Single-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival.

Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.

CD40 forward signalling is a physiological regulator of early sensory axon growth.

Multiple members of the tumour necrosis factor superfamily (TNFSF) regulate the growth and branching of neural processes late in development, when neurons are establishing and refining connections. Here, we present the first evidence that a TNFSF member acts much earlier in development, when axons are growing to their targets. CD40L transiently enhanced axon growth from embryonic mouse DRG neurons cultured at this early stage. Early spinal nerves of embryos lacking the CD40L receptor (Cd40-/- mice) were significantly shorter in vivo than those of Cd40+/+ littermates. CD40L was synthesized in early DRG targets and was co-expressed with CD40 in early DRG neurons. Whereas CD40L enhanced early axon growth independently of neurotrophins, disruption of a CD40L/CD40 autocrine loop impaired early neurotrophin-promoted axon growth. In marked contrast to the widespread regulation of axon and dendrite growth by CD40L reverse signalling later in development, CD40-Fc, which activates reverse signalling, had no effect on early sensory axon growth. These results suggest that CD40 forward signalling is a novel physiological regulator of early axon growth that acts by target-derived and autocrine mechanisms.

Monocarboxylate transporter 1 in Schwann cells contributes to maintenance of sensory nerve myelination during aging.

Schwann cell (SC)-specific monocarboxylate transporter 1 (MCT1) knockout mice were generated by mating MCT1 f/f mice with myelin protein zero (P0)-Cre mice. P0-Cre+/- , MCT1 f/f mice have no detectable early developmental defects, but develop hypomyelination and reduced conduction velocity in sensory, but not motor, peripheral nerves during maturation and aging. Furthermore, reduced mechanical sensitivity is evident in aged P0-Cre+/- , MCT1 f/f mice. MCT1 deletion in SCs impairs both their glycolytic and mitochondrial functions, leading to altered lipid metabolism of triacylglycerides, diacylglycerides, and sphingomyelin, decreased expression of myelin-associated glycoprotein, and increased expression of c-Jun and p75-neurotrophin receptor, suggesting a regression of SCs to a less mature developmental state. Taken together, our results define the contribution of SC MCT1 to both SC metabolism and peripheral nerve maturation and aging.

Regulation of the Natriuretic Peptide Receptor 2 (Npr2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the Npr27A/7A mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the Npr27A/7A mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our in vivo studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth.SIGNIFICANCE STATEMENT The branching of axons is a morphological hallmark of virtually all neurons. It allows an individual neuron to innervate different targets and to communicate with neurons located in different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2), a transmembrane guanylyl cyclase, is essential for the initiation of bifurcation of sensory axons when entering the spinal cord or the hindbrain. By using two genetically engineered mouse lines, we show that phosphorylation of specific serine and threonine residues in juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon bifurcation. These investigations might help to understand the regulation of Npr2 and its integration in intracellular signaling systems.

A unified model of the excitability of mouse sensory and motor axons.

Non-invasive nerve excitability techniques have provided valuable insight into the understanding of neurological disorders. The widespread use of mice in translational research on peripheral nerve disorders and by pharmaceutical companies during drug development requires valid and reliable models that can be compared to humans. This study established a novel experimental protocol that enables comparative assessment of the excitability properties of motor and sensory axons at the same site in mouse caudal nerve, compared the mouse data to data for motor and sensory axons in human median nerve at the wrist, and constructed a mathematical model of the excitability of mouse axons. In a separate study, ischaemia was employed as an experimental manoeuvre to test the translational utility of this preparation. The patterns of mouse sensory and motor excitability were qualitatively similar to human studies under normal and ischaemic conditions. The most conspicuous differences between mouse and human studies were observed in the recovery cycle and the response to hyperpolarization. Modelling showed that an increase in temperature in mouse axons could account for most of the differences in the recovery cycle. The modelling also suggested a larger hyperpolarization-activated conductance in mouse axons. The kinetics of this conductance appeared to be much slower raising the possibility that an additional or different hyperpolarization-activated cyclic-nucleotide gated (HCN) channel isoform underlies the accommodation to hyperpolarization in mouse axons. Given a possible difference in HCN isoforms, caution should be exercised in extrapolating from studies of mouse motor and sensory axons to human nerve disorders.

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors.

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation.

Differences in nerve excitability properties across upper limb sensory and motor axons.

