Cells and organs constantly experience mechanical forces. Neurons, in particular, are exposed to such stimuli during development, aging, disease, and normal activities like movement and homeostasis. Recent studies highlight the key role of microtubules (MTs) in mechanotransduction, adjusting cytoskeletal dynamics in response to mechanical cues. While the effects of acute forces on MTs are known, the impact of repetitive mechanical stimuli over time remains unclear. In this study, we applied repetitive mechanical motion to neurons from the dorsal root ganglia and analyzed responses at varying strain levels. A 10% strain caused MT and organelle damage, leading to cell death. In contrast, a 2.5% strain did not harm cells and instead stabilized MTs. A 5% strain caused damage to the MT structure and leads to MT destabilization, but neurons activate a molecular response to counteract and recover from this damage, suggesting the involvement of the Ras pathway in response to injury. These findings suggest that neurons can adapt to repetitive mechanical stress, maintaining homeostasis when strain is below a certain threshold. Our results improve understanding of how mechanical forces influence neuronal structure and function, and how cells respond to injury by initiating protective pathways.

[This corrects the article DOI: 10.3389/fbioe.2020.00144.].

Axons are sensitive to piconewton (pN) forces, yet the cellular mechanisms underlying this mechanosensitivity remain poorly understood. Axonal mechanotransduction has traditionally been attributed to molecular clutches and mechanosensitive ion channels, whereas cytoskeletal microtubules (MTs) have been regarded primarily as passive load-bearing structures. Here, we propose MTs as active elements in signal mechanotransduction. Using nano-pulling to apply controlled, directional pN forces to hippocampal neurons, we show that force induces axon elongation only when applied along the intrinsic polarity of axonal MTs. Live imaging of the MT plus-end marker EB3 reveals modulation of MT dynamics and enhanced MT growth when force is applied along MT polarity, but not in the opposite direction. Strikingly, restricting force generation to the axolemma, thereby uncoupling mechanical inputs from the axonal cytoskeleton, abolishes force-induced axon elongation.

Axon navigation is guided by spatial patterns of chemical and physical cues in the developing central nervous system. Following injury, these patterns are disrupted, the microenvironment evolves rapidly, and inhibitory molecules create a barrier to the regeneration of severed axons. We have recently developed a technology called nano-pulling designed to stimulate axon growth and regeneration by modulating neuronal mechanotransduction. In this paper, we demonstrate that nano-pulling can induce axon growth in hippocampal neurons even in the presence of repulsive cues, such as chondroitin sulfate proteoglycans, semaphorin 3A, microglial activation, and pro-inflammatory cytokines. Nano-pulling can also enhance the elongation of neural processes in neural progenitors transplanted into an organotypic spinal cord injury model that mimics the tissue complexity and inflammation seen in in vivo models. Our data suggest that nano-pulling could be used as a strategy to manipulate axon growth, overcoming certain extrinsic inhibitory factors.

Genome editing allows for the manipulation of genomic DNA for biotechnology and biomedical applications, but the specificity and control of the editing process remain a challenge. This study introduces a nano-switch for spatiotemporal control of the editing process. The basic module of the nano-switch (the monomer) is composed of a gold nanorod conjugated with the catalytically dead Cas9. Based on mathematical models, we established the design and the mechanism of action of the nano-switch. Briefly, when two monomers, guided by their respective guide RNAs, form a dimer onto the DNA and get irradiated with a near-infrared pulsed laser resonant at the plasmonic properties of the dimer, they generate a localized heat that triggers a thermal break onto the DNA. The nano-switch was generated, validated, and tested in zebrafish embryos at the 1-cell stage. Molecular analysis of irradiated embryos showed targeted DNA mutations, validating the efficacy of the nano-switch as a tool for conditional gene editing that integrates the if-when-where functions.

Mechanical force plays a pivotal role in all aspects of axon development. In this paper, the use of nano-pulling, a technology that enables the intracellular generation of extremely low mechanical forces is explored. It is demonstrated that force-mediated axon growth also exerts global effects that extend to the nuclear level. The mechanistic studies support a model in which exogenous forces induce microtubule stabilization, and significant remodeling of perinuclear microtubules, which preferentially align perpendicularly to the nuclear envelope. An increase in the lateral tension of the nucleus is observed, leading to substantial remodeling of nuclear morphology, characterized by an increase in nuclear grooves and a higher sphericity index (indicating less flattened nuclei). Notably, these changes in nuclear shape are linked to chromatin remodeling, resulting in global transcriptional activation.

