Browsing by Subject "TRPA1"

MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs that play a crucial role in intracellular regulation of gene expression. Emerging evidence indicates that miRNAs can also be found extracellularly in various body fluids, including serum and cerebrospinal fluid (CSF), raising the possibility that these secreted miRNAs could serve as neuromodulators and disease biomarkers. Among these miRNAs, let-7b has been previously identified as a pain inducer through its actions in the peripheral nociceptive system, while TLR7 has been recognized as a critical regulator of pain and itch (pruritus). However, the role of let-7b in spinal cord synaptic transmission and its potential involvement in chronic pain and itch remains unexplored. In this thesis, our investigation commences by demonstrating that let-7b activates a non-canonical pathway of TLR7 in the presence of TRPA1 ion channels. Subsequently, we found that HEK cells and dissociated dorsal root ganglion (DRG) neurons, in which TLR7 and TRPA1 are expressed, exhibit robust calcium responses to extracellular perfusion of let-7b. Furthermore, intrathecal injection of a low dose of let-7b (1 μg) induces short-term (< 24 hours) mechanical and heat hypersensitivity. Mechanistic insights emerge from our observation that synthetic let-7b perfusion of spinal cord slices augments calcium signaling in synaptic terminals and excitatory synaptic transmission (miniature EPSCs) in spinal nociceptive neurons. These effects are contingent on TLR7 and TRPA1 ion channels. Notably, endogenous let-7b is enriched in spinal cord synaptosomes, and its expression is upregulated in DRG neurons, spinal cord tissue, and CSF in response to peripheral inflammation. To explore the role of endogenous let-7b in synaptic transmission and pain, we designed a let-7b antagomir to neutralize secreted let-7b function. Intriguingly, spinal administration of let-7b antagomir attenuates inflammation-induced mechanical pain and synaptic plasticity, suggesting an endogenous role of let-7b in inflammation-induced synaptic plasticity. Additionally, as TLR7 is expressed in spinal microglia, an intrathecal injection of high dose let-7b (10 μg) leads to persistent mechanical allodynia lasting over two weeks, and this effect in abrogated in Tlr7−/− knockout mice. This let-7b induced microgliosis is mitigated by intrathecal administration of minocycline, which suppresses let-7b-induced mechanical allodynia only in male mice but not in female mice. In a mouse cheek model, intradermal injection of let-7b induces both pain (wiping behavior) and itch (scratching behavior). Notably, endosome inhibitors selectively block let-7b-induced itch, without affecting let-7b-induced acute pain. Endosome inhibitors further suppress let-7b-induced persistent calcium increases in cultured trigeminal ganglion neurons. Our findings bring forth the concept that TLR7/TRPA1 axis activation can elicit pain and itch sensations via distinct surface and intracellular calcium signaling pathways within primary sensory neurons. Furthermore, we developed a mouse model of chronic itch induced by cutaneous T cell lymphoma (CTCL). This model is characterized by notable lymphoma growth, chronic scratching for over 60 days, neural innervation of tumor tissue, and elevated levels of let-7b in DRGs. Furthermore, intradermal or systemic administration of let-7b antagomir can alleviate CTCL-induced pruritus. Collectively, this thesis elucidates the novel molecular and cellular mechanisms of pain and itch induced by extracellular miRNA (let-7b), thereby deepening our understanding of the cell biology of primary sensory neurons and neurobiology of pain and itch.

Transient receptor potential channel subfamily A member 1 (TRPA1) is a Ca2+-permeable cation channel that serves as the primary sensor of environmental irritants, noxious substances, and temperature. Many TRPA1 agonists are electrophiles that are recognized by TRPA1 via covalent bond modifications of specific cysteine residues located in the cytoplasmic domains. TRPA1 is also a temperature activated channel displaying unique species-specific thermo sensitivity. Preceding this work, however, a mechanistic understanding of electrophile sensing by TRPA1 has been limited by a lack of structural information. Moreover, the mechanism by which TRPA1 sense temperature has been elusive. Using cryo-electron microscopy, we determined the structures of nanodisc-reconstituted human TRPA1 in ligand free state and in complex with the covalent agonists JT010 or BITC at 2.8, 2.9, and 3.1 Å, respectively. Our structural and functional studies provide the molecular basis for electrophile recognition by the extraordinarily reactive Cys621 in TRPA1 and grant mechanistic insights into electrophile-dependent conformational changes in TRPA1. This work illustrates the fundamental principles of irritant sensing in humans at the molecular level and provides a platform for future drug development targeting TRPA1. Moreover, we determined the cryo-EM structure of rattlesnake TRPA1 in nanodisc-reconstituted condition at 3.3 Å. This structural revealed a novel N-terminal ankyrin repeat domain that was not resolved in previous structures. Our structural and functional studies on rattlesnake TRPA1 provides a framework in understanding the principles of thermo sensitivity in TRPA1.