Optogenetically controlled human functional motor endplate for testing botulinum neurotoxins

Background The lack of physiologically relevant and predictive cell-based assays is one of the major obstacles for testing and developing botulinum neurotoxins (BoNTs) therapeutics. Human-induced pluripotent stem cells (hiPSCs)-derivatives now offer the opportunity to improve the relevance of cellular models and thus the translational value of preclinical data. Methods We investigated the potential of hiPSC-derived motor neurons (hMNs) optical stimulation combined with calcium imaging in cocultured muscle cells activity to investigate BoNT-sensitivity of an in vitro model of human muscle-nerve system. Results Functional muscle-nerve coculture system was developed using hMNs and human immortalized skeletal muscle cells. Our results demonstrated that hMNs can innervate myotubes and induce contractions and calcium transient in muscle cells, generating an in vitro human motor endplate showing dose-dependent sensitivity to BoNTs intoxication. The implementation of optogenetics combined with live calcium imaging allows to monitor the impact of BoNTs intoxication on synaptic transmission in human motor endplate model. Conclusions Altogether, our findings demonstrate the promise of optogenetically hiPSC-derived controlled muscle-nerve system for pharmaceutical BoNTs testing and development.

Human motor endplate remodeling after traumatic nerve injury

OBJECTIVECurrent management of traumatic peripheral nerve injuries is variable with operative decisions based on assumptions that irreversible degeneration of the human motor endplate (MEP) follows prolonged denervation and precludes reinnervation. However, the mechanism and time course of MEP changes after human peripheral nerve injury have not been investigated. Consequently, there are no objective measures by which to determine the probability of spontaneous recovery and the optimal timing of surgical intervention. To improve guidance for such decisions, the aim of this study was to characterize morphological changes at the human MEP following traumatic nerve injury.METHODSA prospective cohort (here analyzed retrospectively) of 18 patients with traumatic brachial plexus and axillary nerve injuries underwent biopsy of denervated muscles from the upper extremity from 3 days to 6 years after injury. Muscle specimens were processed for H & E staining and immunohistochemistry, with visualization via confocal and two-photon excitation microscopy.RESULTSImmunohistochemical analysis demonstrated varying degrees of fragmentation and acetylcholine receptor dispersion in denervated muscles. Comparison of denervated muscles at different times postinjury revealed progressively increasing degeneration. Linear regression analysis of 3D reconstructions revealed significant linear decreases in MEP volume (R = −0.92, R2 = 0.85, p = 0.001) and surface area (R = −0.75, R2 = 0.56, p = 0.032) as deltoid muscle denervation time increased. Surprisingly, innervated and structurally intact MEPs persisted in denervated muscle specimens from multiple patients 6 or more months after nerve injury, including 2 patients who had presented > 3 years after nerve injury.CONCLUSIONSThis study details novel and critically important data about the morphology and temporal sequence of events involved in human MEP degradation after traumatic nerve injuries. Surprisingly, human MEPs not only persisted, but also retained their structures beyond the assumed 6-month window for therapeutic surgical intervention based on previous clinical studies. Preoperative muscle biopsy in patients being considered for nerve transfer may be a useful prognostic tool to determine MEP viability in denervated muscle, with surviving MEPs also being targets for adjuvant therapy.