Spinal Axons Research Articles

Synergistic Activity of Floor-Plate- and Ventricular-Zone-Derived Netrin-1 in Spinal Cord Commissural Axon Guidance

In vertebrates, commissural axons extend ventrally toward the floor plate in the spinal cord and hindbrain. Netrin-1, secreted by floor plate cells, was proposed to attract commissural axons at a distance. However, recent genetic studies in mice have shown that netrin-1 is also produced by ventricular zone (VZ) progenitors and that in the hindbrain, it represents the main source of netrin-1 for commissural axons. Here, we show that genetically deleting netrin-1 either from the VZ or the floor plate does not prevent midline crossing in the spinal cord, although axon pathfinding and fasciculation are perturbed. Strikingly, the VZ and floor plate act synergistically, as the simultaneous ablation of netrin-1 from these two sources severely impedes crossing. These results suggest that floor-plate-derived netrin-1 has a distinct impact on commissuralaxons in the spinal cord and hindbrain.

Identification of a microRNA switch in spinal commissural axon guidance.

Identification of a microRNA switch in spinal commissural axon guidance.

Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation.

Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in vivo characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an "open-book" spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines ex vivo electroporation and "closed-book" spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.

Anatomic conditions for bypass surgery between rostral (T7–T9) and caudal (L2, L4, S1) ventral roots to treat paralysis after spinal cord injury

Severe spinal cord injuries cause permanent neurological deficits and are still considered as inaccessible to efficient therapy. Injured spinal cord axons are unable to spontaneously regenerate. Re-establishing functional activity especially in the lower limbs by reinnervation of the caudal infra-lesional territories might represent an effective therapeutic strategy.Numerous surgical neurotizations have been developed to bridge the spinal cord lesion site and connect the intact supra-lesional portions of the spinal cord to peripheral nerves (spinal nerves, intercostal nerves) and muscles. The major disadvantage of these techniques is the increased hypersensitivity, spasticity and pathologic pain in the spinal cord injured patients, which occur due to the vigorous sprouting of injured afferent sensory fibers after reconstructive surgery.Using micro-surgical instruments and an operation microscope we performed detailed anatomical preparation of the vertebral canal and its content in five human cadavers. Our observations allow us to put forward the possibility to develop a more precise surgical approach, the so called “ventral root bypass” that avoids lesion of the dorsal roots and eliminates sensitivity complications.The proposed kind of neurotization has been neither used, nor put forward. The general opinion is that radix ventralis and radix dorsalis unite to form the spinal nerve inside the dural sac. This assumption is not accurate, because both radices leave the dural sac separately. This neglected anatomical feature allows a reliable intravertebral exposure of the dura-mater ensheathed ventral roots and their damage-preventing end-to-side neurorrhaphy by interpositional nerve grafts.

Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS.

SummarySpinal motor axons traverse large distances to innervate target muscles, thus requiring local control of cellular events for proper functioning. To interrogate axon-specific processes we developed Axon-seq, a refined method incorporating microfluidics, RNA sequencing (RNA-seq), and bioinformatic quality control. We show that the axonal transcriptome is distinct from that of somas and contains fewer genes. We identified 3,500–5,000 transcripts in mouse and human stem cell-derived spinal motor axons, most of which are required for oxidative energy production and ribogenesis. Axons contained transcription factor mRNAs, e.g., Ybx1, with implications for local functions. As motor axons degenerate in amyotrophic lateral sclerosis (ALS), we investigated their response to the SOD1G93A mutation, identifying 121 ALS-dysregulated transcripts. Several of these are implicated in axonal function, including Nrp1, Dbn1, and Nek1, a known ALS-causing gene. In conclusion, Axon-seq provides an improved method for RNA-seq of axons, increasing our understanding of peripheral axon biology and identifying therapeutic targets in motor neuron disease.

