Sodium ion channels in chronic pain: modeling human pain syndromes with induced pluripotent stem cell-derived sensory neurons

Kalia, Anil Kumar; Rothermel, Markus (Thesis advisor); Lampert, Angelika (Thesis advisor); Zimmer-Bensch, Geraldine Marion (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

Inherited mutations in voltage-gated sodium ion channels (Navs) cause disorders of membrane excitability, including chronic pain, epilepsy, myotonia, and cardiac arrhythmias. Navs initiate action potentials in excitable cells and play an important role in the detection and transmission of sensory information from periphery to higher brain centers. Nine different Nav channels (Nav1.1-Nav1.9) have been described in humans. Structurally, Nav channels are composed of one α subunit and one or more β subunits. Nav1.7 is found to be preferentially expressed in the peripheral nervous system and acts as threshold channels. Genetic studies have identified loss-of-function and gain-of-function mutations in Nav1.7 that result in congenital insensitivity to pain and chronic pain syndromes, respectively. Translational challenges from rodents to humans have resulted in failure to develop novel therapeutics for neurological and pain disorders. Human induced pluripotent stem cells (iPSCs) provide an attractive tool to model human diseases. It has also been found that Nav dimerization modulates mutation-induced gating changes in Nav1.5 and Nav1.7 indicating its importance in cardiac arrythmias and pain disorders, respectively. The present thesis work involves understanding of (a) the pharmacological effect of selective Nav1.7 blockers on Nav monomer and dimer complexes, (b) modeling a human pain disorder with iPSC-derived nociceptors and (c) functional characterization of iPSC-derived sensory neurons with a novel accelerated ectodermal differentiation protocol. Peptide toxins have been widely used to understand the complex gating properties of Nav channels. Protoxin-II (ProTx-II) has high affinity and selectivity towards the human Nav1.7 channel. In this project HEK cells stably expressing Nav1.7 were used to investigate the pharmacological effect of ProTx-II in Nav1.7 cells, transfected with the dimerization inhibitor difopein. Patch clamp recordings revealed that difopein treatment significantly reduced the inhibitory effect of ProTx-II on Nav1.7 ion channel currents. This indicates that dimerization of Nav may play an important role in modulating the potency of selective Nav blockers and hence their therapeutic window in treating chronic pain disorders. Inherited erythromelalgia (IEM) is an autosomal dominant neuropathy associated with burning pain, swelling and redness of the extremities. IEM is genetically linked to gain-of-function mutations of the SCN9A gene encoding Nav1.7. In this project, blood cells of an IEM patient carrying the SCN9A mutation were reprogrammed towards iPSCs using the Yamanaka transcription factors. Isogenic controls were generated by CRISPR/Cas9-mediated genome editing. iPSCs were further differentiated into nociceptors with small molecule modulators targeting specific signaling pathways. Our results show increased nociceptor excitability and spontaneous activity in the diseased sensory neurons as compared to Isogenic controls, which is likely the reason why the patient suffers from pain attacks. In addition, as part of the multisensory approach we found that warm and noxious temperature stimuli resulted in increased firing frequency in patient derived sensory neurons. This study highlights the potential of iPSC-derived sensory neurons as model system to study the clinical phenotype of human pain disorders in vitro. Due to variability in efficacy, yield, and reproducibility of current differentiation protocols for the generation of iPSC-derived nociceptors, novel methods are needed to fill the gap and address these challenges. This project involves a novel differentiation method with an ectodermal induction for generation of iPSC-derived sensory neurons. We found that this protocol generates a homogenous dense neuronal network of immature sensory neurons within 7 days as compared to 10-14 days with the more conventional small molecule differentiation protocols, but mature neurons occurred about the same time. This protocol was further validated with diseased cell lines derived from patients suffering from pain disorders displaying cellular hyperexcitability. The results of this thesis will open new opportunities for investigating the mechanisms of pain disorders with in vitro disease modeling and to utilize the platform for identification of novel pharmacological modulators of Navs for personalized treatment to address the unmet medical need for chronic pain.