The central mechanisms through which glial cells regulate whole body metabolism remain poorly understood. Here, we identify Notch signaling in hypothalamic oligodendrocyte lineage cells as a previously unrecognized regulator of systemic energy homeostasis. Pharmacological inhibition of the Notch ligands Jagged1 (Jag1) and Jagged2 (Jag2) induces rapid and reversible weight loss across diverse physiological and metabolic contexts independently of toxicity or caloric intake. Single-nucleus transcriptomic analyses identify hypothalamic oligodendrocyte precursor cells (OPCs) as the principal Notch-responsive population following systemic Jag1/2 inhibition and reveal expansion of a metabolically specialized GPR17⁺ intermediate state characterized by enhanced oxidative metabolism and increased predicted communication with hypothalamic neurons. This glial remodeling is accompanied by fasting-like transcriptional reprogramming of AgRP neurons, reorganization of melanocortin-autonomic circuit activity, and activation of peripheral catabolic programs. Importantly, selective deletion of Notch1/2 in hypothalamic OPCs recapitulates the major physiological and metabolic effects of systemic Jag1/2 inhibition, establishing oligodendrocyte Notch signaling as a causal regulator of whole-body metabolism. Together, our findings establish Notch-dependent oligodendrocyte state transitions as a previously unrecognized mechanism linking glial plasticity to systemic energy homeostasis.

Mature neocortical pyramidal cells functionally express two sodium channel (NaV) isoforms: NaV1.2 and NaV1.6. These isoforms are differentially localized to pyramidal cell compartments, and as such are thought to contribute to different aspects of neuronal excitability. But determining their precise roles in pyramidal cell excitability has been hampered by a lack of tools that allow for selective, acute block of each isoform individually. Here, we leveraged aryl sulfonamide-based molecule (ASC) inhibitors of NaV channels that exhibit state-dependent block of both NaV1.2 and NaV1.6, along with knock-in mice with changes in NaV1.2 or NaV1.6 structure that prevents ASC binding. This allowed for acute, potent, and reversible block of individual isoforms that permitted dissection of the unique contributions of NaV1.2 and NaV1.6 in pyramidal cell excitability. Remarkably, block of each isoform had contrasting—and in some situations, opposing—effects on neuronal action potential output, with NaV1.6 block decreasing and NaV1.2 block increasing output. Thus, NaV isoforms have unique roles in regulating different aspects of pyramidal cell excitability, and our work may help guide the development of therapeutics designed to temper hyperexcitability through selective NaV isoform blockade.

Mature neocortical pyramidal cells functionally express two sodium channel (Na V ) isoforms: Na V 1.2 and Na V 1.6. These isoforms are differentially localized to pyramidal cell compartments, and as such are thought to contribute to different aspects of neuronal excitability. But determining their precise roles in pyramidal cell excitability has been hampered by a lack of tools that allow for selective, acute block of each isoform individually. Here, we leveraged aryl sulfonamide-based molecule (ASC) inhibitors of Na V channels that exhibit state-dependent block of both Na V 1.2 and Na V 1.6, along with knock-in mice with changes in Na V 1.2 or Na V 1.6 structure that prevents ASC binding. This allowed for acute, potent, and reversible block of individual isoforms that permitted dissection of the unique contributions of Na V 1.2 and Na V 1.6 in pyramidal cell excitability. Remarkably, block of each isoform had contrasting-and in some situations, opposing-effects on neuronal action potential output, with Na V 1.6 block decreasing and Na V 1.2 block increasing output. Thus, Na V isoforms have unique roles in regulating different aspects of pyramidal cell excitability, and our work may help guide development of therapeutics designed to temper hyperexcitability through selective Na V isoform blockade.

Background and Aims This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients.Methods We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence.Results Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom.Conclusions Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metabolite─generated by aldehyde oxidase (AO)─possessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.

Sterile alpha and TIR motif containing 1 (SARM1) is an inducible NADase that localizes to mitochondria throughout neurons and senses metabolic changes that occur after injury. Minimal proteomic changes are observed upon either SARM1 depletion or activation, suggesting that SARM1 does not exert broad effects on neuronal protein homeostasis. However, whether SARM1 activation occurs throughout the neuron in response to injury and cell stress remains largely unknown. Using a semiautomated imaging pipeline and a custom-built deep learning scoring algorithm, we studied degeneration in both mixed-sex mouse primary cortical neurons and male human-induced pluripotent stem cell-derived cortical neurons in response to a number of different stressors. We show that SARM1 activation is differentially restricted to specific neuronal compartments depending on the stressor. Cortical neurons undergo SARM1-dependent axon degeneration after mechanical transection, and SARM1 activation is limited to the axonal compartment distal to the injury site. However, global SARM1 activation following vacor treatment causes both cell body and axon degeneration. Context-specific stressors, such as microtubule dysfunction and mitochondrial stress, induce axonal SARM1 activation leading to SARM1-dependent axon degeneration and SARM1-independent cell body death. Our data reveal that compartment-specific SARM1mediated death signaling is dependent on the type of injury and cellular stressor.