OBJECTIVE: The excitability of motor and sensory axons of the main upper limb nerves were compared to characterise the differences between nerves and provide a guide for future studies in human diseases with median neuropathy at the wrist. METHODS: Axonal excitability studies were undertaken on median and ulnar motor (APB and ADM) and sensory axons (D2 and D5) and the superficial radial axons (D1) using a threshold tracking technique. RESULTS: Compared to the median, ulnar motor axons had reduced early depolarising threshold electrotonus (TEd40(10-20 ms) p = 0.02) and superexcitability (p = 0.03). The ulnar sensory axons required a stronger stimulus (p = 0.02) and had a larger rheobase (p = 0.02) than median axons, but were otherwise comparable. The superficial radial axons were "fanned-in" compared to median, and to a lesser degree ulnar axons, with greater resting I/V slope. Mathematical modelling of the radial and median sensory axons suggested that a 15.1% reduction in conductances between nodal and internodal compartments accounted for 82% of this discrepancy. CONCLUSIONS: The excitability parameters of motor and sensory axons are most comparable between median and ulnar nerves. SIGNIFICANCE: The present study demonstrates the feasibility of, and provides normative data for, axonal excitability recordings of the radial and ulnar nerves. We suggest the use of ulnar recordings as an alternative to the median nerve in the setting of compressive neuropathy at the wrist.

Excitability of motor and sensory axons in multifocal motor neuropathy.

OBJECTIVE: To assess excitability differences between motor and sensory axons of affected nerves in patients with multifocal motor neuropathy (MMN). METHODS: We performed motor and sensory excitability tests in affected median nerves of 20 MMN patients and in 20 age-matched normal subjects. CMAPs were recorded from the thenar and SNAPs from the 3rd digit. Clinical tests included assessment of muscle strength, two-point discrimination and joint position. RESULTS: All MMN patients had weakness of the thenar muscle and normal sensory tests. Motor excitability testing in MMN showed an increased threshold for a 50% CMAP, increased rheobase, decreased stimulus-response slope, fanning-out of threshold electrotonus, decreased resting I/V slope, shortened refractory period, and more pronounced superexcitability. Sensory excitability testing in MMN revealed decreased accommodation half-time and S2-accommodation and less pronounced subexcitability. Mathematical modeling indicated increased Barrett-Barrett conductance for motor fibers and increase in internodal fast potassium conductance for sensory fibers. CONCLUSIONS: Excitability findings in MMN suggest myelin sheath or paranodal seal involvement in motor fibers and, possibly, paranodal detachment in sensory fibers. SIGNIFICANCE: Excitability properties of affected nerves in MMN differ between motor and sensory nerve fibers.

Tracking small sensory nerve action potentials in human axonal excitability studies.

BACKGROUND: Excitability studies on normal and diseased human axons in vivo have been greatly enhanced by fast non-invasive threshold-tracking techniques, using surface stimulation and recording. Although sensory axons are often more affected in disease, most studies to date have focussed on motor axons, because of technical difficulties in resolving pathologically small nerve volleys in the presence of noise and stimulus artefact. NEW METHODS: This paper describes techniques for tracking low-amplitude compound action potentials, using a battery-powered, isolated preamplifier of simple construction with high common mode rejection (>125 dB [balanced inputs]) and low noise (<0.4 µV referred to inputs [shorted]). RESULTS: We demonstrate the preamplifier's capability by tracking targets as small as 2 µV for a full range of excitability measurements without the usual distortion due to residual stimulus artefact and without the need for clamping, additional filtering or ensemble averaging. COMPARISON WITH EXISTING METHODS: In practice, threshold-tracking studies have been unable to study sensory axons when the maximal compound sensory action potential was less than about 15 µV. The techniques and amplifier in the present study allow measurements to be made from nerve with maximal responses less than half that size, and we present three recordings in patients with pathologically small nerve action potentials ≤7 µV. CONCLUSIONS: Based on measurements of stimulus artefact distortion, noise and the performance in experiments, we conclude that the techniques described here will facilitate the study of diseased axons for which the sensory potentials have high thresholds and may be only a few microvolts in amplitude.

Excitability of sensory axons in amyotrophic lateral sclerosis.

OBJECTIVE: To evaluate the excitability of sensory axons in patients with amyotrophic lateral sclerosis (ALS). METHODS: Comprehensive sensory nerve excitability studies were prospectively performed on 28 sporadic ALS patients, compared to age-matched controls. Sensory nerve action potentials were recorded from digit 2 following median nerve stimulation at the wrist. Disease severity was measured using motor unit number estimation (MUNE), the revised ALS Functional Rating Scale (ALSFRS-R) and the MRC scale. RESULTS: There were no significant differences in standard and extended measures of nerve excitability between ALS patients and controls. These unchanged excitability measures included accommodation to long-lasting hyperpolarization and the threshold changes after two supramaximal stimuli during the recovery cycle. Excitability parameters did not correlate with MUNE, ALSFRS-R, APB MRC scale or disease duration. CONCLUSIONS: This cross-sectional study has identified normal axonal membrane properties in myelinated sensory axons of ALS patients. Previously described sensory abnormalities could be the result of axonal fallout, possibly due to a ganglionopathy, or to involvement of central sensory pathways rostral to gracile and cuneate nuclei. SIGNIFICANCE: These results demonstrate the absence of generalized dysfunction of the membrane properties of sensory axons in ALS in the face of substantial deficits in motor function.

