Axonal Elongation Research Articles

Studying the Role of P2X7 Receptor in Axonal Growth Using In Utero Electroporation Technique.

The nervous system is formed by a complex network of neuronal connections. During development, neurons elongate their axons through highly stereotyped anatomical pathways to form precise connections. Defects in these mechanisms are related with neurological disorders. Previous studies have reported that inhibition of the P2X7 receptor, an ionotropic purinergic receptor, promotes axonal growth and branching in cultured neurons. However, little is known about the in vivo mechanism of axonal elongation regulated by P2X7. Here, we detailed a step-by-step method to perform in utero cortical electroporation and quantified the electroporated axons employing accessible and open-source image processing software. This effective surgical procedure manipulates in vivo the gene expression in a discrete population of callosal projection neuron. Thus, a better understanding of the involvement of P2X7 in the in vivo establishment of neuronal circuits might help to clarify the basic biology of several neurodevelopmental disorders and axonal regenerative processes.

Magnetically-actuated microposts stimulate axon growth

New strategies to promote neuronal regeneration should aim to increase the speed of axonal elongation. Biochemical signaling is a key factor in axon growth, but recent discoveries have shown that mechanical force, through a process referred to as stretch growth, can significantly influence the elongation rate. Here, we develop a method to apply forces to primary hippocampal neurons from mice using magnetic microposts that actuate in response to an external magnetic field. Neurons are cultured onto these microposts and subjected to an average displacement of 0.2 μm at a frequency of 5 Hz. We find that the mechanical stimulation promotes an increase in the length of the axons compared to control conditions. In addition, there is an increase in the density of microtubules and in the amount of cisternae of the endoplasmic reticulum, providing evidence that stretch growth is accompanied by a mass addition to the neurite. Together, these results indicate that magnetically-actuated microposts can accelerate the rate of axon growth, paving the way for future applications in neuronal regeneration. Video abstract [Display omitted]

Intertissue mechanical interactions shape the olfactory circuit in zebrafish.

While the chemical signals guiding neuronal migration and axon elongation have been extensively studied, the influence of mechanical cues on these processes remains poorly studied invivo. Here, we investigate how mechanical forces exerted by surrounding tissues steer neuronal movements and axon extension during the morphogenesis of the olfactory placode in zebrafish. We mainly focus on the mechanical contribution of the adjacent eye tissue, which develops underneath the placode through extensive evagination and invagination movements. Using quantitative analysis of cell movements and biomechanical manipulations, we show that the developing eye exerts lateral traction forces on the olfactory placode through extracellular matrix, mediating proper morphogenetic movements and axon extension within the placode. Our data shed new light on the key participation of intertissue mechanical interactions in the sculpting of neuronal circuits.

Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F-actin levels in a collagen type I 3D culture.

Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.

Dysregulated miR-29a-3p/PMP22 Modulates Schwann Cell Proliferation and Migration During Peripheral Nerve Regeneration.

Schwann cells switch to a repair phenotype following peripheral nerve injury and create a favorable microenvironment to drive nerve repair. Many microRNAs (miRNAs) are differentially expressed in the injured peripheral nerves and play essential roles in regulating Schwann cell behaviors. Here, we examine the temporal expression patterns of miR-29a-3p after peripheral nerve injury and demonstrate significant up-regulation of miR-29a-3p in injured sciatic nerves. Elevated miR-29a-3p inhibits Schwann cell proliferation and migration, while suppressed miR-29a-3p executes reverse effects. In vivo injection of miR-29a-3p agomir to rat sciatic nerves hinders the proliferation and migration of Schwann cells, delays the elongation and myelination of axons, and retards the functional recovery of injured nerves. Mechanistically, miR-29a-3p modulates Schwann cell activities via negatively regulating peripheral myelin protein 22 (PMP22), and PMP22 extensively affects Schwann cell metabolism. Our results disclose the vital role of miR-29a-3p/PMP22 in regulating Schwann cell phenotype following sciatic nerve injury and shed light on the mechanistic basis of peripheral nerve regeneration.

