The melanocortin-1 receptor gene mediates female- specific mechanisms of analgesia in mice and humans. (2003) Proc. Natl. Acad. Sci USA. Mogil JS, Wilson

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

Dr. Jeffrey S. MogilDr. Jeffrey S. MogilMcGill University, Montreal

Background of Background of QualitativeQualitative Sex Sex

Differences in AnalgesiaDifferences in Analgesia

• 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).

http://www.sci.uidaho.edu/biosci/labs/magnusson/research/




• The same sex-specific effect is observed in analgesia resulting from administration of κ-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).






• 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 κ-opioid analgesia through QTL mapping (2003).


Stress-Induced Pain InhibitionStress-Induced Pain Inhibition

Periaqueductal gray (PAG) of midbrain

Brainstem

Dorsal horn of spinal cord

OW!

Incoming pain information

Nociceptor

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Actual or impending tissue damage in periphery

AbstractAbstract

What was the research question of the Mogil group? Read the abstract above and click to reveal the answer.

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.

AbstractAbstract

What was the research question of the Mogil group? Read the abstract above and click to reveal the answer.

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 gene mediates female-specific κ-opioid analgesia in mice? Is this gene involved in sex differences in analgesia in humans, as well?

HypothesisHypothesisWhat was their hypothesis about the identity of the gene?

HypothesisHypothesisWhat 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 Stress-Induced Analgesia Differs Between SexesBetween Sexes

NMDAR

glutamate& aspartate

Male Mouse: pain inhibition mediated by NMDAR

opioidreceptors

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Stress-Induced Analgesia Differs Stress-Induced Analgesia Differs Between SexesBetween Sexes

??? receptorNMDAR

estrogenreceptors

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Male Mouse: pain inhibition mediated by NMDAR

Female Mouse: an additional pathway mediated by an unknown receptor

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Figure 1: Research QuestionFigure 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?


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 Figure 1: Quantitative Trait Locus (QTL) Mapping(QTL) Mapping

• 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)

“The technique is blind. The disadvantage is it takes a bloody long time.” JSM

Figure 1: Quantitative Trait Locus Figure 1: Quantitative Trait Locus (QTL) Mapping(QTL) Mapping

• 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

“The technique is blind. The disadvantage is it takes a bloody long time.” JSM

Figure 1: Creation of B6D2F2 miceFigure 1: Creation of B6D2F2 mice

B6 D2

DBA/J2: MK-801-insensitive strain

C57BL/6J:MK-801 sensitive

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B6D2F1 B6D2F1X F1 B6D2All heterozygous


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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.

1:2:1

All heterozygous

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D2

Figure 1: Results and ConclusionFigure 1: Results and ConclusionMice were injected with U50,488 (κ-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.

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What can be concluded from these data?

at D8Mit56

Microsatellite Markers

Genotype at D8Mit56

Figure 1: Results and ConclusionFigure 1: Results and ConclusionMice were injected with U50,488 (κ-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.

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.

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Microsatellite Markers

Genotype at D8Mit56

Candidate Gene: Melanocortin-1 Candidate Gene: Melanocortin-1 Receptor (Receptor (Mc1rMc1r))

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 Candidate Gene: Melanocortin-1 Receptor (Receptor (Mc1rMc1r))

eumelanin (black)

phaeomelanin (red/yellow)

α-MSH

MC1R

cAMP

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-melanocyte-stimulating hormone)

Testing the Candidate GeneTesting the Candidate GeneAfter 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?Does MC1R mediate the female-specific stress-induced analgesia pathway?



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?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)


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?



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 MutantFigure 2: Mc1r Null Mutant

e/e “recessive yellow” mouseB6

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 DataFigure 2: Mutant Data

?NMDAR

estrogenreceptors

opioid receptors


NMDAR antagonist (MK-801)

?NMDAR

estrogenreceptors

opioid receptors



Stimulate stress response (U50,488)


?NMDAR

estrogenreceptors

opioid receptors



Stimulate stress response (U50,488)

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.


?NMDAR

estrogenreceptors

opioid receptors

Figure 3: Pharmacological Figure 3: Pharmacological DataDataOutbred Crl:CD-1 “mutt” mice

ANT peptide = MC1R antagonist



Males: ANT has no effect



Males: ANT has no effectMK-801 sensitive




Females: antagonizing MC1R results in MK-801 sensitivity




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

Melanocortin-1 Receptor in HumansMelanocortin-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 HumansMelanocortin-1 Receptor in Humans

eumelanin (black)

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α-MSH

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"The fun thing about this finding was it could have been any gene. It wasn't any gene; it was the redhead gene." JSMPeople with very fair skin and red hair tend to have certain null allele variations of MC1R.

Figure 4: From Mice to HumansFigure 4: From Mice to HumansPentazocine 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.


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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.

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Methodology: Methodology: To clone or not to clone…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: Methodology: To clone or not to clone…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 5Supplementary 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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Documents

The melanocortin-1 receptor gene mediates female- specific mechanisms of analgesia in mice and humans. (2003) Proc. Natl. Acad. Sci USA. Mogil JS, Wilson