A neonatal mouse model of central nervous system infections caused by Coxsackievirus B5

As one of the key members of the coxsackievirus B group, coxsackievirus B5 (CV-B5) can cause many central nervous system diseases, such as viral encephalitis, aseptic meningitis, and acute flaccid paralysis. Notably, epidemiological data indicate that outbreaks of CV-B5-associated central nervous system (CNS) diseases have been reported worldwide throughout history. In this study, which was conducted to promote CV-B5 vaccine and anti-virus drug research, a 3-day-old BALB/c mouse model was established using a CV-B5 clinical isolate (CV-B5/JS417) as the challenge strain. Mice challenged with CV-B5/JS417 exhibited a series of neural clinical symptoms and death with necrosis of neuronal cells in the cerebral cortex and the entire spinal cord, hindlimb muscles, and cardiomyocytes. The viral load of each tissue at various post-challenge time points suggested that CV-B5 replicated in the small intestine and was subsequently transmitted to various organs via viremia; the virus potentially entered the brain through the spinal axons, causing neuronal cell necrosis. In addition, this mouse model was used to evaluate the protective effect of a CV-B5 vaccine. The results indicated that both the inactivated CV-B5 vaccine and anti-CVB5 serum significantly protected mice from a lethal infection of CV-B5/JS417 by producing neutralizing antibodies. In summary, the first CV-B5 neonatal mouse model has been established and can sustain CNS infections in a manner similar to that observed in humans. This model will be a useful tool for studies on pathogenesis, vaccines, and anti-viral drug evaluations.

Connexin 30 Deficiency Attenuates Chronic but Not Acute Phases of Experimental Autoimmune Encephalomyelitis Through Induction of Neuroprotective Microglia.

Glial connexins (Cxs) form gap junction channels through which a pan-glial network plays key roles in maintaining homeostasis of the central nervous system (CNS). In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), expression of astrocytic Cx43 is lost in acute lesions but upregulated in chronic plaques, while astrocytic Cx30 is very low in normal white matter and changes in its expression have not been convincingly shown. In Cx30 or Cx43 single knockout (KO) mice and even in Cx30/Cx43 double KO mice, acute EAE is unaltered. However, the effects of Cx30/Cx43 deficiency on chronic EAE remains to be elucidated. We aimed to clarify the roles of Cx30 in chronic neuroinflammation by studying EAE induced by myelin oligodendrocyte glycoprotein peptide 35–55 in Cx30 KO mice. We found that Cx30 deficiency improved the clinical symptoms and demyelination of chronic but not acute EAE without influencing CD3+ T cell infiltration. Furthermore, increased ramified microglia in the naïve state and induced earlier and stronger microglial activation in the acute and chronic phases of EAE was observed. These activated microglia had an anti-inflammatory phenotype, as shown by the upregulation of arginase-1 and brain-derived neurotrophic factor and the downregulation of nitric oxide synthase 2. In the naïve state, Cx30 deficiency induced modest enlargement of astrocytic processes in the spinal cord gray matter and a partial reduction of Cx43 expression in the spinal cord white matter. These astrocytes in Cx30 KO mice showed earlier and stronger activation during the acute phase of EAE, with upregulated A2 astrocyte markers and a significant decrease in Cx43 in the chronic phases. Spinal cord neurons and axons were more preserved in Cx30 KO mice than in littermates in the chronic phase of EAE. These findings suggest that Cx30 deficiency increased ramified microglia in the CNS in the naïve state and improved chronic EAE through redirecting microglia toward an anti-inflammatory phenotype, suggesting a hitherto unknown critical role of astrocytic Cx30 in regulating microglial number and functional state.