Gongjin-Dan Enhances Neurite Outgrowth of Cortical Neuron by Ameliorating H2O2-Induced Oxidative Damage via Sirtuin1 Signaling Pathway.

Gongjin-dan (GJD) is a multiherbal formula produced from 10 medicinal herbs and has been traditonally used as an oriental medicine to treat cardiovascular diseases, alcoholic hepatitis, mild dementia, and anemia. Additionally, increasing evidence suggests that GJD exerts neuroprotective effects by suppressing inflammation and oxidative stress-induced events to prevent neurological diseases. However, the mechanism by which GJD prevents oxidative stress-induced neuronal injury in a mature neuron remains unknown. Here, we examined the preventive effect and mechanism of GJD on primary cortical neurons exposed to hydrogen peroxide (H2O2). In the neuroprotection signaling pathway, Sirtuin1 is involved in neuroprotective action as a therapeutic target for neurological diseases. After pre-treatment with GJD at three concentrations (10, 25, and 50 µg/mL) and stimulation by H2O2 (30 µM) for 24 h, the influence of GJD on Sirtuin1 activation was assessed using immunocytochemistry, real-time PCR, western blotting, and flow cytometry. GJD effectively ameliorated H2O2-induced neuronal death against oxidative damage through Sirtuin1 activation. In addition, GJD-induced Sirtuin1 activation accelerated elongation of new axons and formation of synapses via increased expression of nerve growth factor and brain-derived neurotrophic factor, as well as regeneration-related genes. Thus, GJD shows potential for preventing neurological diseases via Sirtuin1 activation.

Transthyretin Promotes Axon Growth via Regulation of Microtubule Dynamics and Tubulin Acetylation.

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics. We found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated α-tubulin in peripheral axons. In addition, they failed to modulate MT dynamics in response to sciatic nerve injury, leading to decreased regenerative capacity. Importantly, restoring acetylated α-tubulin levels of TTR KO dorsal root ganglia (DRG) neurons using an HDAC6 inhibitor is sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results reveal a new role for TTR in the modulation of MT dynamics by regulating α-tubulin acetylation via modulation of the acetylase ATAT1, and suggest that this activity underlies TTR neuritogenic function.

RTN4/NoGo-receptor binding to BAI adhesion-GPCRs regulates neuronal development

RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.

Development of 3D culture scaffolds for directional neuronal growth using 2-photon lithography

Conventional applications of transplant technology, applied to severe traumatic injuries of the nervous system, have met limited success in the clinics due to the complexity of restoring function to the damaged tissue. Neural tissue engineering aims to deploy scaffolds mimicking the physiological properties of the extracellular matrix to facilitate the elongation of axons and the repair of damaged nerves. However, the fabrication of ideal scaffolds with precisely controlled thickness, texture, porosity, alignment, and with the required mechanical strength, features needed for effective clinical applications, remains technically challenging. We took advantage of state-of-the-art 2-photon photolithography to fabricate highly ordered and biocompatible 3D nanogrid structures to enhance neuronal directional growth. First, we characterized the physical and chemical properties and proved the biocompatibility of said scaffolds by successfully culturing primary sensory and motor neurons on their surface. Interestingly, axons extended along the fibers with a high degree of alignment to the pattern of the nanogrid, as opposed to the lack of directionality observed on flat glass or polymeric surfaces, and could grow in 3D between different layers of the scaffold. The axonal growth pattern observed is highly desirable for the treatment of traumatic nerve damage occurring during peripheral and spinal cord injuries. Thus, our findings provide a proof of concept and explore the possibility of deploying aligned fibrous 3D scaffold/implants for the directed growth of axons, and could be used in the design of scaffolds targeted towards the restoration and repair of lost neuronal connections.

