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.

Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerves. Moreover, the targeted nanoparticle treatment was correlated with a significantly higher density of myelinated axons in the distal stump of injured nerves, as well as with preservation of unmyelinated axon density as compared with controls and a protective role in injury-denervated muscles, preventing them from denervation. These results highlight the potential of TMCSH-HC nanoparticles as non-viral gene carriers to deliver therapeutic genes into the peripheral neurons and thus, pave the way for their use as an effective therapeutic intervention for peripheral neuropathies.

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Abstract

A novel MEA architecture with excellent electrophysiological recordings is presented, where planar microelectrodes are replaced by localized 3 × 3 arrays of mushroom-shaped microstructures. The micro-mushrooms in this islets configuration are not for membrane engulfment but rather for somata entrapment and neurites embracement. As extracellular signals have a significant contribution from axons initial segment, this MEA design also addresses the electrode-neurites electrical coupling. These islets act as strong physical cues, causing topotaxis and increasing by two-fold the probability for somata to localize in the islets. We carry this topotaxis study not only with rat cortical neurons but also with humanderived SH-SY5Y cells.

By changing the biodegradation rate of trimethyl chitosan-based nanoparticles (NPs) one was able to alter the NP ability to protect or efficiently release DNA and consequently, to modulate their intracellular dynamics. To address the influence of NP degradation rate in their transfection efficiency we took advantage of imaging flow cytometry, a high-throughput bioimaging technique, to unravel some critical aspects about NP formulation such as the distinction between internalized versus cell-associated/adsorbed NP, and even explore NP intracellular localization. Overall, our work provides novel information about the importance of vector degradation rate for gene delivery into cells, as a way to tune gene expression as a function of the desired application, and advances novel approaches to optimize nanoparticle formulation.

Dendrimers have emerged as a powerful class of nanomaterials in the nanomedicine field due to their unique structural features: globular, welldefined, highly branched and controllable structure, nanosizescale, low polydispersity, and the presence of several terminal groups that can be functionalized with different ligands simulating the multivalency present in different biological systems. Although in its infancy, the application of dendrimers as therapeutics or theranostic tools in central nervous system (CNS) disorders is already significant and has opened promising avenues in the treatment of many conditions where the inherent "smartness" of the dendritic structures is being explored to effectively target the CNS. Here we present an overview of the past and future challenges of the use of dendrimers to respond to one of the ultimate challenges in the (nano)medicine field: to attain CNS repair and regeneration.

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