Cdon ablation in motor neurons causes age‐related motor neuron degeneration and impaired sciatic nerve repair

Abstract

Background

The functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging‐related muscle wasting. Motor neuron diseases or aging‐related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule‐downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin‐mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown.

Methods

Hb9‐Cre recombinase‐driven motor neuron‐specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1^G93A) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed.

Results

Mice lacking Cdon in motor neurons exhibited middle age onset lethality and aging‐related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63 ± 0.35 vs. 3.93 ± 0.22 s for f/f, P < 0.01) and physical activity. Consistently, neuromuscular junctions of mnKO muscles were incompletely occupied (49.79 ± 5.74 vs. 79.39 ± 3.77% fully occupied neuromuscular junctions for f/f, P < 0.0001), suggesting an impaired reinnervation. The transmission electron microscopy analysis revealed that mnKO sciatic nerves had smaller axon diameter (0.88 ± 0.13 vs. 1.43 ± 0.48 μm for f/f, P < 0.0001) and myelination defects. RNA sequencing of mnKO lumbar spinal cords showed alteration in genes related to neurogenesis, inflammation and cell death. Among the altered genes, ErbB4 and FgfR expressions were significantly altered in mnKO as well as in Cdon‐depleted NSC34 motor neuron cells. Consistently, Cdon‐depleted NSC34 cells exhibited elevated levels of cleaved Caspase3 and γH2AX proteins, as well as Bax transcription. Cdon‐depleted NSC34 cells also exhibited impaired activation of Akt in response to neuregulin‐1 (NRG1) treatment.

Conclusions

Our current data demonstrate the functional importance of Cdon in motor neuron function and nerve repair. Cdon ablation causes alterations in neurotrophin signalling that leads to motor neuron degeneration.

Introduction

Aging is one of the main risk factors of neurodegenerative diseases such as amyotrophic lateral sclerosis. ¹ The functional decline of motor neurons that occurs with age has been shown to be tightly linked with muscle wasting and frailty. ² Reduced synaptic transmission or decreased nerve conduction velocity leads to muscle wasting, ³ and muscle function is in turn critical for the trophic support of motor neurons and axon growth. ⁴ As a result, the maintenance of motor neuron function and the neuromuscular junction (NMJ), which is the site where presynaptic motor neuron terminus and myofibre interacts, appears to be critical for the prevention of neurodegenerative diseases. The impairment in axonal regeneration, remyelination and reinnervation after injury or denervation is another critical feature of motor neuron diseases. Among diverse pathways, perturbations in neurotrophic signalling, oxidative stress, inflammation and mitochondrial dysfunction seem to be closely associated with the aging‐related motor neuron degeneration, neuromuscular junction stability and regenerative capacity of motor neurons. ⁴ , ⁵ , ⁶

Cell adhesion molecules have been implicated in multiple stages of central nervous system development including cell differentiation, synapse formation and neuron–glia interaction to ensure synaptic transmission and plasticity. ⁷ , ⁸ Cell adhesion molecules, such as NCAM, L1 and N‐cadherin are important in supporting axonal regeneration and remyelination. ⁹ , ¹⁰ Cdon (cell adhesion molecule‐downregulated oncogene; also known as Cdo) is closely related to N‐CAM and L1 and plays important roles in multiple signalling pathways, including Sonic hedgehog (Shh), netrin and cadherin‐mediated signalling. Along with Boc (BOC cell adhesion associated, oncogene regulated) and Gas1 (growth arrest‐specific 1), Cdon functions as Shh coreceptor and is implicated in the specification of ventral neural cell types such as dopaminergic or motor neurons during embryonic development. ¹¹ , ¹² Multiple studies have also proposed the eminent roles of Shh signalling in nerve regeneration after peripheral nerve injury. ¹³ , ¹⁴ Shh is transiently upregulated upon nerve injury and facilitate motor neuron survival and axonal regrowth, likely through neurotrophin regulation. ¹⁴ Although the role of Cdon as a Shh coreceptor in embryonic ventral neuronal specification is well studied, their role in nerve regeneration or motor neuron function is uncharacterized.

