The New England Journal of Medicine

Clinical Implications of Basic Research

Volume 347:362-364

August 1, 2002

Number 5

Knocking Out the DREAM to Study Pain

Although some mechanisms by which acute pain evolves into a chronic syndrome
are known, many others, including changes in the brain stem, thalamus, and
cerebral cortex, are not understood. Moreover, even though chronic
neuropathic and inflammatory pain can be relieved to some extent with opiate
compounds, these syndromes are still poorly understood. The treatment
options for pain with a central cause are even more bleak. Now, however, the
revolution in genomic engineering has opened up new possibilities for
studying the mechanisms of acute and chronic pain and designing novel and
more selective drugs.
Prodynorphin is the precursor of dynorphin, an opioid neuropeptide.
Expression of the prodynorphin gene is controlled by a calcium-regulated
transcription factor that binds to a downstream regulatory element (DRE) of
the gene and is termed DRE antagonistic modulator (DREAM). When DREAM binds
to DRE, it inhibits transcription of the prodynorphin gene ( Figure 1A
<http://content.nejm.org/cgi/content/full/347/5/#F1> ). 1
<http://content.nejm.org/cgi/content/full/347/5/#R1>  In view of the role of
dynorphin in spinal and supraspinal modulation of afferent nociceptive
signals and its likely involvement in chronic-pain syndromes, the DREAM
transcriptional regulator should be an important target for assessing
nociceptive transmission.

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Figure 1. Effect of the Presence (Panel A) and Absence (Panel B) of
Downstream Regulatory Element Antagonistic Modulator (DREAM) on Nociceptive
Processing after Noxious Stimulation of the Skin with a Pin.
In Panel A, the prodynorphin gene is blocked by DREAM, and the nociceptive
signal is transmitted by substance P (SP)–containing dorsal-root neurons
essentially unaltered through the spinal cord. This activated state is
characterized by a large depolarizing potential and multiple spike
discharges. Such activity subsequently activates systems governing
descending regulatory control and thalamic nuclei, triggering the perception
of pain. In Panel B, DREAM has been knocked out, and the release of
dynorphin from spinal cord interneurons becomes a prominent part of the
nociceptive response. This inactivates spinal-projection neurons, leading to
a greatly reduced excitatory response. Subsequently, there is almost
complete inactivation of descending regulatory and pain-processing systems.
In addition to raising issues related to the specific functions of DREAM,
this simple scheme emphasizes the importance of neurophysiological research
involving this model and hypotheses to guide drug design.