Haploinsufficiency of ARFGEF1 is associated with developmental delay, intellectual disability, and epilepsy with variable expressivity

PurposeADP ribosylation factor guanine nucleotide exchange factors (ARFGEFs) are a family of proteins implicated in cellular trafficking between the Golgi apparatus and the plasma membrane through vesicle formation. Among them is ARFGEF1/BIG1, a protein involved in axon elongation, neurite development, and polarization processes. ARFGEF1 has been previously suggested as a candidate gene for different types of epilepsies, although its implication in human disease has not been well characterized. MethodsInternational data sharing, in silico predictions, and in vitro assays with minigene study, western blot analyses, and RNA sequencing. ResultsWe identified 13 individuals with heterozygous likely pathogenic variants in ARFGEF1. These individuals displayed congruent clinical features of developmental delay, behavioral problems, abnormal findings on brain magnetic resonance image (MRI), and epilepsy for almost half of them. While nearly half of the cohort carried de novo variants, at least 40% of variants were inherited from mildly affected parents who were clinically re-evaluated by reverse phenotyping. Our in silico predictions and in vitro assays support the contention that ARFGEF1-related conditions are caused by haploinsufficiency, and are transmitted in an autosomal dominant fashion with variable expressivity. ConclusionWe provide evidence that loss-of-function variants in ARFGEF1 are implicated in sporadic and familial cases of developmental delay with or without epilepsy.

Schwann cell delivery via a novel 3D collagen matrix conduit improves outcomes in critical length nerve gap repairs.

The current clinical standard of harvesting a nerve autograft for repair of long-segment peripheral nerve injuries (PNIs) is associated with many potential complications. Guidance channels offer an alternative therapy. The authors investigate whether autologous Schwann cells (SCs) implanted within a novel collagen-glycosaminoglycan conduit will improve axonal regeneration in a long-segment PNI model. Novel NeuraGen 3D collagen matrix conduits were implanted with autologous SCs to investigate axonal regeneration across a critical size defect (13 mm) in male Fischer rat sciatic nerve. Reversed sciatic nerve autografts served as positive controls, and conduits filled with serum only as negative controls. Electrophysiological assessments were made in vivo. Animals were killed at 4 or 16 weeks postinjury, muscle weights were measured, and grafts underwent immunohistochemical and morphometric analysis. SC survival was confirmed by the presence of green fluorescent protein-labeled SCs within regenerated fibers. Regeneration and elongation of myelinated axons in all segments of the graft were significantly enhanced at 16 weeks in the SC-filled conduits compared to the conduit alone and were statistically similar to those of the autograft. Nerves repaired with SC-filled conduits exhibited onset latencies and nerve conduction amplitudes similar to those of the contralateral controls and autograft (p < 0.05). Adding SCs to the conduit also significantly reduced muscle atrophy compared to conduit alone (p < 0.0001). Repair of long-segment PNI of rat sciatic nerve is significantly enhanced by SC-filled NeuraGen 3D conduits. Improvements in the total number of myelinated axons, axon diameter, and myelin thickness throughout SC-filled conduits allow for significant recovery in nerve conduction and a decrease in muscle atrophy.

Adipose tissue-derived neurotrophic factor 3 regulates sympathetic innervation and thermogenesis in adipose tissue

Activation of brown fat thermogenesis increases energy expenditure and alleviates obesity. Sympathetic nervous system (SNS) is important in brown/beige adipocyte thermogenesis. Here we discover a fat-derived “adipokine” neurotrophic factor neurotrophin 3 (NT-3) and its receptor Tropomyosin receptor kinase C (TRKC) as key regulators of SNS growth and innervation in adipose tissue. NT-3 is highly expressed in brown/beige adipocytes, and potently stimulates sympathetic neuron neurite growth. NT-3/TRKC regulates a plethora of pathways in neuronal axonal growth and elongation. Adipose tissue sympathetic innervation is significantly increased in mice with adipocyte-specific NT-3 overexpression, but profoundly reduced in mice with TRKC haploinsufficiency (TRKC +/−). Increasing NT-3 via pharmacological or genetic approach promotes beige adipocyte development, enhances cold-induced thermogenesis and protects against diet-induced obesity (DIO); whereas TRKC + /− or SNS TRKC deficient mice are cold intolerant and prone to DIO. Thus, NT-3 is a fat-derived neurotrophic factor that regulates SNS innervation, energy metabolism and obesity.

