The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII). We show that active NMII light chains are colocalized with actin rings and organized in a circular periodic manner throughout the axon shaft. In contrast, NMII heavy chains are mostly positioned along the longitudinal axonal axis, being able to crosslink adjacent rings. NMII filaments can play contractile or scaffolding roles determined by their position relative to actin rings and activation state. We also show that MPS destabilization through NMII inactivation affects axonal electrophysiology, increasing action potential conduction velocity. In summary, our findings open new perspectives on axon diameter regulation, with important implications in neuronal biology.

Microfluidic-based platform to mimic the in vivo peripheral administration of neurotropic nanoparticles.

AIM: Propose a nanoparticle for neuron-targeted retrograde gene delivery and describe a microfluidic-based culture system to provide insight into vector performance and safety. METHODS: Using compartmentalized neuron cultures we dissected nanoparticle bioactivity upon delivery taking advantage of (quantitative) bioimaging tools. RESULTS: Targeted and nontargeted nanoparticles were internalized at axon terminals and retrogradely transported to cell bodies at similar average velocities but the former have shown an axonal flux 2.7-times superior to nontargeted nanoparticles, suggesting an improved cargo-transportation efficiency. The peripheral administration of nanoparticles to axon terminals is nontoxic as compared with their direct administration to the cell body or whole neuron. CONCLUSION: A neuron-targeted nanoparticle system was put forward. Microfluidic-based neuron cultures are proposed as a powerful tool to investigate nanoparticle bio-performance.

In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles.

A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies.