Analgesic effect of Botulinum toxin in neuropathic pain is sodium channel independent.

Botulinum neurotoxin type A BoNT/A is used off-label as a third line therapy for neuropathic pain. However, the mechanism of action remains unclear. In recent years, the role of voltage-gated sodium channels (Nav) in neuropathic pain became evident and it was suggested that block of sodium channels by BoNT/A would contribute to its analgesic effect. We assessed sodium channel function in the presence of BoNT/A in heterologously expressed Nav1.7, Nav1.3, and the neuronal cell line ND7/23 by high throughput automated and manual patch-clamp. We used both the full protein and the isolated catalytic light chain LC/A for acute or long-term extracellular or intracellular exposure. To assess the toxin's effect in a human cellular system, we differentiated induced pluripotent stem cells (iPSC) into sensory neurons from a healthy control and a patient suffering from a hereditary neuropathic pain syndrome (inherited erythromelalgia) carrying the Nav1.7/p.Q875E-mutation and carried out multielectrode-array measurements. Both BoNT/A and the isolated catalytic light chain LC/A showed limited effects in heterologous expression systems and the neuronal cell line ND7/23. Spontaneous activity in iPSC derived sensory neurons remained unaltered upon BoNT/A exposure both in neurons from the healthy control and the mutation carrying patient. BoNT/A may not specifically be beneficial in pain syndromes linked to sodium channel variants. The favorable effects of BoNT/A in neuropathic pain are likely based on mechanisms other than sodium channel blockage and new approaches to understand BoNT/A's therapeutic effects are necessary.

The pain target NaV1.7 is expressed late during human iPS cell differentiation into sensory neurons as determined in high-resolution imaging.

Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.