Reconstructing the nigrostriatal pathway is one of the major challenges in cell replacement therapies for Parkinson's disease due to the lack of enabling technologies capable of guiding the reinnervation of dopaminergic precursors transplanted into the substantia nigra toward the striatum. This paper examines nano-pulling, as a technology to enable the remote manipulation of axonal growth. Specifically, an organotypic model consisting of co-cultures of the substantia nigra and the striatum is developed to demonstrate that when cortical neural progenitors are transplanted into the substantia nigra, nano-pulling can guide and enhance the elongation of neural projections toward the striatum. To provide additional evidence, induced pluripotent stem cell-derived dopaminergic progenitor neurospheres are generated and it is shown that nano-pulling can induce guided growth and promote the maturation of their neural processes. Altogether, this study demonstrates the potential of nano-pulling as an emerging technique to promote directed reinnervation within the central nervous system.

Alzheimer’s disease (AD) and related tauopathies such as frontotemporal dementia (FTD) or traumatic brain injury (TBI) are neurodegenerative disorders characterized by progressive loss of memory and cognitive function. The main histopathological features of AD are amyloid-β plaques and Tau neurofibrillary tangles, suggested to interfere with neuronal function and to cause microtubule (MT) destabilization. We recently demonstrated that low mechanical forces promote MT stabilization, which in turn promotes axon growth and neuronal maturation. As neurites may become dystrophic due to MT destabilization in tauopathies, we hypothesized that force-induced MT stabilization is neuroprotective in cell models subjected to tauopathy-like stress. We set up two different pathological cellular models subjecting them to AD-related Tau pathology stressors. We found that exposure of mouse primary neurons to Tau oligomers and neurons derived from human induced pluripotent stem cell (hiPSC) to amyloid-β oligomers resulted in neurotoxic effects such as axonal shortening, reduction in dendrite number, and MT destabilization. Mechanical stimulation (i) prevented delays in axonal extensions and dendrite sprouting, restoring axon outgrowth to physiological levels, and (ii) compensated for axonal MT destabilization by increasing MT stability to levels higher than in control conditions. In summary, we here demonstrate that low mechanical force can be used as a neuroprotective extrinsic factor to prevent MT destabilization and axon degeneration caused by AD-like or tauopathy-like stressors.

A novel Cas13d-based gene-editing approach has been developed to target viral RNAs in infected cells, reducing the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Zika virus (ZIKV) by up to 90% compared with controls. Despite its potential, the use of Cas13d as an antiviral faces several challenges that limit its effectiveness before reaching target cells. This study presents a proof-of-concept strategy for constructing Cas13d with gold nanoparticles (Au_NPs) to destroy SARS-CoV-2 and ZIKV genomes into cells. The Au_NPs Cas13d complexes were administered to Huh-7 cells infected with either virus, in single or multiple doses. The study demonstrated that Au_NPs Cas13d cuts target RNAs with comparable efficiency as lipofected ribonucleoprotein (RNP). Additionally, we found that Au_NPs Cas13d can spontaneously enter cells by endocytosis or diffusion, before the first 4 h of treatment. Au_NPs Cas13d co-localized with SARS-CoV-2 virions in early endosomes and reduced SARS-CoV-2 replication after a single administration, unlike RNPs, which showed no antiviral activity. However, Au_NPs Cas13d was less efficient at reducing ZIKV replication compared with lipofected Cas13d-RNPs, likely due to different intracellular localization. These results suggest that Au_NPs can be adapted as a new antiviral strategy, highlighting an innovative delivery method of Cas13d against viruses without the need for transfecting, providing a new gene-editing-based approach against emerging RNA viruses.

