Capacity For Axonal Regeneration Research Articles

CX3CL1-CX3CR1 axis protects retinal ganglion cells by inhibiting microglia activation in a distal optic nerve trauma model

BackgroundThe chemokine CX3CL1 has been reported to play an important role in optic nerve protection, but the underlying mechanism is still unclear. CX3CR1, the only receptor of CX3CL1, is specifically expressed on retinal microglia, whose activation plays a role in the pathological process of optic nerve injury. This study aimed to evaluate whether CX3CL1 exerts optic neuroprotection by affecting the activation of microglia by combining with CX3CR1.MethodsA mouse model of distal optic nerve trauma (ONT) was used to evaluate the effects of the CX3CL1-CX3CR1 axis on the activation of microglia and survival or axonal regeneration of retinal ganglion cells (RGCs). The activation of microglia, loss of RGCs, and damage to visual function were detected weekly till 4 weeks after modeling. CX3CL1 was injected intravitreally immediately or delayed after injury and the status of microglia and RGCs were examined.ResultsIncreases in microglia activation and optic nerve damage were accompanied by a reduced production of the CX3CL1-CX3CR1 axis after the distal ONT modeling. Both immediate and delayed intravitreal injection of CX3CL1 inhibited microglia activation, promoted survival of RGCs, and improved axonal regenerative capacity. Injection with CX3CL1 was no longer effective after 48 h post ONT. The CX3CL1-CX3CR1 axis promotes survival and axonal regeneration, as indicated by GAP43 protein and gene expression, of RGCs by inhibiting the microglial activation after ONT.ConclusionsThe CX3CL1-CX3CR1 axis could promote survival and axonal regeneration of RGCs by inhibiting the microglial activation after optic nerve injury. The CX3CL1-CX3CR1 axis may become a potential target for the treatment of optic nerve injury. Forty-eight hours is the longest time window for effective treatment after injury. The study is expected to provide new ideas for the development of targeted drugs for the repair of optic nerve.

Multifunctional Hierarchical Nanoplatform with Anisotropic Bimodal Mesopores for Effective Neural Circuit Reconstruction after Spinal Cord Injury.

A persistent inflammatory response, intrinsic limitations in axonal regenerative capacity, and widespread presence of extrinsic axonal inhibitors impede the restoration of motor function after a spinal cord injury (SCI). A versatile treatment platform is urgently needed to address diverse clinical manifestations of SCI. Herein, we present a multifunctional nanoplatform with anisotropic bimodal mesopores for effective neural circuit reconstruction after SCI. The hierarchical nanoplatform features of a Janus structure consist of dual compartments of hydrophilic mesoporous silica (mSiO2) and hydrophobic periodic mesoporous organosilica (PMO), each possessing distinct pore sizes of 12 and 3 nm, respectively. Unlike traditional hierarchical mesoporous nanomaterials with dual-mesopores interlaced with each other, the two sets of mesopores in this Janus nanoplatform are spatially independent and possess completely distinct chemical properties. The Janus mesopores facilitate controllable codelivery of dual drugs with distinct properties: the hydrophilic macromolecular enoxaparin (ENO) and the hydrophobic small molecular paclitaxel (PTX). Anchoring with CeO2, the resulting mSiO2&PMO-CeO2-PTX&ENO nanoformulation not only effectively alleviates ROS-induced neuronal apoptosis but also enhances microtubule stability to promote intrinsic axonal regeneration and facilitates axonal extension by diminishing the inhibitory effect of extracellular chondroitin sulfate proteoglycans. We believe that this functional dual-mesoporous nanoplatform holds significant potential for combination therapy in treating severe multifaceted diseases.

Transcriptome and methylome of the supraoptic nucleus provides insights into the age-dependent loss of neuronal plasticity.