In the current study, we determined the in vivo function of Cdon in neuromuscular function and motor neuron regeneration after peripheral nerve injury. Due to the early lethality of whole body Cdon knockout mice and multiple developmental defects including holoprosencephaly, we generated mice with motor neuron‐specific Cdon ablation (mnKO) by breeding Cdon^f/f mice with HB9‐Cre recombinase carrying mice. The significance of Cdon in motor neurons was revealed by evaluations of motor function via multiple behavioural tests and histological analysis of motor neurons and NMJ. To investigate Cdon's role in the context of motor neuron diseases, Cdon was ablated in motor neurons of amyotrophic lateral sclerosis mouse model and evaluated how Cdon ablation further exacerbate the disease phenotype. To determine Cdon's function in nerve regeneration, sciatic nerve of mnKO mice was crushed to find impaired peripheral nerve repair after crush injury. Transcriptome analysis of mnKO mice spinal cord and Cdon depletion in NSC34 motor neuron‐like cells revealed reduction in Erb‐B2 receptor tyrosine kinase 4 (ErbB4) expression and impaired response to neuregulin 1, likely contributing to the cellular stress and death of the motor neurons. In summary, these data suggest that Cdon is a critical factor for the maintenance of motor neuron function during aging and nerve repair post‐injury.

Methods

Animal studies

Mice bearing Cdon‐floxed allele (Cdon^f/f) were maintained as previously described. ¹⁵ To generate mice with a motor neuron‐specific ablation of Cdon (mnKO), Cdon^f/f (f/f) were crossed with transgenic mice expressing Cre under the control of the Hb9 promoter (Hb9‐Cre, Jackson Laboratory). Littermates of f/f and mnKO mice were employed for the phenotype studies. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at Sungkyunkwan University (SKKU) and complied with the animal experiment guidelines of SKKU Ethics Committee (SKKUIACUC2022‐05‐45‐1). All experimental mice were housed in the animal facility at 23°C with a 12:12 h light–dark cycle with ad libitum access to food and water. All animal experiments were performed in a blinded fashion.

Gait, grip strength and open field analysis

To analyse the physical activities of f/f and mnKO mice, mice were subjected to gait test, grip strength test, and open field test in a blinded fashion. All behaviour tests were performed during the light cycle (11 am to 5 pm). To obtain footprints, hindlimb of the mice were stamped with black or purple nontoxic paint. The animals were then allowed to walk along a 50‐cm‐long and 10‐cm‐wide runway surrounded by 10‐cm‐height walls within an enclosed box. Under the runway, a sheet of white paper was placed. Stride, sway and stance were measured for the gait test.

The grip strength test was measured using a grip strength meter (Bioseb). As a mouse grasp the bar with forelimb, the peak pull force in grams was recorded on a digital force transducer. The mice were pulled gently with consistent force until the forelimb was detached from the grid. The maximal strength was recorded when the forelimb detached from the grid. We performed three consecutive measurements per day. When the grip tests were performed on the same day, mice were allowed to rest for at least 30 min between the tests. The order of mice tested on each day was randomized, and the experimenters were blinded to the results of the previous tests.

The open field test was performed in an open plastic container (30 × 30 × 30 cm). At the beginning of each trial, mice were placed in a corner of the plastic container, and their free movement was recorded for 20 min. The Noldus EthoVision 14.0 tracking software was used to analyse moved distance and velocity with heatmap.

Compound muscle action potentials (CMAPs)

Measurement of compound muscle action potentials was performed as previously described ³ with minor modification. Mice were anesthetized with 5% isoflurane and maintained by continuous inhalation of 2% isoflurane mixed with oxygen. For sciatic nerve stimulation and tracing CMAPs, a portable electromyography unit (PowerLab, AD instruments) was used. The active electrodes were positioned at tibialis anterior (TA) muscle. Sciatic nerve stimulation was performed using a voltage of 5, 10 and 20 mV and repeated 10 times each. Four parameters (Amplitude, Duration, Latency, and amplitude change) after each stimulation were recorded by the LabChart software version 8.0 (AD instruments).

