Shim J, Tanaka B, Taub DG, Mis MA, Schulman BR , et al.
Brain : a journal of neurology •
Inherited erythromelalgia, small fibre neuropathy and paroxysmal extreme pain disorder are caused by gain-of-function mutations in the voltage-gated sodium channel Nav1.7. It remains unknown how different mutations in the same channel enhancing electrogenesis in sensory neurons results in such distinct disease presentations. Most of the work analysing the impact of these mutations on electrophysiological properties has used overexpression systems in cell lines and rodent sensory neurons, which might differ from the natural context. We have differentiated sensory neurons from induced pluripotent stem cells derived from patient samples that have the Nav1.7 A1632G mutation. This strategy reveals changes in electrophysiological properties, not previously observed in cell lines, that might be important for disease presentation. Furthermore, using CRISPR/Cas9, we corrected this mutation, which reduced the underlying hyperexcitability, providing a path for personalized medicine to treat these disorders, and we introduced the mutation into control induced pluripotent stem cells, which generated hyperexcitability, providing causality. Induced pluripotent stem cell sensory neurons are a robust, scalable and relevant model to study the effects of gain-of-function mutations in ion channels in pain-related disorders.
Wimalasena NK, Taub DG, Shim J, Hakim S, Kawaguchi R , et al.
Experimental neurology •
Gain-of-function mutations in Scn9a, which encodes the peripheral sensory neuron-enriched voltage-gated sodium channel Na1.7, cause paroxysmal extreme pain disorder (PEPD), inherited erythromelalgia (IEM), and small fiber neuropathy (SFN). Conversely, loss-of-function mutations in the gene are linked to congenital insensitivity to pain (CIP). These mutations are evidence for a link between altered sodium conductance and neuronal excitability leading to somatosensory aberrations, pain, or its loss. Our previous work in young adult mice with the Na1.7 gain-of-function mutation, I228M, showed the expected DRG neuron hyperexcitability, but unexpectedly the mice had normal mechanical and thermal behavioral sensitivity. We now show that with aging both male and female mice with this mutation unexpectedly develop a profound insensitivity to noxious heat and cold, as well skin lesions that span the body. Electrophysiology demonstrates that, in contrast to young mice, aged I228M mouse DRGs have a profound loss of sodium conductance and changes in activation and slow inactivation dynamics, representing a loss-of-function. Through RNA sequencing we explored how these age-related changes may produce the phenotypic changes and found a striking and specific decrease in C-low threshold mechanoreceptor- (cLTMR) associated gene expression, suggesting a potential contribution of this DRG neuron subtype to Na1.7 dysfunction phenotypes. A GOF mutation in a voltage-gated channel can therefore produce over a prolonged time, highly complex and unexpected alterations in the nervous system beyond excitability changes.
Alich TC, Röderer P, Szalontai B, Golcuk K, Tariq S , et al.
Frontiers in cellular neuroscience •
Human induced pluripotent stem cells (hiPSCs) are a promising approach to study neurological and neuropsychiatric diseases. Most methods to record the activity of these cells have major drawbacks as they are invasive or they do not allow single cell resolution. Genetically encoded voltage indicators (GEVIs) open the path to high throughput visualization of undisturbed neuronal activity. However, conventional GEVIs perturb membrane integrity through inserting multiple copies of transmembrane domains into the plasma membrane. To circumvent large add-ons to the plasma membrane, we used a minimally invasive novel hybrid dark quencher GEVI to record the physiological and pathological firing patterns of hiPSCs-derived sensory neurons from patients with inherited erythromelalgia, a chronic pain condition associated with recurrent attacks of redness and swelling in the distal extremities. We observed considerable differences in action potential firing patterns between patient and control neurons that were previously overlooked with other recording methods. Our system also performed well in hiPSC-derived forebrain neurons where it detected spontaneous synchronous bursting behavior, thus opening the path to future applications in other cell types and disease models including Parkinson's disease, Alzheimer's disease, epilepsy, and schizophrenia, conditions associated with disturbances of neuronal activity and synchrony.