In recent years, the CRISPR/Cas9 technology has emerged as a highly efficient tool for cell gene editing. However, the delivery of the CRISPR/Cas9 system into cells remains a significant challenge, drastically limiting in vivo gene therapy applications. In this study, we present a transfection/transduction-free tool for intracellular delivery of the Cas9:gRNA ribonucleoprotein. The Cas9 enzyme is conjugated to a 12 nm gold nanoparticle through affinity binding between the 6x His-tag of the protein and the NTA-Ni²LJ groups on the nanoparticles. This link chemistry allows a fine control of the density of the enzymes decorating the particle surface, the orientation of the bonding and the stability of the interaction. Importantly, the surface chemistry of this nanoformulation has been precisely engineered to modulate the cellular internalization and localization. Thanks to this approach of precision chemistry, this nanoformulation demonstrated the ability to spontaneously enter human melanoma cells as monodispersed particles that localize in cell cytoplasm, endosomes, and nucleus. It also shows effective gene editing efficiency similarly to conventional transfection tools. This gold-based formulation of Cas9 represents a ready-to-use biotech editing tool, and a promising solution for direct in vivo gene editing applications. ### Competing Interest Statement The authors have declared no competing interest.

Genome editing comprises the most promising work in 21st century genetics, with molecular surgery as its practical horizon. For a successful clinical application of molecular surgery, e.g. to treat cancer, safe and efficient editing is crucial. Light-induced molecular surgery is the perfect tool for its excellent control over wavelength, power, and exposure time. Light is also an excellent tool for the detection of (living) cells. These modalities are ideal for opto-fluidics: combining integrated photonics and microfluidics in a chip. In this work, we used the TriPleX((R)) waveguide platform, comprised of silicon nitride and silicon oxide, to control high power (>1W) visible light. Living cells are inserted into a 100 micrometer wide microfluidic channel after which they are focused into a 25 micrometer wide section in its center using side-, back-and lift sheath flows. The cells can be readily recovered at the microfluidic channel's output with >90% survival rate. Chemically deactivated CRISPR/Cas9 molecules are activated by the laser light for safe molecular surgery. In parallel, it is possible to detect living cells flowing in the microfluidic channel. Measuring light absorption by the analyte makes it possible to detect each individual cell passing by the laser light, and to microscopically verify that >97% of the cells are correctly centered in the microfluidic channel. This device represents a first step to a fully integrated on-chip flow cytometer. Early results demonstrate its efficacy in cell detection and controllable exposure, paving the way to safe molecular surgery.

This paper details the comprehensive design and prototyping of a 3D-printed wearable device tailored for mouse models which addresses the need for non-invasive applications in spinal cord studies and therapeutic treatments. Our work was prompted by the increasing demand for wearable devices in preclinical research on freely behaving rodent models of spinal cord injury. We present an innovative solution that employs compliant 3D-printed structures for stable device placement on the backs of both healthy and spinal cord-injured mice. In our trial, the device was represented by two magnets that applied passive magnetic stimulation to the injury site. This device was designed to be combined with the use of magnetic nanoparticles to render neurons or neural cells sensitive to an exogenous magnetic field, resulting in the stimulation of axon growth in response to a pulling force. We show different design iterations, emphasizing the challenges faced and the solutions proposed during the design process. The iterative design process involved multiple phases, from the magnet holder (MH) to the wearable device configurations. The latter included different approaches: a “Fitbit”, “Belt”, “Bib”, and ultimately a “Cape”. Each design iteration was accompanied by a testing protocol involving healthy and injured mice, with qualitative assessments focusing on animal wellbeing. Follow-up lasted for at least 21 consecutive days, thus allowing animal welfare to be accurately monitored. The final Cape design was our best compromise between the need for a thin structure that would not hinder movement and the resistance required to maintain the structure at the correct position while withstanding biting and mechanical stress. The detailed account of the iterative design process and testing procedures provides valuable insights for researchers and practitioners engaged in the development of wearable devices for mice, particularly in the context of spinal cord studies and therapeutic treatments. Finally, in addition to describing the design of a 3D-printed wearable holder, we also outline some general guidelines for the design of wearable devices.

IntroductionAxonal plasticity is strongly related to neuronal development as well as regeneration. It was recently demonstrated that active mechanical tension, intended as an extrinsic factor, is a valid contribution to the modulation of axonal plasticity.MethodsIn previous publications, our team validated a the “nano-pulling” method used to apply mechanical forces to developing axons of isolated primary neurons using magnetic nanoparticles (MNP) actuated by static magnetic fields. This method was found to promote axon growth and synaptic maturation. Here, we explore the use of nano-pulling as an extrinsic factor to promote axon regeneration in a neuronal tissue explant.ResultsWhole dorsal root ganglia (DRG) were thus dissected from a mouse spinal cord, incubated with MNPs, and then stretched. We found that particles were able to penetrate the ganglion and thus become localised both in the somas and in sprouting axons. Our results highlight that nano-pulling doubles the regeneration rate, and this is accompanied by an increase in the arborizing capacity of axons, an accumulation of cellular organelles related to mass addition (endoplasmic reticulum and mitochondria) and pre-synaptic proteins with respect to spontaneous regeneration. In line with the previous results on isolated hippocampal neurons, we observed that this process is coupled to an increase in the density of stable microtubules and activation of local translation.DiscussionOur data demonstrate that nano-pulling enhances axon regeneration in whole spinal ganglia exposed to MNPs and external magnetic fields. These preliminary data represent an encouraging starting point for proposing nano-pulling as a biophysical tool for the design of novel therapies based on the use of force as an extrinsic factor for promoting nerve regeneration.