Sciatic nerve crush injury and sampling

For sciatic nerve crush, mice were anesthetized with 5% isoflurane, and the skin was incised about 1 cm posterior and parallel to the femur. The sciatic nerve was exposed at the mid‐tight level and crushed with smooth forceps for 10 s between the proximal and distal site of the nerve. Following denervation, the crushed nerve was repositioned, and the muscle and skin were sutured by dissolvable surgical nylon sutures. After the injury, the mice were allowed to recover under a heating lamp in the cage. Before and after surgery, the surgical site was sterilized by the povidone‐iodine solution. Carprofen (5 mg/kg) was administered subcutaneously for post‐operative pain control. Following recovery, sciatic nerve was sampled by dissecting the region just proximal from the injury site for immunostaining and electron microscopy. For RNA analysis, the whole region of sciatic nerve was harvested and processed. For spinal cord sampling, lumbar spinal cord segment L1–L6 were dissected out and proceeded for cryosection and biochemical analysis.

Cell culture experiments

NSC34 motor neuron‐like cells were cultured in growth medium (DMEM high glucose, Gibco) containing 10% fetal bovine serum (Gibco), 1% glutamax (Gibco), 10 units/mL penicillin and 10 μg/mL streptomycin (Welgene Inc.) at 37°C, 5% CO₂. Cells were induced to differentiate with Advanced DMEM/F12 (Gibco), 1% fetal bovine serum (Gibco), 1% MEM NEAA (Gibco), 10 units/mL penicillin and 10 μg/mL streptomycin (Welgene Inc.), and 1 μM retinoic acid (Sigma). For transfection experiments, cells were transfected using TransIT‐X2 System (Mirus, MIR 6010). To delete or overexpress Cdon, the expression vectors for Cdon or Cdon shRNA with matched control vectors were utilized as previously described. ¹⁵

Protein experiments

Immunoblot experiments were performed as previously described. ¹⁶ Briefly, cells or homogenized tissue samples were dissolved in RIPA buffer (1× PBS, 1% IGEPAL CA‐630 (v/v), 0.1% sodium dodecyl sulfate (SDS) (w/v), 0.5% sodium deoxycholate (w/v) and 50 mM sodium fluoride). The primary antibodies used in this study are provided in Table S1.

Histology and immunostaining

Spinal cord, sciatic nerve and hindlimb muscle tissues were embedded in Tissue‐Tek OCT Compound (Sakura), and 7‐μm thick serial sections were prepared using a cryomicrotome. The sections were fixed, permeabilized and incubated with primary antibodies provided in Table S1. The brightfield images were captured and processed with a Nikon ECLIPSE TE‐2000U inverted microscope using NIS‐Elements F software (Nikon). The fluorescent images were obtained using LSM‐710 confocal microscope system (Carl Zeiss) and ZEN software (Carl Zeiss).

Electron microscopy and toluidine staining

For sciatic nerve isolation, the mice were perfused by 4% PFA before sacrifice. A segment of the peripheral nerve was excised 1 mm distal to the point of separation from the main branch point of the sciatic nerve and drop‐fixed in modified Karnovsky's fixative (20% PFA and 8% glutaraldehyde in 0.2 M sodium cacodylate, pH 7.4) at 4°C overnight. Tissue was washed with 0.2 M sodium cacodylate buffer (pH 7.2) then post‐fixed with 2% aqueous osmium tetroxide for 2 h. Samples were washed with cacodylate buffer then dehydrated through ascending alcohols, washed with propylene oxide and embedded in Spurr's resin (Electron Microscopy Sciences). For light microscopy, 500 nm semi‐thin sections were cut on an EM U7 microtome (Leica) and counterstained with toluidine blue and viewed on a light microscope AxioImager M2 (Carl Zeiss). Images were recorded with an AxioCam MRm. For transmission electron microscopy (TEM), ultrathin sections (70–80 nm) were cut on an EM UC7 microtome (Leica) and collected on 1 × 2 mm formvar coated copper slot grids. Images were captured with a Talos L120C (FEI). Data were analysed using ImageJ software.

