Tetracycline transactivator overexpression in keratinocytes triggers a TRPV1 primary sensory neuron-dependent neuropathic itch
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Abstract
Mouse models that combine tetracycline-controlled gene expression systems and conditional genetic activation can tightly regulate transgene expression in discrete cell types and tissues. However, the commonly used Tet-Off variant, tetracycline transactivator (tTA), when overexpressed and fully active, can lead to developmental lethality, disease, or more subtle behavioral phenotypes. Here we describe a profound itch phenotype in mice expressing a genetically encoded tTA that is conditionally activated within the Phox2a lineage. Phox2a; tTA mice develop intense, localized scratching and regional skin lesions that can be controlled by the tTA inhibitor, doxycycline. As gabapentin, but not morphine, relieved the scratching, we consider this phenotype to result from chronic neuropathic itch, not pain. In contrast to the Phox2a lineage, mice with tTA activated within the Phox2b lineage, which has many similar areas of recombination within the nervous system, did not recapitulate the scratching phenotype. In Phox2a-Cre mice, but not Phox2b-Cre, intense Cre-dependent reporter expression was found in skin keratinocytes which formed the area at which skin lesions developed. Most interestingly, repeated topical application of the DREADD agonist, CNO, which chronically induced G i signaling in Phox2a-keratinocytes, completely reversed the localized scratching and skin lesions. Furthermore, ablation of TRPV1-expressing, primary afferent neurons reduced the scratching with a time course comparable to that produced by G i -DREADD inhibition. These temporal properties suggest that the neuropathic itch condition arises not only from localized keratinocyte activation of peripheral nerves but also from a persistent, gabapentin-sensitive state of central sensitization.
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Sure, np. Let's connect by email.
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I would love to see the results of the nalfurafine and difelikefalin experiments in this model! Would you be open to contacting me when you have those results? My email is jasmine.neal@arcadiascience.com. :)
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Great question. We like the nalfurafine experiment to test whether central KOR activation reduces spontaneous scratching in our model. I hope to provide an update on that soon. If nalfurafine is effective, introducing difelikefalin, a peripherally restricted drug, would strengthen the conclusion of central effects, if ineffective in comparison. On the other hand, clearly, an ongoing peripheral drive is important clinically, and how much that is happening in our transgenic mouse model is interesting. Thanks for bringing up the translational perspective and the reference to the CKD study.
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Thanks. It is interesting to consider the dynamics of skin recovery without those neurons and in the hM4Di experiment. However, as your second question implies, we are eager to use transcriptomics to identify factors that initiate the itch, not necessarily those factors that maintain the itch. As tTA is an exogenous protein, we don't know what to expect. Transcriptomic analysis may reveal a novel peripheral signal that provokes primary sensory neurons to drive central sensitization.
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Robust scratching starts at 7 weeks, yeah. Before that time, it's hard to tell the mice apart from WT controls. Though, a slight prodromal skin/hair phenotype is noticeable.
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transcriptomic analysis of keratinocytes
Do you think it could be helpful to do temporal transcriptomic analysis to reveal the dynamic nature of keratinocytes given that there was a two-month delay to reverse the scratching phenotype after TRPV1 ablation? Perhaps that wouldn't be useful since itch, in this case, is hypothesized to be sustained in the CNS rather than in the periphery?
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intraperitoneal injection of morphine
Did you consider testing kappa opioid receptor agonists such as nalfurafine and difelikefalin since KORs may be associated with itch attenuation while MORs may be associated with itch intensification? This would be especially interesting from a translation perspective since difelikefalin is usually used for chronic kidney disease-associated pruritus but is in the pipeline for AD.
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mice developed significant spontaneous scratching that increased over time
Wow, the results look profound. Just curious about the onset of the scratching behavior. I'm looking at the time course data in Sup Fig 1b... why did you start measuring scratching at 7 weeks? Is that when you started to notice scratching?
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