Loss of the mitochondrial i‐AAA protease YME1L leads to ocular dysfunction and spinal axonopathy

Disturbances in the morphology and function of mitochondria cause neurological diseases, which can affect the central and peripheral nervous system. The i‐AAA protease YME1L ensures mitochondrial proteostasis and regulates mitochondrial dynamics by processing of the dynamin‐like GTPase OPA1. Mutations in YME1L cause a multi‐systemic mitochondriopathy associated with neurological dysfunction and mitochondrial fragmentation but pathogenic mechanisms remained enigmatic. Here, we report on striking cell‐type‐specific defects in mice lacking YME1L in the nervous system. YME1L‐deficient mice manifest ocular dysfunction with microphthalmia and cataracts and develop deficiencies in locomotor activity due to specific degeneration of spinal cord axons, which relay proprioceptive signals from the hind limbs to the cerebellum. Mitochondrial fragmentation occurs throughout the nervous system and does not correlate with the degenerative phenotype. Deletion of Oma1 restores tubular mitochondria but deteriorates axonal degeneration in the absence of YME1L, demonstrating that impaired mitochondrial proteostasis rather than mitochondrial fragmentation causes the observed neurological defects.

Spinal Progenitor-Laden Bridges Support Earlier Axon Regeneration Following Spinal Cord Injury.

Spinal cord injury (SCI) results in loss of tissue innervation below the injury. Spinal progenitors have a greater ability to repair the damage and can be injected into the injury, but their regenerative potential is hampered by their poor survival after transplantation. Biomaterials can create a cell delivery platform and generate a more hospitable microenvironment for the progenitors within the injury. In this work, polymeric bridges are used to deliver embryonic spinal progenitors to the injury, resulting in increased progenitor survival and subsequent regeneration and functional recovery, thus demonstrating the importance of combined therapeutic approaches for SCI.

Motor Exit Point (MEP) Glia: Novel Myelinating Glia That Bridge CNS and PNS Myelin.

Oligodendrocytes (OLs) and Schwann cells (SCs) have traditionally been thought of as the exclusive myelinating glial cells of the central and peripheral nervous systems (CNS and PNS), respectively, for a little over a century. However, recent studies demonstrate the existence of a novel, centrally-derived peripheral glial population called motor exit point (MEP) glia, which myelinate spinal motor root axons in the periphery. Until recently, the boundaries that exist between the CNS and PNS, and the cells permitted to cross them, were mostly described based on fixed histological collections and static lineage tracing. Recent work in zebrafish using in vivo, time-lapse imaging has shed light on glial cell interactions at the MEP transition zone and reveals a more complex picture of myelination both centrally and peripherally.

Proprioception 2.0: novel functions for muscle spindles.

Muscle spindles are encapsulated mechanosensory receptors within skeletal muscle tissue that inform the central nervous system about the contractile status of each muscle. This information is required for any coordinated movement and for stable posture. This review summarizes recent findings regarding novel functions for proprioceptive feedback information, muscle spindle disease and the molecular basis of mechanoreception. Muscle spindle function is not limited to regulating motor control but is also required for appropriate realignment of fractured bones, successful regeneration of spinal cord axons after injury and spinal alignment. Several proteins responsible for or modulating mechanotransduction in proprioceptive sensory neurons have been identified, including the Piezo2 channel as a candidate for the principal mechanotransduction channel. Many neuromuscular diseases are known to be accompanied by an impaired function of muscle spindles, resulting in a decline of motor performance and coordination in the patients. Our knowledge regarding the molecular basis of muscle spindle function is still incomplete. However, increasing our understanding of mechanotransduction in muscle spindles is a prerequisite for finding appropriate strategies to prevent injuries due to unstable gait and frequent falls.

Nck2 is essential for limb trajectory selection by spinal motor axons

The development of a functioning nervous system requires precise assembly of neuronal connections, which can be achieved by the guidance of axonal growth cones to their proper targets. How axons are guided by signals transmitted to the cytoskeleton through cell surface-expressed guidance receptors remains unclear. We investigated the function of Nck2 adaptor protein as an essential guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory into the limb. Nck2 mRNA and protein are preferentially expressed in the medial subgroups of chick LMC neurons during axon trajectory into the limb. Nck2 loss- and gain-of-function in LMC neurons using in ovo electroporation perturb LMC axon trajectory selection demonstrating an essential role of Nck2 in motor axon guidance. We also showed that Nck2 knockdown and overexpression perturb the growth preference of LMC neurites against ephrins in vitro and Eph-mediated redirection of LMC axons in vivo. Finally, the significant changes of LMC neurite growth preference against ephrins in the context of Nck2 and α2-chimaerin loss- and gain-of-function implicated Nck2 function to modulate α2-chimaerin activity. Here, we showed that Nck2 is required for Eph-mediated axon trajectory selection from spinal motor neurons through possible interaction with α2-chimaerin. Developmental Dynamics 247:1043-1056, 2018. © 2018 Wiley Periodicals, Inc.