Segregation of motor and sensory axons regenerating through bicompartmental tubes by combining extracellular matrix components with neurotrophic factors.

Segregation of regenerating motor and sensory axons may be a good strategy to improve selective functionality of regenerative interfaces to provide closed-loop commands. Provided that extracellular matrix components and neurotrophic factors exert guidance effects on different neuronal populations, we assessed in vivo the potential of separating sensory and motor axons regenerating in a bicompartmental Y-type tube, with each branch prefilled with an adequate combination of extracellular matrix and neurotrophic factors. The severed rat sciatic nerve was repaired using a bicompartmental tube filled with a collagen matrix enriched with fibronectin (FN) and brain-derived neurotrophic factor (BDNF) encapsulated in poly-lactic co-glycolic acid microspheres (FN + MP.BDNF) in one compartment to preferentially attract motor axons and collagen enriched with laminin (LM) and nerve growth factor (NGF) and neurotrophin-3 (NT-3) in microspheres (LM + MP.NGF/NT-3) in the other compartment for promoting sensory axons regeneration. Control animals were implanted with the same Y-tube with a collagen matrix with microspheres (MP) containing PBS (Col + MP.PBS). By using retrotracer labelling, we found that LM + MP.NGF/NT-3 did not attract higher number of regenerated sensory axons compared with controls, and no differences were observed in sensory functional recovery. However, FN + MP.BDNF guided a higher number of regenerating motor axons compared with controls, improving also motor recovery. A small proportion of sensory axons with large soma size, likely proprioceptive neurons, was also attracted to the FN + MP.BDNF compartment. These results demonstrate that muscular axonal guidance can be modulated in vivo by the addition of fibronectin and BDNF.

In vivo analysis of axonal transport in zebrafish.

Intracellular transport of proteins and organelles in neurons plays an essential role in nervous system development and maintenance. Axon outgrowth, synapse formation, and synapse function, among other physiological processes, require active transport of these cargos between the neuronal soma and axon terminals. Abnormalities in this axonal transport are associated with a number of neurodevelopmental and neurodegenerative disorders, such as Charcot-Marie-Tooth disease, Alzheimer disease, and amyotrophic lateral sclerosis. Despite its importance for nervous system development and health, methods for visualizing axonal transport in an intact vertebrate have been lacking. Using the advantages of the zebrafish system, we have developed a straightforward approach to visualize axonal transport of various cargos and motor proteins in intact zebrafish embryos and larvae. Here, we describe this approach in detail and discuss how it can be applied to address questions related to cargo-specific transport regulation and its effects on axon morphology and function in the developing and mature nervous system.

Focal release of neurotrophic factors by biodegradable microspheres enhance motor and sensory axonal regeneration in vitro and in vivo.

Neurotrophic factors (NTFs) promote nerve regeneration and neuronal survival after peripheral nerve injury. However, drawbacks related with administration and bioactivity during long periods limit their therapeutic application. In this study, PLGA microspheres (MPs) were used to locally release different NTFs and evaluate whether they accelerate axonal regeneration in comparison with free NTFs or controls. ELISA, SEM, UV/visible light microscopy, organotypic cultures of DRG explants and spinal cord slices were used to characterize MP properties and the bioactivity of the released NTFs. Results of organotypic cultures showed that encapsulated NTFs maintain longer bioactivity and enhance neurite regeneration of both sensory and motor neurons compared with free NTFs. For in vivo assays, the rat sciatic nerve was transected and repaired with a silicone tube filled with collagen gel or collagen mixed with PBS encapsulated MPs (control groups) and with free or encapsulated NGF, BDNF, GDNF or FGF-2. After 20 days, a retrotracer was applied to the regenerated nerve to quantify motor and sensory axonal regeneration. NTF encapsulation in MPs improved regeneration of both motor and sensory axons, as evidenced by increased numbers of retrolabeled neurons. Hence, our results show that slow release of NTFs with PLGA MP enhance nerve regeneration.