NaV1.7, also known as the sodium voltage-gated channel alpha subunit 9 (SCN9A), plays a critical role in action potential induction and conduction in pain-causing nociceptors. Accordingly, NaV1.7 blockers may be effective non-opioids analgesics. SCN9A is also expressed in autonomic neurons, but its functional role in the autonomic system is less established. We employed three highly selective NaV1.7 inhibitors to investigate the role of NaV1.7 in action potential conduction in postganglionic sympathetic nerves and in sympathetic adrenergic contractions of blood vessels. Our single neuron rt-PCR analysis revealed that nearly all neurons (32/39) isolated from guinea pig stellate ganglia expressed NaV1.7 mRNA. Genentech Pharmaceuticals provided a highly selective NaV1.7 blocker, GNE8493, which exhibits a selectivity ranging from 500 to 5000-fold for NaV1.7 over all other NaV subtypes. SiteOne Pharmaceuticals contributed another mechanistically distinct and highly selective NaV1.7 blocker, ST2262, with a selectivity of 1000-fold for human NaV1.7 over other NaV subtypes (as reported in Sci Rep. 10:14791, 2020). Unfortunately, ST2262 has little affnity for rodent and guinea pig NaV1.7. We quantified the effect of GNE8493 on postganglionic compound actional potentials (CAP) within the sympathetic trunk of superior cervical ganglia (SCG). GNE8493 inhibited the CAP in the postganglionic neurons of SCG by ~ 70% (n=5, p<0.01). With such a significant impact of the NaV1.7 blocker on post-ganglionic actional potential conduction, we were prompted to explore its potential physiological relevance. Our study involved the isolated pulmonary arteries from anonymous human lung donors (obtained from IIAM) and isolated pulmonary arteries and the abdominal aorta from guinea pigs. We measured smooth muscle tension using standard tissue bath techniques. Stimulation of intrinsic nerves was achieved through electrical field stimulation (EFS) at 12V, 1msec, and 10Hz for 30 seconds, resulting in rapid contractions in all tissues. These contractions were completely prevented by the nonselective NaV1 blocker tetrodotoxin and by the alpha adrenoceptor antagonist prazosin. The contractions were quantified as a percentage of the maximum adrenergic contraction achievable, induced by the addition of 100 μM phenylephrine at the end of the experiment. In guinea pigs, GNE8493 completely blocked sympathetic contractions of the isolated pulmonary arteries (IC50 of -log (M) 5.8) and abdominal aorta (IC50 of -log (M) 6.4 ± 0.3). In human pulmonary arteries, GNE8493 (1 μM) and ST2262 inhibited the sympathetic contractions 94% and 87%, respectively (p<0.01, n=6). These findings support the hypothesis that pharmacological inhibition of NaV1.7 using selective inhibitors has the potential to reduce sympathetic function in specific vascular beds. R35HL155671 - BJU F32HL170490 - JSK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NaV1.7 plays a crucial role in inducing and conducting action potentials in pain-transducing sensory nociceptor fibres, suggesting that NaV1.7 blockers could be effective non-opioid analgesics. While SCN9A is expressed in both sensory and autonomic neurons, its functional role in the autonomic system remains less established. Our single neuron rt-PCR analysis revealed that 82% of sympathetic neurons isolated from guinea-pig stellate ganglia expressed NaV1.7 mRNA, with NaV1.3 being the only other tetrodotoxin-sensitive channel expressed in approximately 50% of neurons. We investigated the role of NaV1.7 in conducting action potentials in postganglionic sympathetic nerves and in the sympathetic adrenergic contractions of blood vessels using selective NaV1.7 inhibitors. Two highly selective NaV1.7 blockers, GNE8493 and PF 05089771, significantly inhibited postganglionic compound action potentials by approximately 70% (P < 0.01), with residual activity being blocked by the NaV1.3 inhibitor, ICA 121431. Electrical field stimulation (EFS) induced rapid contractions in guinea-pig isolated aorta, pulmonary arteries, and human isolated pulmonary arteries via stimulation of intrinsic nerves, which were inhibited by prazosin or the NaV1 blocker tetrodotoxin. Our results demonstrated that blocking NaV1.7 with GNE8493, PF 05089771, or ST2262 abolished or strongly inhibited sympathetic adrenergic responses in guinea-pigs and human vascular smooth muscle. These findings support the hypothesis that pharmacologically inhibiting NaV1.7 could potentially reduce sympathetic and parasympathetic function in specific vascular beds and airways. KEY POINTS: 82% of sympathetic neurons isolated from the stellate ganglion predominantly express NaV1.7 mRNA. NaV1.7 blockers inhibit action potential conduction in postganglionic sympathetic nerves. NaV1.7 blockade substantially inhibits sympathetic nerve-mediated adrenergic contractions in human and guinea-pig blood vessels. Pharmacologically blocking NaV1.7 profoundly affects sympathetic and parasympathetic responses in addition to sensory fibres, prompting exploration into the broader physiological consequences of NaV1.7 mutations on autonomic nerve activity.