The age-dependent loss of neuronal plasticity is a well-known phenomenon that is poorly understood. The loss of this capacity for axonal regeneration is emphasized following traumatic brain injury, which is a major cause of disability and death among adults in the US. We have previously shown the intrinsic capacity of magnocellular neurons within the supraoptic nucleus to undergo axonal regeneration following unilateral axotomization in an age-dependent manner. The aim of this research was to determine the age-dependent molecular mechanisms that may underlie this phenomenon. As such, we characterized the transcriptome and DNA methylome of the supraoptic nucleus in uninjured 35-day old rats and 125-day old rats. Our data indicates the downregulation of a large number of axonogenesis related transcripts in 125-day old rats compared to 35-day old rats. Specifically, several semaphorin and ephrin genes were downregulated, as well as growth factors including FGF's, insulin-like growth factors (IGFs), and brain-derived neurotrophic factor (BDNF). Differential methylation analysis indicates enrichment of biological processes involved in axonogenesis and axon guidance. Conversely, we observed a robust and specific upregulation of MHCI related transcripts. This may involve the activator protein 1 (AP-1) transcription factor complex as motif analysis of differentially methylated regions indicate enrichment of AP-1 binding sites in hypomethylated regions. Together, our data suggests a loss of pro-regenerative capabilities with age which would prevent axonal growth and appropriate innervation following injury.

Drosophila Laser Axotomy Injury Model to Investigate RNA Repair and Splicing in Axon Regeneration.

The limited axon regeneration capacity of mature neurons often leads to insufficient functional recovery after damage to the central nervous system (CNS). To promote CNS nerve repair, there is an urgent need to understand the regeneration machinery in order to develop effective clinical therapies. To this aim, we developed a Drosophila sensory neuron injury model and the accompanying behavioral assay to examine axon regeneration competence and functional recovery after injury in the peripheral and central nervous systems. Specifically, we used a two-photon laser to induce axotomy and performed live imaging to assess axon regeneration, combined with the analysis of the thermonociceptive behavior as a readout of functional recovery. Using this model, we found that the RNA 3'-terminal phosphate cyclase (Rtca), which acts as a regulator for RNA repair and splicing, responds to injury-induced cellular stress and impedes axon regeneration after axon breakage. Here we describe how we utilize our Drosophila model to assess the role of Rtca during neuroregeneration.

Brain-derived neurotrophic factor (BDNF) induces antagonistic action to Nogo signaling by the upregulation of lateral olfactory tract usher substance (LOTUS) expression.

Neurons in the central nervous system (CNS) have limited capacity for axonal regeneration after trauma and neurological disorders due to an endogenous nonpermissive environment for axon regrowth in the CNS. Lateral olfactory tract usher substance (LOTUS) contributes to axonal tract formation in the developing brain and axonal regeneration in the adult brain as an endogenous Nogo receptor-1 (NgR1) antagonist. However, how LOTUS expression is regulated remains unclarified. This study examined molecular mechanism of regulation in LOTUS expression and found that brain-derived neurotrophic factor (BDNF) increased LOTUS expression in cultured hippocampal neurons. Exogenous application of BDNF increased LOTUS expression at both mRNA and protein levels in a dose-dependent manner. We also found that pharmacological inhibition with K252a and gene knockdown by siRNA of tropomyosin-related kinase B (TrkB), BDNF receptor suppressed BDNF-induced increase in LOTUS expression. Further pharmacological analysis of the TrkB signaling pathway revealed that BDNF increased LOTUS expression through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades, but not phospholipase C-γ (PLCγ) cascade. Additionally, treatment with c-AMP response element binding protein (CREB) inhibitor partially suppressed BDNF-induced LOTUS expression. Finally, neurite outgrowth assay in cultured hippocampal neurons revealed that BDNF treatment-induced antagonism for NgR1 by up-regulating LOTUS expression. These findings suggest that BDNF may acts as a positive regulator of LOTUS expression through the TrkB signaling, thereby inducing an antagonistic action for NgR1 function by up-regulating LOTUS expression. Also, BDNF may synergistically affect axon regrowth through the upregulation of LOTUS expression.

Revisiting the immune landscape post spinal cord injury: More than black and white.