Hyperphosphorylated Tau as a Novel Biomarker for Traumatic Axonal Injury in the Spinal Cord.

Current biomarker research in spinal cord injury (SCI) and traumatic brain injury has focused on a number of structural protein candidates, including the microtubule-associated protein tau. Evidence from models of traumatic brain injury has demonstrated that hyperphosphorylation of tau (p-tau) occurs in injured axons and demonstrates its utility as a biomarker for brain injury; however, the potential of p-tau as a biomarker for SCI is not yet known. Therefore, the present study determined whether tau is hyperphosphorylated in injured spinal cord axons, and then examined cerebrospinal fluid (CSF) and serum concentrations of p-tau and total-tau protein after a clinically relevant severe impact-compression SCI in rats. We found that severe SCI at T8 showed the presence of p-tau in damaged axons with a similar time course and distribution pattern to β-APP, a biomarker of axonal injury. The presence of p-tau and β-APP positive axons extended no farther than 5000 μm rostral and caudal to the injury epicenter, and was at its maximum at one day post-SCI. CSF levels of p-tau and total-tau significantly increased at one day post-SCI; however, only serum p-tau levels were significantly elevated in rats with SCI compared with naïve rats. These results suggest that CSF and serum p-tau may be a useful biomarker for severe traumatic SCI.

Counteracting roles of MHCI and CD8+ T cells in the peripheral and central nervous system of ALS SOD1G93A mice

BackgroundThe major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononucleated cells with CD8+T lymphocytes. We previously showed that MHCI is upregulated in the spinal cord microglia and motor axons of transgenic SOD1G93A mice.MethodsTo assess the role of MHCI in the disease, we examined transgenic SOD1G93A mice crossbred with β2 microglobulin-deficient mice, which express little if any MHCI on the cell surface and are defective for CD8+ T cells.ResultsThe lack of MHCI and CD8+ T cells in the sciatic nerve affects the motor axon stability, anticipating the muscle atrophy and the disease onset. In contrast, MHCI depletion in resident microglia and the lack of CD8+ T cell infiltration in the spinal cord protect the cervical motor neurons delaying the paralysis of forelimbs and prolonging the survival of SOD1G93A mice.ConclusionsWe provided straightforward evidence for a dual role of MHCI in the peripheral nervous system (PNS) compared to the CNS, pointing out regional and temporal differences in the clinical responses of ALS mice. These findings offer a possible explanation for the failure of systemic immunomodulatory treatments and suggest new potential strategies to prevent the progression of ALS.

Evidence of axon connectivity across a spinal cord transection in rats treated with epidural stimulation and motor training combined with olfactory ensheathing cell transplantation

Olfactory ensheathing cells (OECs) are unique glia that support axon outgrowth in the olfactory system, and when used as cellular therapy after spinal cord injury, improve recovery and axon regeneration. Here we assessed the effects of combining OEC transplantation with another promising therapy, epidural electrical stimulation during a rehabilitative motor task. Sprague-Dawley rats received a mid-thoracic transection and transplantation of OECs or fibroblasts (FBs) followed by lumbar stimulation while climbing an inclined grid. We injected pseudorabies virus (PRV) into hindlimb muscles 7 months post-injury to assess connectivity across the transection. Analyses showed that the number of serotonergic (5-HT) axons that crossed the rostral scar border and the area of neurofilament-positive axons in the injury site were both greater in OEC- than FB-treated rats. We detected PRV-labeled cells rostral to the transection and remarkable evidence of 5-HT and PRV axons crossing the injury site in 1 OEC- and 1 FB-treated rat. The axons that crossed suggested either axon regeneration (OEC) or small areas of probable tissue sparing (FB). Most PRV-labeled thoracic neurons were detected in laminae VII or X, and ~25% expressed Chx10, a marker for V2a interneurons. These findings suggest potential regeneration or sparing of circuits that connect thoracic interneurons to lumbar somatic motor neurons. Despite evidence of axonal connectivity, no behavioral changes were detected in this small-scale study. Together these data suggest that when supplemented with epidural stimulation and climbing, OEC transplantation can increase axonal growth across the injury site and may promote recovery of propriospinal circuitry.