Advances in olfactory ensheathing cells for the treatment of spinal cord injury

Spinal cord injury is a highly disabled neurological disease, and there is still a lack of effective treatments. Studies have proved that olfactory ensheathing cells are one of the ideal seed cells for promoting nerve regeneration after spinal cord injury. Olfactory ensheathing cells can promote axonal germination and elongation through secretion, interaction with astrocytes, regulation of inflammatory reaction, migration characteristics, myelination, anti-oxidation, lipid regulation and other channels. Thus olfactory ensheathing cells play the role of neuroprotection and nerve repair. In recent years, some studies have used bioengineering, tissue engineering, reprogramming and other technologies to enhance the efficacy of olfactoryensheathing cells from different aspects, thereby providing new therapeutic strategies for optimizing the cell therapy of spinal cord injury. This article will summarize the mechanism of olfactory ensheathing cells in repairing spinal cord injury, and review the progress of optimizing strategy of olfactory ensheathing cells in treating spinal cord injury recently, so as to provide new research ideas for the further developing the repair potential of olfactory ensheathing cells and optimize the cell therapy effect of spinal cord injury.

Effects of Surface Chemistry Interaction on Primary Neural Stem Cell Neurosphere Responses.

The characteristics of a material’s surface are extremely important when considering their interactions with biological species. Despite surface chemistry playing a critical role in mediating the responses of cells, there remains no single rule which dictates absolute performance; this is particularly challenging when considering the response of differing cell types to a range of materials. Here, we highlight the functional behavior of neural stem cells presented as neurospheres, with respect to a range of alkane-based self-assembled monolayers presenting different functional groups: OH, CO2H, NH2, phenyl, CH3, SH, and laminin. The influence of chemical cues was examined in terms of neurosphere spreading on each of these defined surfaces (cell adhesion and migration capacity) and neuronal versus glial marker expression. Measurements were made over a time series of 3, 5, and 7 days, showing a dynamic nature to the initial responses observed after seeding. While OH surfaces presented an excellent platform for glial migration, larger proportions of cells expressing neuronal β3-tubulin were found on SH- and laminin-coated surfaces. Axonal elongation was found to be initially similar on all surfaces with neurite lengths having a wider spread predominantly on NH2- and laminin-presenting surfaces. A generalized trend could not be found to correlate cellular responses with surface wettability, lipophilicity (log P), or charge/ionizability (pKa). These results highlight the potential for chemical cues to direct primary neural stem cell responses in contact with the defined materials. New biomaterials which control specific cell culture characteristics in vitro will streamline the up-scale manufacture of cellular therapies, with the enrichment of the required populations resulting from a defined material interaction.

Stressed axons craving for glial sugar: links to regeneration?

Stressed axons craving for glial sugar: links to regeneration?

Coactosin Promotes F-Actin Protrusion in Growth Cones Under Cofilin-Related Signaling Pathway.

During brain development, axon outgrowth and its subsequent pathfinding are reliant on a highly motile growth cone located at the tip of the axon. Actin polymerization that is regulated by actin-depolymerizing factors homology (ADF-H) domain-containing family drives the formation of lamellipodia and filopodia at the leading edge of growth cones for axon guidance. However, the precise localization and function of ADF-H domain-containing proteins involved in axon extension and retraction remain unclear. We have previously shown that transcripts and proteins of coactosin-like protein 1 (COTL1), an ADF-H domain-containing protein, are observed in neurites and axons in chick embryos. Coactosin overexpression analysis revealed that this protein was localized to axonal growth cones and involved in axon extension in the midbrain. We further examined the specific distribution of coactosin and cofilin within the growth cone using superresolution microscopy, structured illumination microscopy, which overcomes the optical diffraction limitation and is suitable to the analysis of cellular dynamic movements. We found that coactosin was tightly associated with F-actin bundles at the growth cones and that coactosin overexpression promoted the expansion of lamellipodia and extension of growth cones. Coactosin knockdown in oculomotor neurons resulted in an increase in the levels of the inactive, phosphorylated form of cofilin and dysregulation of actin polymerization and axonal elongation, which suggests that coactosin promoted axonal growth in a cofilin-dependent manner. Indeed, the application of a dominant-negative form of LIMK1, a downstream effector of GTPases, reversed the effect of coactosin knockdown on axonal growth by enhancing cofilin activity. Combined, our results indicate that coactosin functions promote the assembly of protrusive actin filament arrays at the leading edge for growth cone motility.