Sensory neurons of the vagal ganglia (VG) innervate lungs and play a critical role in maintaining airway homeostasis. However, the specific VG neurons that innervate lungs, and the mechanisms by which these neurons sense and respond to airway insults, are not well understood. Here, we identify a subpopulation of lung-innervating VG neurons defined by their expression of Tmc3 . Single cell transcriptomics illuminated several subpopulations of Tmc3+ sensory neurons, revealing distinct Piezo2 - and Trpv1 -expressing subclusters. Furthermore, Tmc3 deficiency in VG neurons leads to global and subcluster specific transcriptional changes related to metabolic and ion channel function. Importantly, we show that broncho-constriction and dilation can be modulated through inhibition or activation of Tmc3+ VG neurons resulting in a decrease or increase of end-expiratory lung volume, respectively. Together, our data show that Tmc3 is a marker of lung-innervating neurons and may play a pivotal role in maintaining fundamental inspiratory and expiratory processes. Significance Harnessing the neuronal mechanisms that regulate lung function offers potential alternatives to existing corticosteroid treatment regimens for respiratory illness associated with acute bronchoconstriction including asthma, COPD, and emphysema. Our findings define Transmembrane channel-like 3 , Tmc3 , as a marker of lung-innervating sensory neurons, identify distinct subpopulations of Tmc3 + neurons with unique transcriptional profiles, and show that activation or inhibition of these neurons has a significant impact on airway function. Our work highlights potential avenues of novel targeted intervention in respiratory conditions driven by dysfunctional neuronal reflexes.

The voltage-gated sodium (Na V ) channel Na V 1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available Na V channel-blocking drugs are not selective among the nine Na V channel subtypes, Na V 1.1–Na V 1.9. Moreover, the two currently known classes of Na V 1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure–activity relationships of the acylsulfonamide class of Na V 1.7 inhibitors, exemplified by the clinical development candidate GDC-0310 , has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the Na V 1.7 channel, we pursued high-resolution ligand-bound Na V 1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the Na V 1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in Na V channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective Na V channel modulators targeting VSD4.

We performed single-nucleus RNA-sequencing on adult human spinal cord using a neuronal nuclei enrichment strategy. We obtained transcriptomic profiles of >14,000 spinal neurons, including a small population of motor neurons that shares similarities with mouse motor neurons and can be subdivided into alpha and gamma subtypes. We sought to compare our results to those from a recent study by Yadav and colleagues, which provides a single-nucleus transcriptomic atlas of the human spinal cord. While most neuronal nuclei from both studies share similar features, our results from motor neurons differ substantially. We reanalyzed their RNA-sequencing data and provide evidence that the authors incorrectly identified cholinergic cellular debris as motor neuron nuclei in their dataset, raising doubts about their conclusions regarding motor neurons. Our findings underscore the challenges associated with transcriptionally profiling motor neurons from the spinal cord because of their rarity. We propose specific enrichment strategies and recommend important quality control measures for future transcriptional profiling studies involving human spinal cord tissue and rare cell types.