Cheng et al. 2 <http://content.nejm.org/cgi/content/full/347/5/#R2>  deleted
DREAM in mice by targeted disruption of the gene and performed a number of
groundbreaking studies of the mechanisms by which DREAM regulates
nociception. The DREAM-knockout animals had essentially normal motor
control, spatial learning, and anxiety in open field tests but significantly
reduced responses to acute thermal and mechanical stimulation of the skin
and viscera and decreased responses in neuropathic and inflammatory models
of chronic pain ( Figure 1B
<http://content.nejm.org/cgi/content/full/347/5/#F1> ). These features make
DREAM-knockout mice ideal for assessing transmitter systems that regulate
nociception and reduce pain behavior mediated by kappa-opioid receptors.
Opioids interact with receptors at all levels of nociceptive and pain
processing. The principal opioid receptors are the mu, kappa, and delta
subtypes. The effectiveness of compounds such as morphine, which are
selective for mu-opioid receptors, reflects the wide distribution of
mu-opioid receptors and the ability of such compounds to block the
transmission of the nociceptor to the spinal cord and to control the
perception of pain in the cerebral cortex. Dynorphin is released by
interneurons in the spinal cord, binds to kappa-opioid receptors on
spinal-projection neurons, and has analgesic effects. It appears, however,
that dynorphin is not expressed by nociceptors, is elevated in chronic-pain
syndromes, and through its actions at N-methyl-D-aspartate (NMDA) receptors,
3 <http://content.nejm.org/cgi/content/full/347/5/#R3>  may actually enhance
nociceptive transmission, under some circumstances, rather than reduce it.
For these reasons, the expression of dynorphins must be under the tight
control of DREAM.
Critical evidence of a specific role of DREAM in pain processing 2
<http://content.nejm.org/cgi/content/full/347/5/#R2>  was provided by
gel-shift assays showing calcium- and dose-dependent binding of prodynorphin
DRE to DREAM and a lack of DREAM in the DREAM-knockout mice. The
DREAM-knockout mice had elevated levels of messenger RNA (mRNA) for
prodynorphin in the spinal cord, normal levels of mRNA for the opioids
pro-opiomelanocortin and proenkephalin and the DRE-containing c-fos, and no
changes in the levels of kappa-opioid or NMDA receptors in the spinal cord.
The decreased responses to pain in DREAM-knockout mice were mediated by
kappa-opioid receptors, as was shown with the kappa-selective antagonist
norbinaltorphimine dihydrochloride.
These studies also assessed the NMDA receptors in DREAM-knockout mice. The
NMDA receptor mediates the entry of calcium into spinal neurons through the
activation of glutamatergic nociceptors. In the DREAM-knockout mice,
however, a selective NMDA antagonist (MK-801) had no effect on the
tail-flick test, which measures the interval between noxious heating of the
tail and withdrawal of the tail from the stimulus. Although MK-801 increases
the threshold for tail withdrawal in normal animals, the drug did not change
the response in knockout mice, which suggests that the actions of dynorphin
in the knockout mice are not mediated by an NMDA mechanism, as they are in
normal animals. Since DREAM may also be regulated independently of nuclear
calcium levels, 4 <http://content.nejm.org/cgi/content/full/347/5/#R4>  the
actions of dynorphin in this model may not be directly linked to central
sensitization.
Many new questions arise from these pivotal findings. First, what is the
specificity of DREAM in nociceptive and pain processing? All pain-induced
behavior appears to be interrupted at a nodal point in the spinal cord of
these knockout animals. Although this could prove to be an important feature
of DREAM, it means that descending inhibitory functions in the brain stem
and other functions of central-pain processing will not be available for
study because they have been permanently blocked. Regulatory specificity is
being uncovered in animals with other knockout targets. For example,
deletion of the dopamine-{beta} hydroxylase gene widely blocks expression of
noradrenaline, yet it inhibits hyperalgesia for thermal but not mechanical
stimulation. 5 <http://content.nejm.org/cgi/content/full/347/5/#R5>  Second,
what is the involvement of the supraspinal midbrain and medial thalamus in
altered pain processing in the DREAM-deficient animals? Although the
hippocampus was analyzed for prodynorphin levels, there was no assessment of
pain mechanisms in the forebrain in regions established as nociceptive.
Third, what agents, processes, and genes regulate the expression of DREAM,
and how is it regulated during acute pain in normal animals? Fourth, how can
this gene serve as a target for therapy? Compounds that regulate the
expression of neuronal genes are not yet available. Indeed, we must evaluate
other systems involved in establishing chronic pain using microarray
analysis in DREAM-knockout animals during acute and chronic pain.
There are some difficulties inherent in the use of the DREAM-knockout model
to study pain processing. Since all nociceptive processing is blocked at a
nodal point in the spinal cord, this model may not tell us much about the
unique causes of neuropathic and inflammatory pain. DREAM-knockout mice
express prodynorphin in the ventral horns, but this has not been observed in
normal adult mice. Moreover, chronic pain usually begins in adulthood, yet
the effect in knockout mice begins very early in life. Further investigation
of these neurons could provide insight into dormant systems that may be
useful for targeting drugs. It should also be noted that chronic pain
reduces nociceptive responses of the anterior insular and cingulate
cortices. Such cortical alterations must be identified in animal models
because these are the sites of pain-induced stress and anxiety. Although the
direct therapeutic value of the knockout model is limited, targeted delivery
of DNA complexes to the central nervous system is possible. 6
<http://content.nejm.org/cgi/content/full/347/5/#R6>
The DREAM-knockout model may be useful for designing drugs that can directly
regulate genes, and the side effects of such an approach may be reduced by
the use of site-specific expression vectors. There is no doubt that merging
drug design with genetic engineering has great potential. The dream is that
these engineering techniques will elucidate the mechanisms of the least
understood pain syndromes and provide selective targets for effective relief
of pain, even for the most intransigent pain syndromes, such as central
pain.

Brent A. Vogt, Ph.D.
State University of New York Upstate Medical University
Syracuse, NY 13210
Supported by a grant (NS38485) from the National Institutes of Health.
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Edward E. Rylander, M.D.
Diplomat American Board of Family Practice.
Diplomat American Board of Palliative Medicine.