Spinal cord injury (SCI) induced catastrophic neurological disability is currently incurable, especially in elderly patients. Due to the limited axon regeneration capacity and hostile microenvironment in the lesion site, essential neural network reconstruction remains challenging. Owing to the blood-spinal cord barrier (BSCB) created immune cells and cytokines isolation, the immune elements were incorrectly recognized as innocent bystanders during the SCI pathological process traditionally. Emerging evidence demonstrated that the central nervous system (CNS) is an "immunological quiescent" rather than "immune privileged" area, and the CNS-associated immune response played mixed roles which dedicate beneficial and detrimental contributions throughout the SCI process. Consequently, coordinating double-edged immunomodulation is vital to promote tissue repair and neurological recovery post-SCI. The comprehensive exploration and understanding of the immune landscape post-SCI are essential in establishing new avenues for further basic and clinical studies. In this context, this review summarizes the recent significant breakthroughs in key aspects of SCI-related immunomodulation, including innate and adaptive immune response, immune organ changes, and holistic immune status modification. Moreover, the currently existing immune-oriented therapies for SCI will be outlined.

Microfluidic devices as model platforms of CNS injury-ischemia to study axonal regeneration by regulating mitochondrial transport and bioenergetic metabolism

Central nervous system (CNS) neurons typically fail to regenerate their axons after injury leading to neurological impairment. Axonal regeneration is a highly energy-demanding cellular program that requires local mitochondria to supply most energy within injured axons. Recent emerging lines of evidence have started to reveal that injury-triggered acute mitochondrial damage and local energy crisis contribute to the intrinsic energetic restriction that accounts for axon regeneration failure in the CNS. Characterizing and reprogramming bioenergetic signaling and mitochondrial maintenance after axon injury-ischemia is fundamental for developing therapeutic strategies that can restore local energy metabolism and thus facilitate axon regeneration. Therefore, establishing reliable and reproducible neuronal model platforms is critical for assessing axonal energetic metabolism and regeneration capacity after injury-ischemia. In this focused methodology article, we discuss recent advances in applying cutting-edge microfluidic chamber devices in combination with state-of-the-art live-neuron imaging tools to monitor axonal regeneration, mitochondrial transport, bioenergetic metabolism, and local protein synthesis in response to injury-ischemic stress in mature CNS neurons.

Coordinated NADPH oxidase/hydrogen peroxide functions regulate cutaneous sensory axon de- and regeneration

Tissue wounding induces cutaneous sensory axon regeneration via hydrogen peroxide (H2O2) that is produced by the epithelial NADPH oxidase, Duox1. Sciatic nerve injury instead induces axon regeneration through neuronal uptake of the NADPH oxidase, Nox2, from macrophages. We therefore reasoned that the tissue environment in which axons are damaged stimulates distinct regenerative mechanisms. Here, we show that cutaneous axon regeneration induced by tissue wounding depends on both neuronal and keratinocyte-specific mechanisms involving H2O2 signaling. Genetic depletion of H2O2 in sensory neurons abolishes axon regeneration, whereas keratinocyte-specific H2O2 depletion promotes axonal repulsion, a phenotype mirrored in duox1 mutants. Intriguingly, cyba mutants, deficient in the essential Nox subunit, p22Phox, retain limited axon regenerative capacity but display delayed Wallerian degeneration and axonal fusion, observed so far only in invertebrates. We further show that keratinocyte-specific oxidation of the epidermal growth factor receptor (EGFR) at a conserved cysteine thiol (C797) serves as an attractive cue for regenerating axons, leading to EGFR-dependent localized epidermal matrix remodeling via the matrix-metalloproteinase, MMP-13. Therefore, wound-induced cutaneous axon de- and regeneration depend on the coordinated functions of NADPH oxidases mediating distinct processes following injury.

The role of mitochondria in the recovery of neurons after injury.

The role of mitochondria in the recovery of neurons after injury.

Mitochondrial function in spinal cord injury and regeneration.

Many people around the world suffer from some form of paralysis caused by spinal cord injury (SCI), which has an impact on quality and life expectancy. The spinal cord is part of the central nervous system (CNS), which in mammals is unable to regenerate, and to date, there is a lack of full functional recovery therapies for SCI. These injuries start with a rapid and mechanical insult, followed by a secondary phase leading progressively to greater damage. This secondary phase can be potentially modifiable through targeted therapies. The growing literature, derived from mammalian and regenerative model studies, supports a leading role for mitochondria in every cellular response after SCI: mitochondrial dysfunction is the common event of different triggers leading to cell death, cellular metabolism regulates the immune response, mitochondrial number and localization correlate with axon regenerative capacity, while mitochondrial abundance and substrate utilization regulate neural stem progenitor cells self-renewal and differentiation. Herein, we present a comprehensive review of the cellular responses during the secondary phase of SCI, the mitochondrial contribution to each of them, as well as evidence of mitochondrial involvement in spinal cord regeneration, suggesting that a more in-depth study of mitochondrial function and regulation is needed to identify potential targets for SCI therapeutic intervention.