Oligodendrocytic but not neuronal Nogo restricts corticospinal axon sprouting after CNS injury

Recovery from injury to the central nervous system (CNS) is limited in the mammalian adult. Nonetheless, some degree of spontaneous recovery occurs after partial CNS injury. Compensatory axonal growth from uninjured neurons, termed sprouting, contributes to this naturally occurring recovery process and can be modulated by molecular intervention. Extensive studies have depicted a long-held hypothesis that oligodendrocyte-derived Nogo restricts axonal sprouting and functional recovery after CNS injury. However, cell type-specific function of Nogo in compensatory sprouting, spinal axon repair or functional recovery after CNS injury has not been reported. Here we present data showing that inducible, cell type-specific deletion of Nogo from oligodendrocytes led to a ~50% increase in the compensatory sprouting of corticospinal tract (CST) axons in the cervical spinal cord after unilateral pyramidotomy in mice. In contrast to a previously proposed growth-promoting role of neuronal Nogo in the optic nerve, deleting neuronal Nogo did not significantly affect CST axon sprouting in the spinal cord. Sprouting axons were associated with the expression of synaptic marker VGLUT1 in both the oligodendrocytic Nogo deletion and control mice. However, we did not detect any functional improvement in fine motor control associated with the increased sprouting in oligodendrocytic Nogo deletion mice. These data show for the first time with genetic evidence that Nogo specifically expressed by oligodendrocytes restricts compensatory sprouting after CNS injury, supporting a longstanding but heretofore untested hypothesis. While implicating a focus on sprouting as a repair mechanism in the translational potential of targeting the myelin inhibitory pathway, our study illustrates the challenge to harness enhanced structural plasticity for functional improvement.

Aligned fibrous PVDF-TrFE scaffolds with Schwann cells support neurite extension and myelination in vitro

Objective. Polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE), which is a piezoelectric, biocompatible polymer, holds promise as a scaffold in combination with Schwann cells (SCs) for spinal cord repair. Piezoelectric materials can generate electrical activity in response to mechanical deformation, which could potentially stimulate spinal cord axon regeneration. Our goal in this study was to investigate PVDF-TrFE scaffolds consisting of aligned fibers in supporting SC growth and SC-supported neurite extension and myelination in vitro. Approach. Aligned fibers of PVDF-TrFE were fabricated using the electrospinning technique. SCs and dorsal root ganglion (DRG) explants were co-cultured to evaluate SC-supported neurite extension and myelination on PVDF-TrFE scaffolds. Main results. PVDF-TrFE scaffolds supported SC growth and neurite extension, which was further enhanced by coating the scaffolds with Matrigel. SCs were oriented and neurites extended along the length of the aligned fibers. SCs in co-culture with DRGs on PVDF-TrFE scaffolds promoted longer neurite extension as compared to scaffolds without SCs. In addition to promoting neurite extension, SCs also formed myelin around DRG neurites on PVDF-TrFE scaffolds. Significance. This study demonstrated PVDF-TrFE scaffolds containing aligned fibers supported SC-neurite extension and myelination. The combination of SCs and PVDF-TrFE scaffolds may be a promising tissue engineering strategy for spinal cord repair.

