The primary goal of this project is to study, using human induced-nociceptor neurons, the cellular mechanisms contributing to peripheral sensory diseases that cause pain and small fiber neuropathy. The induced-nociceptors (i-nociceptors) will be generated by directly converting (transdifferentiating) a somatic cell (in this case fibroblasts) into a cell with a different and distinct lineage (in this case, nociceptor neurons). e have established a protocol for efficiently transdifferentiating mouse and human fibroblasts into nociceptors by expression of a small set of defined transcription factors. These i-nociceptors have the morphology and marker expression patterns expected of adult nociceptors. The cells also respond with a robust calcium influx to capsaicin a TRPV1 agonist, mustard oil, a TRPA1 agonist and a ?-methylene ATP, a P2X3 agonist. The neurons have, moreover, the broad action potentials typical of nociceptors, which are contributed to by a tetrodotoxin-resistant sodium current and express transcripts for quintessential nocicepter markers Nav1.7, Nav1.8, TRPV1, and P2X3. The induced neurons have, therefore, sufficient features of native mature nociceptors to enable us to model key aspects of nociceptive transduction, membrane excitability and neuropathy. We now plan to characterize the function and expression profiles of human i-nociceptors. We will also use newly developed gene-targeting techniques to introduce gain- and loss-of-function mutations of Nav1.7 known from human genetic studies to produce pain or congenital analgesia into mouse and human i-nociceptors. These nociceptors will be compared with i-nociceptors derived from patients with inherited erythromelalgia (IEM) due to Nav1.7 mutations. Furthermore, we will compare i-nociceptors that are isogenic except for a defined Nav1.7 mutation, by correcting the mutation in a patient-derived iPSC line. We anticipate measuring a clear hyperexcitability phenotype in nociceptors with natural or engineered Nav1.7 gain-of-function mutations, and a loss of this phenotype when the mutation is corrected. Finally, we will use human i-nociceptors to study how a cancer chemotherapeutic agent oxaliplatin may cause pain and neuropathy. The proposal will enable exploitation of human nociceptors to study human disease conditions and screen for novel treatment strategies.
The goal of this grant is to generate in a dish, those human sensory neurons that initiate pain, in order to identify the mechanisms responsible for peripheral nervous system sensory diseases. The pain sensory neurons will be converted from fibroblasts by changing the set of gene instructions that determine cell fate, converting a cell from one determined fate (fibroblast) to another (pain sensory neuron). The function of the induced human sensory neurons will be fully- characterized. This will provide a novel and unique opportunity to study the function of human pain neurons at a molecular and cellular level, and to determine what changes occur in sensory neurons derived from patients with specific diseases, and how. Furthermore, we will create mutations in genes known to cause pain, and explore the effects of this on human and mouse sensory neuron excitability and growth. The approach will enable detailed analysis of the cellular biology of pain and small fiber neuropathy, and can be used to screen for new treatments.
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