Histomorphometric Analysis of Femoral and Sciatic Nerve Regeneration in a Rat Hindlimb Allotransplantation Model

Peripheral nerve regeneration is essential for functional recovery after traumatic limb injury or limb transplantation. With the use of immunosuppression, it has the capacity to provide regeneration and function equal to that of an autograft. The purpose of this study was to determine whether there is any difference in regeneration and rejection response between peripheral femoral and sciatic nerve in rat hindlimb allotransplantation model. The hindlimbs of recipient Fischer 344 rats were amputated at the mid-thigh and the donor and recipient femurs were joined by an intramedullary rod. The femoral and sciatic nerves were repaired with 10-O nylon end-to-end suture followed by vessel, muscle, and skin closure. The control group received auto-transplantation and the experimental group received allo-transplantation from Sprague-Dawley donor rats. The recipients were treated with an immunosuppressive agent, FK506 (2 mg/kg), for the observation period. Both sciatic and femoral nerves were harvested 10 weeks after operation and histomorphometric analysis was conducted between these 2 nerves and control group. The sciatic nerve showed better regeneration, with significantly increased percentage nerve, fiber count, and density (P < .05), but demonstrated more immune cell proliferation relative to femoral nerve. The data showed that there are some differences in axonal regeneration capacity and immune response between large peripheral nerves.

An exception to the rule? Regeneration of the injured spinal cord in the spiny mouse.

The capacity for long-distance axon regeneration and functional recovery after spinal cord injury in the adult has long been thought to be a unique feature of certain non-mammalian vertebrates. However, in this issue of Developmental Cell, Nogueira-Rodrigues etal. report an astonishingly high regenerative ability in the spiny mouse.

Epidural oscillating field stimulation increases axonal regenerative capacity and myelination after spinal cord trauma.

Oscillating field stimulation (OFS) with regular alterations in the polarity of electric current is a unique, experimental approach to stimulate, support, and potentially guide the outgrowth of both sensory and motor nerve fibers after spinal cord injury (SCI). In previous experiments, we demonstrated the beneficial effects of OFS in a 4-week survival period after SCI. In this study, we observed the major behavioral, morphological, and protein changes in rats after 15 minutes of T9 spinal compression with a 40 g force, followed by long-lasting OFS (50 µA), over a 8-week survival period. Three groups of rats were analyzed: rats after T9 spinal compression (SCI group); SCI rats subjected to implantation of active oscillating field stimulator (OFS + SCI group); and SCI rats subjected to nonfunctional OFS (nOFS + SCI group). Histopathological analysis of spinal tissue indicated a strong impact of epidural OFS on the reduction of tissue and myelin loss after SCI in the segments adjacent to the lesion site. Quantitative fluorescent analysis of the most affected areas of spinal cord tissue revealed a higher number of spared axons and oligodendrocytes of rats in the OFS + SCI group, compared with rats in the SCI and nOFS + SCI groups. The protein levels of neurofilaments (NF-l), growth-associated protein-43 (marker for newly sprouted axons), and myelin basic protein in rats were signifiantly increased in the OFS + SCI group than in the nOFS + SCI and SCI groups. This suggests a supporting role of the OFS in axonal and myelin regeneration after SCI. Moreover, rats in the OFS + SCI group showed great improvements in sensory and motor functions than did rats in the nOFS + SCI and SCI groups. All these findings suggest that long-lasting OFS applied immediately after SCI can provide a good microenviroment for recovery of damaged spinal tissue by triggering regenreative processes in the acute phase of injury.

Identification of key genes involved in axon regeneration and Wallerian degeneration by weighted gene co-expression network analysis.