Axonal Transport and Local Translation of mRNA in Neurodegenerative Diseases.

Since neurons have long neurites including axons, it is crucial for the axons to transport many intracellular substances such as proteins and mitochondria in order to maintain their morphology and function. In addition, mRNAs have also been shown to be transported within axons. RNA-binding proteins form complexes with mRNAs, and regulate transport of the mRNAs to axons, as well as locally translate them into proteins. Local translation of mRNAs actively occurs during the development and damage of neurons, and plays an important role in axon elongation, regeneration, and synapse formation. In recent years, it has been reported that impaired axonal transport and local translation of mRNAs may be involved in the pathogenesis of some neurodegenerative diseases. In this review, we discuss the significance of mRNA axonal transport and their local translation in amyotrophic lateral sclerosis/frontotemporal dementia, spinal muscular atrophy, Alzheimer’s disease, and fragile X syndrome.

Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids.

SummaryThe development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway.

Efficacy of sliced nerves of different thickness in a biodegradable nerve conduit to promote Schwann cell migration and axonal growth: An experimental study in the rat model.

Using the rat sciatic nerve model, sliced nerves of different thickness was combined to a biodegradable nerve conduit and the amount of nerve fragment necessary to promote nerve regeneration was investigated. Harvested sciatic nerve (n=6) was processed in sliced nerve of the different width; 2, 1, 0.5 mm, respectively. Western blot analysis was carried out to determine protein expression of Erk1/2. Subsequently, a total of 246 rats were used to create a 10 mm gap in the sciatic nerve. A polyglycolic acid-based nerve conduit was used to bridge the gap, with one sliced (width; 2, 1, 0.5 mm) or two (width; 1 mm × 2) incorporated within the conduit (n=6 at each point in each group). At 2, 4, 8, and 20 weeks after surgery, samples were resected and subjected to immune-histological, transmission electron microscopic, and motor functional evaluation for nerve regeneration. Western blot analysis demonstrated Erk1/2 expressions were significantly increased in the groups of 2-mm and 1-mm width and attenuated in the 0.5-mm width group (p < .05). The immune-histological study showed the migration of Schwann cells and axon elongation were significantly extended in the groups of 2-mm, 1-mm, and 1 mm × 2 width at 4 weeks (p < .01), in which nerve conduction velocity was marked at 20 weeks (p < .01) after implantation. When nerve tissue was inserted in the biodegradable nerve conduit as a sliced nerve, the method of inserting two sheets with a slice width of 1 mm most strongly accelerated motor function.

Axon-enriched lincRNA ALAE is required for axon elongation via regulation of local mRNA translation.

Long intergenic noncoding RNAs (lincRNAs) are critical regulators involved in diverse biological processes. However, the roles and related mechanisms of lincRNAs in axon development are largely unknown. Here we report an axon-enriched lincRNA regulating axon elongation, referred to as ALAE. Profiling of highly expressed lincRNAs detected abundant and enriched ALAE in the axons of dorsal root ganglion (DRG) neurons, where it locally promoted axon elongation. Mechanically, ALAE directly interacted with the KH3-4 domains of KH-type splicing regulatory protein (KHSRP) and impeded its association with growth-associated protein 43 (Gap43) mRNA. Knockdown of ALAE reduced the protein but not the mRNA level of GAP43 in the axons of DRG neurons. Furthermore, local disruption of the interaction between ALAE and KHSRP in the axon significantly inhibited Gap43 mRNA translation, impairing axon elongation. This study implies crucial roles of axon-enriched lincRNAs in spatiotemporal regulation of local translation during axon development.