Langerhans cells regulate cutaneous innervation density and mechanical sensitivity in mouse footpad.

Langerhans cells are epidermal dendritic cells responsible for antigen presentation during an immune response. Langerhans cells associate intimately with epidermal sensory axons. While there is evidence that Langerhans cells may produce neurotrophic factors, a role in regulating cutaneous innervation has not been established. We used genetically engineered mice in which the diphtheria toxin (DT) receptor is targeted to Langerhans cells (Lang-DTR mice) to assess sensory axon-dendritic cell interactions. Diphtheria toxin administration to wild type mice did not affect epidermal structure, Langerhans cell content, or innervation density. A DT administration regimen supramaximal for completely ablating epidermal Langerhans cells in Lang-DTR mice reduced PGP 9.5-immunoreactive total innervation and calcitonin gene related peptide-immunoreactive peptidergic nociceptor innervation. Quantitative real-time polymerase chain reaction showed that epidermal gene expression of brain derived neurotrophic factor was unchanged, but nerve growth factor and glial cell line-derived neurotrophic factor mRNAs were reduced. Behavioral testing showed that, while thermal sensitivity was unaffected, mice depleted of Langerhans cells displayed mechanical hypersensitivity. These findings provide evidence that Langerhans cells play an important role in determining cutaneous sensory innervation density and mechanical sensitivity. This may involve alterations in neurotrophin production by Langerhans or other epidermal cells, which in turn may affect mechanical sensitivity directly or as a result of neuropathic changes.

A multifunctional neurotrophin with reduced affinity to p75NTR enhances transplanted Schwann cell survival and axon growth after spinal cord injury.

The lack of regeneration of axonal pathways after SCI is associated with the presence of inhibitory molecules within the glial scar, the loss of the neuron's intrinsic capacity to grow and the absence of growth factors. The NGF family of neurotrophins is a potent growth factor for several types of supraspinal and sensory axons. It is unclear, however, whether the neurotrophin's axon growth-promoting activities after central nervous system (CNS) injuries are mediated through the Trk receptors or p75 neurotrophin receptor (p75(NTR)) or both. To investigate the role of these receptors in the re-growth of specific fiber tracts after SCI, we created a series of neurotrophins that preferentially bind to either TrkB/C or p75(NTR) receptors. All the mutations were made on the NT-3/D15A backbone, a multifunctional neurotrophin that can bind TrkB, TrkC and p75(NTR). To test the mutants' axon growth-promoting activity after rat contusion SCI, we examined several spinal cord fiber projections after transplanting Schwann cells (SCs) expressing the different multi-neurotrophins. Grafts expressing the NT-3/D15A with reduced binding affinity to p75(NTR) contained more surviving SCs, and sensory as well as supra-spinal fibers, within the transplant than the NT-3/D15A neurotrophin-SC grafts. These data support the idea that neurotrophins lacking p75 activity can be more effective in promoting axon growth after CNS injury.

Bioenergetic deficits in peripheral nerve sensory axons during chemotherapy-induced neuropathic pain resulting from peroxynitrite-mediated post-translational nitration of mitochondrial superoxide dismutase.

Many of the widely used anticancer drugs induce dose-limiting peripheral neuropathies that undermine their therapeutic efficacy. Animal models of chemotherapy-induced painful peripheral neuropathy (CIPN) evoked by a variety of drug classes, including taxanes, vinca alkaloids, platinum-complexes, and proteasome-inhibitors, suggest that the common underlying mechanism in the development of these neuropathies is mitotoxicity in primary nerve sensory axons (PNSAs) arising from reduced mitochondrial bioenergetics [eg adenosine triphosphate (ATP) production deficits due to compromised respiratory complex I and II activity]. The causative mechanisms of this mitotoxicity remain poorly defined. However, peroxynitrite, an important pro-nociceptive agent, has been linked to mitotoxicity in several disease states and may also drive the mitotoxicity associated with CIPN. Our findings reveal that the development of mechano-hypersensitivity induced by paclitaxel, oxaliplatin, and bortezomib was prevented by administration of the peroxynitrite decomposition catalyst Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) without interfering with their anti-tumor effects. Peak CIPN was associated with the nitration and inactivation of superoxide dismutase in the mitochondria, but not in the cytosol, as well as a significant decrease in ATP production within the PNSAs; all of these events were attenuated by MnTE-2-PyP(5+). Our results provide continued support for the role of mitotoxicity in the development of CIPN across chemotherapeutic drug classes, and identify peroxynitrite as a key mediator in these processes, thereby providing the rationale towards development of "peroxynitrite-targeted" therapeutics for CIPN.