Peripheral nerve injury repair requires a certain degree of cooperation between axon regeneration and Wallerian degeneration. Therefore, investigating how axon regeneration and degeneration work together to repair peripheral nerve injury may uncover the molecular mechanisms and signal cascades underlying peripheral nerve repair and provide potential strategies for improving the low axon regeneration capacity of the central nervous system. In this study, we applied weighted gene co-expression network analysis to identify differentially expressed genes in proximal and distal sciatic nerve segments from rats with sciatic nerve injury. We identified 31 and 15 co-expression modules from the proximal and distal sciatic nerve segments, respectively. Functional enrichment analysis revealed that the differentially expressed genes in proximal modules promoted regeneration, while the differentially expressed genes in distal modules promoted neurodegeneration. Next, we constructed hub gene networks for selected modules and identified a key hub gene, Kif22, which was up-regulated in both nerve segments. In vitro experiments confirmed that Kif22 knockdown inhibited proliferation and migration of Schwann cells by modulating the activity of the extracellular signal-regulated kinase signaling pathway. Collectively, our findings provide a comparative framework of gene modules that are co-expressed in injured proximal and distal sciatic nerve segments, and identify Kif22 as a potential therapeutic target for promoting peripheral nerve injury repair via Schwann cell proliferation and migration. All animal experiments were approved by the Institutional Animal Ethics Committee of Nantong University, China (approval No. S20210322-008) on March 22, 2021.

Know How to Regrow—Axon Regeneration in the Zebrafish Spinal Cord

The capacity for long-distance axon regeneration and functional recovery after spinal cord injury is poor in mammals but remarkable in some vertebrates, including fish and salamanders. The cellular and molecular basis of this interspecies difference is beginning to emerge. This includes the identification of target cells that react to the injury and the cues directing their pro-regenerative responses. Among existing models of successful spinal cord regeneration, the zebrafish is arguably the most understood at a mechanistic level to date. Here, we review the spinal cord injury paradigms used in zebrafish, and summarize the breadth of neuron-intrinsic and -extrinsic factors that have been identified to play pivotal roles in the ability of zebrafish to regenerate central nervous system axons and recover function.

Ascending dorsal column sensory neurons respond to spinal cord injury and downregulate genes related to lipid metabolism

Regeneration failure after spinal cord injury (SCI) results in part from the lack of a pro-regenerative response in injured neurons, but the response to SCI has not been examined specifically in injured sensory neurons. Using RNA sequencing of dorsal root ganglion, we determined that thoracic SCI elicits a transcriptional response distinct from sciatic nerve injury (SNI). Both SNI and SCI induced upregulation of ATF3 and Jun, yet this response failed to promote growth in sensory neurons after SCI. RNA sequencing of purified sensory neurons one and three days after injury revealed that unlike SNI, the SCI response is not sustained. Both SCI and SNI elicited the expression of ATF3 target genes, with very little overlap between conditions. Pathway analysis of differentially expressed ATF3 target genes revealed that fatty acid biosynthesis and terpenoid backbone synthesis were downregulated after SCI but not SNI. Pharmacologic inhibition of fatty acid synthase, the enzyme generating palmitic acid, decreased axon growth and regeneration in vitro. These results support the notion that decreased expression of lipid metabolism-related genes after SCI, including fatty acid synthase, may restrict axon regenerative capacity after SCI.

Inhibition of Nav1.8 Transport Promotes Axon Regeneration Via Regulating Dynamic Changes in Macrophage Phenotype after Sciatic Nerve Injury

Peripheral nerve injury induces the forward transport of Nav1.8 driven by KIF5b, however, little is known regarding the role of Nav1.8 transport in peripheral nerve regeneration. Here, we reported the beneficial effects of inhibition of Nav1.8 transport on functional recovery and nerve regeneration following experimental sciatic nerve transection (SNT), and characterized the impact of dynamic changes of M1/M2 macrophage polarization phenotypes in this regeneration process. Sequential behavioral data demonstrated a significant improvement of sensory and motor functions over time in rats with inhibition of Nav1.8 transport. Moreover, electrophysiological and histological analysis collectively indicated that inhibition of Nav1.8 transport promoted axonal regrowth after SNT without affecting remyelination of axons. We also discovered that inhibition of Nav1.8 transport facilitates the early recruitment of pro-phagocytic M1 macrophages and subsequently accelerate phenotypic switching from M1 to reparative M2 phenotype, which might be related to the increased secretion of CCL2 and SP acting on macrophage receptor CCR2 and NK-1R respectively. In contrast, enhancing Nav1.8 forward transport by overexpressing KIF5b in DRG neurons substantially impaired the release of CCL2 and SP, the M1/M2 macrophage phenotype switch, and axonal regenerative capacity. Notably, we also found that continuous nerve block with ropivacaine, a non-selective sodium channels blocker, can promote functional recovery and nerve regeneration by inhibiting Nav1.8 transport after SNT. Together, our studies revealed a constructive role of Nav1.8 transport in axonal regrowth after SNT and supported a potential clinical application of continuous blocking with ropivacaine in in peripheral nerve repair.