Abstract Background Dose-limiting toxicities significantly impact the benefit/risk profile of many drugs. Whole genome sequencing (WGS) in patients receiving drugs with dose-limiting toxicities can identify therapeutic hypotheses to prevent these toxicities. Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting neurological toxicity of chemotherapies with no effective approach for prevention. Methods We conducted a genetic study of time-to-first peripheral neuropathy event using 30× germline WGS data from whole blood samples from 4900 European-ancestry cancer patients in 14 randomized controlled trials. A substantial number of patients in these trials received taxane and platinum-based chemotherapies as part of their treatment regimen, either standard of care or in combination with the PD-L1 inhibitor atezolizumab. The trials spanned several cancers including renal cell carcinoma, triple negative breast cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, and melanoma. Results We identified a locus consisting of low-frequency variants in intron 13 of GRID2 associated with time-to-onset of first peripheral neuropathy (PN) indexed by rs17020773 (p = 2.03 × 10−8, all patients, p = 6.36 × 10−9, taxane treated). Gene-level burden analysis identified rare coding variants associated with increased PN risk in the C-terminus of GPR68 (p = 1.59 × 10−6, all patients, p = 3.47 × 10−8, taxane treated), a pH-sensitive G-protein coupled receptor (GPCR). The variants driving this signal were found to alter predicted arrestin binding motifs in the C-terminus of GPR68. Analysis of snRNA-seq from human dorsal root ganglia (DRG) indicated that expression of GPR68 was highest in mechano-thermo-sensitive nociceptors. Conclusions Our genetic study provides insight into the impact of low-frequency and rare coding genetic variation on PN risk and suggests that further study of GPR68 in sensory neurons may yield a therapeutic hypothesis for prevention of CIPN.

Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overex-pression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low -threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action poten-tials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.

Single-nucleus RNA sequencing (snRNA-seq) is a powerful approach to study cellular heterogeneity within and across tissues. We and others have previously performed snRNA-seq on adult mouse spinal cord. 1 Blum J.A. Klemm S. Shadrach J.L. Guttenplan K.A. Nakayama L. Kathiria A. Hoang P.T. Gautier O. Kaltschmidt J.A. Greenleaf W.J. Gitler A.D. Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons. Nat. Neurosci. 2021; 24: 572-583 Crossref PubMed Scopus (78) Google Scholar ,2 Alkaslasi M.R. Piccus Z.E. Hareendran S. Silberberg H. Chen L. Zhang Y. Petros T.J. Le Pichon C.E. Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord. Nat. Commun. 2021; 12: 2471https://doi.org/10.1038/s41467-021-22691-2 Crossref PubMed Scopus (45) Google Scholar To begin to investigate human motor neurons, we performed a pilot snRNA-seq experiment on adult human spinal cord and found discrepancies with Yadav et al., who recently performed snRNA-seq on human spinal cord. 3 Yadav A. Matson K.J.E. Li L. Hua I. Petrescu J. Kang K. Alkaslasi M.R. Lee D.I. Hasan S. Galuta A. et al. A cellular taxonomy of the adult human spinal cord. Neuron. 2023; 111: 328-344.e7https://doi.org/10.1016/j.neuron.2023.01.007 Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar A cellular taxonomy of the adult human spinal cordYadav et al.NeuronFebruary 01, 2023In BriefYadav, Matson, et al. use single-nucleus RNA sequencing, spatial transcriptomics, and immunohistochemistry to profile the cell types of the adult human spinal cord, identifying 64 glial and neuronal populations. This resource reveals how the unique molecular environments of specific cell types could contribute to chronic pain or neurodegeneration. Full-Text PDF Open AccessA reproducible signature of cytoskeletal and ALS-related genes in human motoneuronsYadav et al.NeuronDecember 06, 2023In BriefWe recently created an atlas of the adult human lumbar spinal cord.1 As part of this work, we found that motoneurons are distinguished by gene expression related to cytoskeletal structure, cell size, and amyotrophic lateral sclerosis (ALS). We observed this pattern through single-nucleus RNA sequencing (snRNA-seq) (seven donors), validated it with spatial transcriptomics (five donors), and demonstrated it at the protein level for several key factors (four donors). Full-Text PDF

Sensory neurons of the dorsal root ganglion (DRG) are critical for maintaining tissue homeostasis by sensing and initiating responses to stimuli. While most preclinical studies of DRGs are conducted in rodents, much less is known about the mechanisms of sensory perception in primates. We generated a transcriptome atlas of mouse, guinea pig, cynomolgus monkey, and human DRGs by implementing a common laboratory workflow and multiple data-integration approaches to generate high-resolution cross-species mappings of sensory neuron subtypes. Using our atlas, we identified conserved core modules highlighting subtype-specific biological processes related to inflammatory response. We also identified divergent expression of key genes involved in DRG function, suggesting species-specific adaptations specifically in nociceptors that likely point to divergent function of nociceptors. Among these, we validated that TAFA4, a member of the druggable genome, was expressed in distinct populations of DRG neurons across species, highlighting species-specific programs that are critical for therapeutic development.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.