Genome editing allows for the manipulation of genomic DNA for biotechnology and biomedical applications, but the specificity and control of the editing process remain a challenge. This study introduces a nano-switch for spatiotemporal control of the editing process. The basic module of the nano-switch (the monomer) is composed of a gold nanorod conjugated with the dead Cas9. Based on mathematical models, we established the design and the mechanism of action of the nano-switch. Briefly, when two monomers, guided by their respective guide RNAs, form a dimer onto the DNA and get irradiated with a Near-Infrared pulsed laser resonant at the plasmonic properties of the dimer, they generate a localized heat that triggers a thermal break onto the DNA. The nano-switch was generated, validated, and tested in zebrafish embryos at the 1-cell stage. Molecular analysis of irradiated embryos showed targeted DNA mutations, validating the efficacy of the nano-switch as a tool for conditional gene editing that integrates the if-when-where functions. ### Competing Interest Statement The authors have declared no competing interest. * dCas9, dead Cas9; AuNR : gold nanorod AuNR-dCas9 : gold nanorod functionalized with the enzyme dCas9 LSPR : Localized Surface Plasmon Resonance NHS : N-Hydroxysuccinimide NTA : Nitrilotriacetic acid HRM : High-Resolution Melting TEM : Transmission Electron Microscopy

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs. Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (≥30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs. Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (≥30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

Biological structures have evolved to very efficiently generate, transmit, and withstand mechanical forces. These biological examples have inspired mechanical engineers for centuries and led to the development of critical insights and concepts. However, progress in mechanical engineering also raises new questions about biological structures. The past decades have seen the increasing study of failure of engineered structures due to repetitive loading, and its origin in processes such as materials fatigue. Repetitive loading is also experienced by some neurons, for example in the peripheral nervous system. This perspective, after briefly introducing the engineering concept of mechanical fatigue, aims to discuss the potential effects based on our knowledge of cellular responses to mechanical stresses. A particular focus of our discussion are the effects of mechanical stress on axons and their cytoskeletal structures. Furthermore, we highlight the difficulty of imaging these structures and the promise of new microscopy techniques. The identification of repair mechanisms and paradigms underlying long-term stability is an exciting and emerging topic in biology as well as a potential source of inspiration for engineers.

Abstract Microtubules are highly polar structures and are characterized by high anisotropy and stiffness. In neurons, they play a key role in the directional transport of vesicles and organelles. In the neuronal projections called axons, they form parallel bundles, mostly oriented with the plus-end towards the axonal termination. Their physico-chemical properties have recently attracted attention as a potential candidate in sensing, processing and transducing physical signals generated by mechanical forces. Here, we discuss the main evidence supporting the role of microtubules as a signal hub for axon growth in response to a traction force. Applying a tension to the axon appears to stabilize the microtubules, which, in turn, coordinate a modulation of axonal transport, local translation and their cross-talk. We speculate on the possible mechanisms modulating microtubule dynamics under tension, based on evidence collected in neuronal and non-neuronal cell types. However, the fundamental question of the causal relationship between these mechanisms is still elusive because the mechano-sensitive element in this chain has not yet been identified.

We developed a fluorescent molecular probe based on gold nanoparticles functionalized with N,N′-bis(2-(1-piperazino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide dihydrochloride, and these probes exhibit potential for applications in microscopic thermometry. The intensity of fluorescence was affected by changes in temperature. Chemical environments, such as different buffers with the same pH, also resulted in different fluorescence intensities. Due to the fluorescence intensity changes exhibited by modified gold nanoparticles, these materials are promising candidates for future technologies involving microscopic temperature measurements.