Axon Injury-Induced Autophagy Activation Is Impaired in a C. elegans Model of Tauopathy.

Autophagy is a conserved pathway that plays a key role in cell homeostasis in normal settings, as well as abnormal and stress conditions. Autophagy dysfunction is found in various neurodegenerative diseases, although it remains unclear whether autophagy impairment is a contributor or consequence of neurodegeneration. Axonal injury is an acute neuronal stress that triggers autophagic responses in an age-dependent manner. In this study, we investigate the injury-triggered autophagy response in a C. elegans model of tauopathy. We found that transgenic expression of pro-aggregant Tau, but not the anti-aggregant Tau, abolished axon injury-induced autophagy activation, resulting in a reduced axon regeneration capacity. Furthermore, axonal trafficking of autophagic vesicles were significantly reduced in the animals expressing pro-aggregant F3ΔK280 Tau, indicating that Tau aggregation impairs autophagy regulation. Importantly, the reduced number of total or trafficking autophagic vesicles in the tauopathy model was not restored by the autophagy activator rapamycin. Loss of PTL-1, the sole Tau homologue in C. elegans, also led to impaired injury-induced autophagy activation, but with an increased basal level of autophagic vesicles. Therefore, we have demonstrated that Tau aggregation as well as Tau depletion both lead to disruption of injury-induced autophagy responses, suggesting that aberrant protein aggregation or microtubule dysfunction can modulate autophagy regulation in neurons after injury.

MicroRNA-19a-PTEN Axis Is Involved in the Developmental Decline of Axon Regenerative Capacity in Retinal Ganglion Cells

Irreversible blindness from glaucoma and optic neuropathies is attributed to retinal ganglion cells (RGCs) losing the ability to regenerate axons. While several transcription factors and proteins have demonstrated enhancement of axon regeneration after optic nerve injury, mechanisms contributing to the age-related decline in axon regenerative capacity remain elusive. In this study, we show that microRNAs are differentially expressed during RGC development and identify microRNA-19a (miR-19a) as a heterochronic marker; developmental decline of miR-19a relieves suppression of phosphatase and tensin homolog (PTEN), a key regulator of axon regeneration, and serves as a temporal indicator of decreasing axon regenerative capacity. Intravitreal injection of miR-19a promotes axon regeneration after optic nerve crush in adult mice, and it increases axon extension in RGCs isolated from aged human donors. This study uncovers a previously unrecognized involvement of the miR-19a-PTEN axis in RGC axon regeneration, and it demonstrates therapeutic potential of microRNA-mediated restoration of axon regenerative capacity in optic neuropathies.

Growth-Promoting Treatment Screening for Corticospinal Neurons in Mouse and Man

Neurons of the central nervous system (CNS) that project long axons into the spinal cord have a poor axon regenerative capacity compared to neurons of the peripheral nervous system. The corticospinal tract (CST) is particularly notorious for its poor regeneration. Because of this, traumatic spinal cord injury (SCI) is a devastating condition that remains as yet uncured. Based on our recent observations that direct neuronal interleukin-4 (IL-4) signaling leads to repair of axonal swellings and beneficial effects in neuroinflammation, we hypothesized that IL-4 acts directly on the CST. Here, we developed a tissue culture model for CST regeneration and found that IL-4 promoted new growth cone formation after axon transection. Most importantly, IL-4 directly increased the regenerative capacity of both murine and human CST axons, which corroborates its regenerative effects in CNS damage. Overall, these findings serve as proof-of-concept that our CST regeneration model is suitable for fast screening of new treatments for SCI.