Non-viral-mediated suppression of AMIGO3 promotes disinhibited NT3-mediated regeneration of spinal cord dorsal column axons

After injury to the mature central nervous system (CNS), myelin-derived inhibitory ligands bind to the Nogo-66 tripartite receptor complex expressed on axonal growth cones, comprised of LINGO-1 and p75NTR/TROY and induce growth cone collapse through the RhoA pathway. We have also shown that amphoterin-induced gene and open reading frame-3 (AMIGO3) substitutes for LINGO-1 and can signal axon growth cone collapse. Here, we investigated the regeneration of dorsal root ganglion neuron (DRGN) axons/neurites after treatment with a short hairpin RNA (sh) AMIGO3 plasmid delivered with a non-viral in vivo-jetPEI vector, and the pro-survival/axogenic neurotrophin (NT) 3 in vitro and in vivo. A bicistronic plasmid, containing both shAMIGO3 and NT3 knocked down >75% of AMIGO3 mRNA in cultured DRGN and significantly overexpressed NT3 production. In vivo, intra-DRG injection of in vivo-jetPEI plasmids containing shAMIGO3/gfp and shAMIGO3/nt3 both knocked down AMIGO3 expression in DRGN and, in combination with NT3 overexpression, promoted DC axon regeneration, recovery of conduction of compound action potentials across the lesion site and improvements in sensory and locomotor function. These findings demonstrate that in vivo-jetPEI is a potential non-viral, translatable DRGN delivery vehicle in vivo and that suppression of AMIGO3 disinhibits the growth of axotomised DRGN enabling NT3 to stimulate the regeneration of their DC axons and enhances functional recovery.

Identification of the extracellular matrix protein Fibulin-2 as a regulator of spinal nerve organization

During amniote peripheral nervous system development, segmentation ensures the correct patterning of the spinal nerves relative to the vertebral column. Along the antero-posterior (rostro-caudal) axis, each somite-derived posterior half-sclerotome expresses repellent molecules to restrict axon growth and neural crest migration to the permissive anterior half-segment. To identify novel regulators of spinal nerve patterning, we investigated the differential gene expression of anterior and posterior half-sclerotomes in the chick embryo by RNA-sequencing. Several genes encoding extracellular matrix proteins were found to be enriched in either anterior (e.g. Tenascin-C, Laminin alpha 4) or posterior (e.g. Fibulin-2, Fibromodulin, Collagen VI alpha 2) half-sclerotomes. Among them, the extracellular matrix protein Fibulin-2 was found specifically restricted to the posterior half-sclerotome. By using in ovo ectopic expression in chick somites, we found that Fibulin-2 modulates spinal axon growth trajectories in vivo. While no intrinsic axon repellent activity of Fibulin-2 was found, we showed that it enhances the growth cone repulsive activity of Semaphorin 3A in vitro. Some molecules regulating axon growth during development are found to be upregulated in the adult central nervous system (CNS) following traumatic injury. Here, we found increased Fibulin-2 protein levels in reactive astrocytes at the lesion site of a mouse model of CNS injury. Together, these results suggest that the developing vertebral column and the adult CNS share molecular features that control axon growth and plasticity, which may open up the possibility for the identification of novel therapeutic targets for brain and spinal cord injury.

Binding of EphrinA5 to RET receptor tyrosine kinase: An in vitro study.

Eph/Ephrin signaling pathways are crucial in regulating a large variety of physiological processes during development, such as cell morphology, proliferation, migration and axonal guidance. EphrinA (efn-A) ligands, in particular, can be activated by EphA receptors at cell-cell interfaces and have been proposed to cause reverse signaling via RET receptor tyrosine kinase. Such association has been reported to mediate spinal motor axon navigation, but conservation of the interactive signaling pathway and the molecular mechanism of the interaction are unclear. Here, we found Danio rerio efn-A5b bound to Mus musculus EphA4 with high affinity, revealing structurally and functionally conserved EphA/efn-A signaling. Interestingly, we observed no interaction between efn-A5b and RET from zebrafish, unlike earlier cell-based assays. Their lack of association indicates how complex efn-A signaling is and suggests that there may be other molecules involved in efn-A5-induced RET signaling.