The melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans.

1
The melanocortin-1 receptor gene mediates
female-specific 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.
2
Dr. Jeffrey S. Mogil
McGill University, Montreal
3
Background of Qualitative Sex Differences in
Analgesia
http//www.sci.uidaho.edu/biosci/labs/magnusson/re
search/

• 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).

4
Background of Qualitative Sex Differences in
Analgesia
http//www.sci.uidaho.edu/biosci/labs/magnusson/re
search/

• 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).

5
Background of Qualitative Sex Differences in
Analgesia
http//www.sci.uidaho.edu/biosci/labs/magnusson/re
search/

• 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 stress-induced analgesia in mice
  (7 1997).

6
Background of Qualitative Sex Differences in
Analgesia
http//www.sci.uidaho.edu/biosci/labs/magnusson/re
search/

• 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 stress-induced analgesia in mice
  (7 1997).
• This paper looks at sex-specific ?-opioid
  analgesia through QTL mapping (2003).

7
Stress-Induced Pain Inhibition
OW!
Periaqueductal gray (PAG) of midbrain
Modified pain information
Brainstem
Dorsal horn of spinal cord
Nociceptor
Actual or impending tissue damage in periphery
Incoming pain information
8
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 ?-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 ?-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 ?-opioid analgesia. We found
that women with two variant MC1R alleles
displayed significantly greater analgesia from
the ?-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.

9
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 ?-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 ?-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 ?-opioid analgesia. We found
that women with two variant MC1R alleles
displayed significantly greater analgesia from
the ?-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 ?-opioid
analgesia in mice? Is this gene involved in sex
differences in analgesia in humans, as well?
10
Hypothesis
What was their hypothesis about the identity of
the gene?
11
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
12
Stress-Induced Analgesia Differs Between Sexes
opioid receptors
Male Mouse pain inhibition mediated by NMDAR
Modified pain information
glutamate aspartate
NMDAR
13
Stress-Induced Analgesia Differs Between Sexes
opioid receptors
Male Mouse pain inhibition mediated by
NMDAR Female Mouse an additional pathway
mediated by an unknown receptor
estrogen receptors
Modified pain information
glutamate aspartate
??? receptor
NMDAR
14
Figure 1 Research Question
Looking at Figure 1, what question was the Mogil
group trying to answer when they designed this
study?
15
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?
16
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
17
Figure 1 Quantitative Trait Locus (QTL) Mapping
The technique is blind. 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)

18
Figure 1 Quantitative Trait Locus (QTL) Mapping
The technique is blind. 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

19
Figure 1 Creation of B6D2F2 mice
X
B6
D2
C57BL/6J MK-801 sensitive
DBA/J2 MK-801-insensitive strain
20
Figure 1 Creation of B6D2F2 mice
X
B6
D2
C57BL/6J MK-801 sensitive
DBA/J2 MK-801-insensitive strain
X
F1
B6D2
B6D2F1
All heterozygous
B6D2F1
21
Figure 1 Creation of B6D2F2 mice
X
B6
D2
C57BL/6J MK-801 sensitive
DBA/J2 MK-801-insensitive strain
X
F1
B6D2
B6D2F1
All heterozygous
B6D2F1
F2
B6
D2
121
B6D2F2
B6D2
B6
B6B6
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.
22
Figure 1 Results and Conclusion
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. A statistical
linkage analysis was performed for the
microsatellite genotypes and the analgesia
phenotypes.
likelihood of QTL
Microsatellite Markers
What can be concluded from these data?
at D8Mit56
Genotype at D8Mit56
23
Figure 1 Results and Conclusion
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. A statistical
linkage analysis was performed for the
microsatellite genotypes and the analgesia
phenotypes.
likelihood of QTL
Microsatellite Markers
What can be concluded from these data?
at D8Mit56
?-opiod analgesia is linked to the distal portion
of chromosome 8 (near 67cM) in females. There is
no linkage of ?-opioid analgesia in this region
for males.
Genotype at D8Mit56
24
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.
25
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.
(a-melanocyte-stimulating hormone)
eumelanin (black)
phaeomelanin (red/yellow)
26
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.
27
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
28
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?
29
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)
30
Figure 2 Research Question
What method did they use to answer their question?
31
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?
32
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.
33
Figure 2 Mc1r Null Mutant
e/e recessive yellow mouse
B6
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.
34
Figure 2 Mutant Data
opioid receptors
estrogen receptors
?
NMDAR
35
Figure 2 Mutant Data
opioid receptors
NMDAR antagonist (MK-801)
estrogen receptors
?
NMDAR
36
Figure 2 Mutant Data
Stimulate stress response (U50,488)
opioid receptors
NMDAR antagonist (MK-801)
estrogen receptors
?
NMDAR
What can be concluded from these data?
37
Figure 2 Mutant Data
Stimulate stress response (U50,488)
opioid receptors
NMDAR antagonist (MK-801)
estrogen receptors
?
NMDAR
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.
38
Figure 3 Pharmacological Data
Outbred CrlCD-1 mutt mice
ANT peptide MC1R antagonist
39
Figure 3 Pharmacological Data
Outbred CrlCD-1 mutt mice
ANT peptide MC1R antagonist Males ANT has no
effect
40
Figure 3 Pharmacological Data
Outbred CrlCD-1 mutt mice
ANT peptide MC1R antagonist Males ANT has no
effect MK-801 sensitive
41
Figure 3 Pharmacological Data
Outbred CrlCD-1 mutt mice
ANT peptide MC1R antagonist Males ANT has no
effect MK-801 sensitive Females antagonizing
MC1R results in MK-801 sensitivity
42
Figure 3 Pharmacological Data
Outbred CrlCD-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 female-specific pathway, causing mouse to
switch systems and have male-type NMDAergic
analgesia profile
43
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
44
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.
eumelanin (black)
phaeomelanin (red/yellow)
45
Figure 4 From Mice to Humans
Pentazocine analgesia by sex and MC1R genotype in
humans
Pentazocine analgesic. ?-opioid receptor agonist.
Thermal pain
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
What can be concluded from these data?
Ischemic pain
less analgesia
46
Figure 4 From Mice to Humans
Pentazocine analgesia by sex and MC1R genotype in
humans
redhead
Pentazocine analgesic. ?-opioid receptor agonist.
Thermal pain
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
What can be concluded from these data?
Ischemic pain
less analgesia
47
Figure 4 From Mice to Humans
Pentazocine analgesia by sex and MC1R genotype in
humans
redhead
Pentazocine analgesic. ?-opioid receptor agonist.
Thermal pain
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
What can be concluded from these data?
Ischemic pain
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.
less analgesia
48
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
49
Methodology To clone or not to clone
I dont really care why C57/B6 mice are
different than DBA2 mice. Thats not why were
doing this. Were doing this to find out why
some people are different from other people. . .
. Once you get good enough evidence that its
likely to be a particular gene in humans, simply
start the human study. . . . And as far as Im
concerned once you have the human finding, its
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
Im permanently leaving that left untied and that
drives most mouse geneticists absolutely insane.
JSM
50
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 414 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 (OVXE2
5.0 µg/day, i.p., in sesame oil vehicle, for 67
days), following our original protocol (1). After
baseline testing on the 49?C 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 lt 0.05. ,
Significantly different from intact male and OVX
female group, P lt 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,
1725.
Supplementary materials are from
http//www.pnas.org/cgi/content/full/0730053100/DC
1/2
51
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