; it was the redhead gene." JSM
People with very fair skin and red hair tend to have certain null allele
variations of MC1R.
α-MSH
MC1R
cAMP
phaeomelanin
(red/yellow)
eumelanin
(black)
Figure 4: From Mice to Humans
Pentazocine: analgesic. κ-opioid receptor
agonist.
Humans were genotyped for their MC1R
alleles and were tested for thermal and
ischemic pain before and after administration
of pentazocine or saline.
more analgesia
Pentazocine analgesia by sex and MC1R genotype in humans
What can be concluded from these data?
Thermal pain
less analgesia
Ischemic pain
Figure 4: From Mice to Humans
Pentazocine analgesia by sex and MC1R genotype in humans
Pentazocine: analgesic. κ-opioid receptor
agonist.
Humans were genotyped for their MC1R
alleles and were tested for thermal and
ischemic pain before and after administration
of pentazocine or saline.
more analgesia
“redhead”
What can be concluded from these data?
Thermal pain
less analgesia
Ischemic pain
Figure 4: From Mice to Humans
Pentazocine analgesia by sex and MC1R genotype in humans
Pentazocine: analgesic. κ-opioid receptor
agonist.
Humans were genotyped for their MC1R
alleles and were tested for thermal and
ischemic pain before and after administration
of pentazocine or saline.
more analgesia
“redhead”
What can be concluded from these data?
Ischemic pain
less analgesia
Women with two variant alleles of
MC1R were more sensitive to
pentazocine analgesia than women
of other genotypes. There was no
significant effect of MC1R genotype
in males.
Thermal pain
Methodology:
To clone or not to clone…
"We didn't do what a lot of people in the mouse genetics field think is
the final step: [positional cloning]. What I did instead is regarded by
some as cheating. I was able to get my philosophy about [the
methodology] down on paper into a fairly prominent journal. Had this
gotten into Science or Nature, I wouldn't have had the space, and
there's no way they would have accepted such heresy" JSM
Methodology:
To clone or not to clone…
“I don’t really care why C57/B6 mice are different than DBA2 mice.
That’s not why we’re doing this. We’re doing this to find out why
some people are different from other people. . . . Once you get good
enough evidence that it’s likely to be a particular gene in humans,
simply start the human study. . . . And as far as I’m concerned once
you have the human finding, it’s completely moot now to find the
actual C that changed to a T that makes the difference between one
mouse strain and another mouse strain. So I’m permanently leaving
that left untied and that drives most mouse geneticists absolutely
insane.” JSM
Supplementary Figure 5
Fig. 5. Switching of analgesia mechanisms in female B6 and e/e mice depending on estrogenic status. C57BL/6 (B6) and mutant (e/e) mice
of both sexes (n = 4–14 per genotype per sex per status per drug) were either left intact or ovariectomized (OVX), via surgical removal
(dorsal incision) of both ovaries under isoflurane/oxygen anesthesia. No testing occurred for at least 2 weeks after surgery. Some OVX mice
were given chronic estrogen replacement (OVX+E2; 5.0 μg/day, i.p., in sesame oil vehicle, for 6–7 days), following our original protocol (1).
After baseline testing on the 49ΊC tail-withdrawal test, all mice received a s.c. injection of MK-801 (0.075 mg/kg) or saline (10 ml/kg),
followed immediately by an i.p. injection of U50,488 (50 mg/kg). Percent analgesia scores were calculated as described. *, Significant
blockade of analgesia by MK-801, P < 0.05. †, Significantly different from intact male and OVX female group, P < 0.05. Note that both B6
and e/e females display evidence of estrogenic control of analgesic magnitude. Only B6 females display evidence of neurochemical
switching of analgesia (i.e., MK-801 sensitivity), because e/e mice appear to be using the male-like, MK-801 sensitive system regardless of
hormonal status.
1. Mogil, J. S., Sternberg, W. F., Kest, B., Marek, P. & Liebeskind, J. C. (1993) Pain 53, 17–25.
Supplementary materials are from: http://www.pnas.org/cgi/content/full/0730053100/DC1/2
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