Involvement of TRPV4 in Serotonin-Evoked Scratching.

synergistic antipruritic effects of gamma aminobutyric acid A and B agonists ina a mouse model of atopic dermatitis

Ferda Cevikbas, PhD,a,b* Joao M. Braz, PhD,a* Xidao Wang, PhD,a Carlos Solorzano, PhD,a Mathias Sulk, MD,b,c
Timo Buhl, MD,b,d Martin Steinhoff, PhD,b,e,f,g,h and Allan I. Basbaum, PhDa

Atopic dermatitis (AD), an inflammatory, relapsing chronic
pruritic skin disease, is an often intractable form of chronic itch
that negatively impacts the quality of life of millions of patients.1
Unfortunately, because chronic itch conditions have very
different etiologies, most treatments have poor outcomes and
are accompanied by unacceptable adverse side effects, notably
sedation.2 Clearly, a better understanding of the pathophysiology
of these chronic itch conditions is critical to designing successful
therapeutic strategies.
Studies of the etiology of chronic itch3 generally focus on
changes in skin and immune dysfunction. However, there is
now considerable evidence for a contribution of primary afferent
pruritoceptors that transmit itch messages to spinal cord and
brainstem circuits engaged by and that regulate these messages.4
Of particular interest are studies demonstrating commonalities in
the mechanisms underlying nerve injury-induced neuropathic
pain and itch and the possibility that comparable approaches
may be appropriate for their management.5
Although there is evidence for specificity in the transmission of
itch and pain messages at the level of the primary afferent
nociceptor and pruritoceptor,6,7 both pain and itch are under
spinal cord inhibitory interneuron-mediated control. For example,
loss of spinal cord gamma aminobutyric acid (GABA) or glycinergic
function is a major contributor to the spontaneous pain and hypersensitivity
that develops following nerve injury.8-10 Moreover,
persistent scratching, a manifestation of chronic itch, occurs in the
Bhlhb5 mutant mouse, in which there is dramatic loss of dorsal
horn GABAergic inhibitory interneurons.11 Ablation of glycinergic
interneurons also induces excessive scratching and pain.12
And in a model of dry skin–induced scratching in the mouse,
GABA and glycine receptor antagonists can block scratchinginduced
inhibition of firing in superficial dorsal horn neurons.13
Finally, in patients, acute withdrawal of intrathecal baclofen, a
GABA-B receptor agonist, can induce pruritus.14
Given the evidence for a potential contribution of GABA
agonists in the management of pruritus, it is surprising that there
are no studies that assessed their utility in preclinical or clinical
conditions. Here, we demonstrate that both GABA-A and

GABA-B agonists are not only effective in models of acute itch,
but we also show that systemic administration of very low doses
of these agonists has synergistic antipruritic effects in IL-31
overexpressing transgenic mouse, a model of AD15 that is refractory
to antihistamines1,16 and thus particularly difficult to
manage. Most importantly, the antipruritic synergy could be produced
without concomitant sedation. Finally, we show that sustaining
high levels of GABA inhibition can be achieved using
intraspinal transplantation of cortical GABAergic interneuron
precursor cells. The transplants not only attenuated spontaneous
scratching but also dramatically reduced skin lesions in the IL-
31 overexpressing transgenic mouse (IL-31Tg) mice.

Interleukin 31 receptor (IL-31RA) is a novel Type I cytokine
receptor that pairs with oncostatinMreceptor to mediate IL-31
signaling. Binding of IL-31 to its receptor results in the phosphorylation
and activation of STATs, MAPK, and JNK signaling
pathways. IL-31 plays a pathogenic role in tissue inflammation,
particularly in allergic diseases. Recent studies demonstrate
IL-31RA expression and signaling in non-hematopoietic cells,
but this receptor is poorly studied in immune cells. Macrophages
are key immune-effector cells that play a critical role in
Th2-cytokine-mediated allergic diseases. Here, we demonstrate
that Th2 cytokines IL-4 and IL-13 are capable of up-regulating
IL-31RA expression on both peritoneal and bone marrowderived
macrophages from mice. Our data also demonstrate
that IL-4R-driven IL-31RA expression is STAT6 dependent
in macrophages. Notably, the inflammation-associated genes
Fizz1 and serum amyloid A (SAA) are significantly up-regulated
inM2macrophages stimulated with IL-31, but not in IL-4 receptor-
deficient macrophages. Furthermore, the absence of Type II
IL-4 receptor signaling is sufficient to attenuate the expression
of IL-31RA in vivo during allergic asthma induced by soluble egg
antigen, which may suggest a role for IL-31 signaling in Th2
cytokine-driven inflammation and allergic responses. Our study
reveals an important counter-regulatory role between Th2 cytokine
and IL-31 signaling involved in allergic diseases.Th2 Cytokines Augment IL-31IL-31RA Interactions via

Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice
Akira Machida1, Hiroya Kuwahara1,4, Azat Mayra1, Takayuki Kubodera1, Takashi Hirai2, Fumiko Sunaga1, Mio Tajiri1, Yukihiko Hirai3, Takashi Shimada3, Hidehiro Mizusawa1 and Takanori Yokota1,4,5*

Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice

Abstract
Background: There is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA–expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA).
Results: Intraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA.
Conclusions: Although we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.
Keywords: RNA interference, Adeno-associated virus 9, Dorsal root ganglia, Blood–nerve barrier

Leukotriene B(4) mediates sphingosylphosphorylcholine-induced itch-associated responses in mouse skin.

A central neural circuit for itch sensation.

Overexpression of TRPC3 reduces the content of intracellular calcium stores in HEK-293 cells.

Substitution with a Single Cysteine in the Green Fluorescent Protein-Based Calcium Indicator GCaMP3 Enhances Calcium Sensitivity.

A novel TRPM8 agonist relieves dry eye discomfort

A novel TRPM8 agonist relieves dry eye discomfort S

A novel TRPM8 agonist relieves dry eye discomfort

Jee Myung Yang1,2†, Fengxian Li3,4†, Qin Liu4, Marco Rüedi5, Edward Tak Wei6, Michael Lentsman7, Hyo Seok Lee2, Won Choi2, Seong Jin Kim8*† and Kyung Chul Yoon2*†

Abstract
Background: Physical cooling of the eye surface relieves ocular discomfort, but translating this event to drug treatment of dry eye discomfort not been studied. Here, we synthesized a water-soluble TRPM8 receptor agonist called cryosim-3 (C3, 1-diisopropylphosphorylnonane) which selectively activates TRPM8 (linked to cooling) but not TRPV1 or TRPA1 (linked to nociception) and tested C3 in subjects with mild forms of dry eye disease.
Methods: A set of 1-dialkylphosphoryalkanes were tested for activation of TRPM8, TRPV1 and TRPA1 receptors in transfected cells. The bioactivity profiles were compared by perioral, topical, and intravenous delivery to anesthetized rats. The selected lead candidate C3 or vehicle (water) was applied with a cotton gauze pad to upper eyelids of patients with dry eye disease (n = 30). Cooling sensation, tear film break-up time (TBUT), basal tear secretion, and corneal staining were evaluated. C3 was then applied four times daily for 2 weeks to patients using a pre-loaded single unit applicator containing 2 mg/mL of C3 in water (n = 20) or water only. TBUT, basal tear secretion, and corneal staining, and three questionnaires surveys of ocular discomfort (VAS scale, OSDI, and CVS symptoms) were analyzed before and at 1 and 2 weeks thereafter.
Results: C3 was a selective and potent TRPM8 agonist without TRPV1 or TRPA1 activity. In test animals, the absence of shaking behavior after C3 perioral administration made it the first choice for further study. C3 increased tear secretion in an animal model of dry eye disease and did not irritate when wiped on eyes of volunteers. C3 singly applied (2 mg/ml) produced significant cooling in <5 min, an effecting lasting 46 min with an increase in tear secretion for 60 min. C3 applied for 2 weeks also significantly increased basal tear secretion with questionnaire surveys of ocular discomfort indices clearly showing improvement of symptoms at 1 and 2 weeks. No complaints of irritation or pain were reported by any subject.
Conclusions: C3 is a promising candidate for study of TRPM8 function on the eye surface and for relief of dry eye discomfort.
Trial registration: ISRCTN24802609 and ISRCTN13359367. Registered 23 March 2015 and 2 September 2015. Keywords: Dry eye, Eyelid, Ocular discomfort, TRPM8

1-s2.0-S0091674913017107-main

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Abstract