RNA analysis

Quantitative RT‐PCR analysis was performed as previously described. ¹⁶ , ¹⁷ Briefly, the tissues were homogenized by FastPrepR‐24 (MP Biomedicals) and extracted using an easy‐spin Total RNA Extraction Kit (iNtRON Biotechnology). The fold change in gene expression was normalized against the expression of ribosomal gene L32 or 18S RNA. The sequences of the primers used in this study are provided in Table S2.

To assess transcriptome of mnKO spinal cords, RNA sequencing was performed with Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies). RNA sequencing data were analysed by using ExDEGAv1.61 (e‐Biogen). The unbiased Gene Set Enrichment Analysis (GSEA; http://www.broadinstitute.org/gsea) was performed as described in previous studies. ¹⁶ All GSEA plots including GSEA enrichment plot and Enrichment Map were generated with the GSEA software (Broad Institute; software.broadinstitute.org/gsea/).

Open database analysis

For the transcriptomic analysis of single nuclei RNA sequencing, the SeqGeqTM program (Flowjo) was used to analyse the gene‐barcode matrices of the BAM file for PHATE clustering. The datasets analysed in this study obtained from NCBI GEO database were deposited with the Status Omnibus (GEO) as ‘GSE167597’.

Statistical analysis

Values are expressed as either mean ± SD or mean ± SEM, as indicated in the figure legends. The statistical significance was calculated using Student's t‐test (paired, two‐tailed). Differences were considered statistically significant if P < 0.05. For comparisons between multiple groups, the statistical significance was tested by analysis of variance test using SPSS v12.0.

Results

Cdon is expressed in the motor neurons and neuromuscular junction

To determine the role of Cdon in neuromuscular function, we investigated the expression of Cdon in motor neurons and NMJ using the GEO open database. Single‐nuclei RNA sequencing data of adult mouse spinal cord showed that Cdon expressed in various clusters of choline acetyltransferase (ChAT)‐positive motor neurons (Figure 1A,B). The comparison of Cdon expression between synaptic and non‐synaptic region of skeletal muscle revealed that the Cdon expression is distinctively higher in the synaptic regions than in the non‐synaptic region of skeletal muscle, correlating with the expression levels of Chrna1 and myelin binding protein (Figure 1C). To verify the Cdon expression in the motor neurons and NMJs, isolated extensor digitorum longus (EDL) myofibres of wild type mice were immunostained for Cdon and NMJ markers. Cdon colocalized with alpha‐bungarotoxin (BTX) that labels NMJ (Figure 1D) and to some degree with Neurofilament (NF) and Synaptophsin (SV) that labels presynaptic endplate (Figure 1E). Cdon expression was also found in the periphery of sciatic nerve axons partially overlapping with myelin binding protein (Figure 1F). These data suggest Cdon's potential role in neuromuscular function and axon‐myelin interaction.

Figure 1.

Cdon is expressed in the motor neurons and neuromuscular junction. (A) Potential of heat‐diffusion for affinity‐based trajectory embedding (PHATE) plot from single‐nuclei RNA sequencing data of adult mouse spinal cord colour‐coded for cluster density (left) and feature plot depicting the choline acetyltransferase (ChAT) expression in the spinal cord (right). (GEO accession: GSE167597). (B) PHATE plot of the ChAT‐positive clusters colour‐coded for cholinergic interneurons, visceral motor neurons (MNs), and skeletal MNs (left) and feature plot showing the expression of Cdon in the ChAT‐positive motor neuron clusters (right). (GEO accession: GSE167597). (C) A heat map of NMJ‐related genes and Cdon expression in synaptic and non‐synaptic region of diaphragm muscle from the GEO open database (GEO accession: GSE2873). (D) Representative confocal image of extensor digitorum longus (EDL) myofibre immunostained with indicated antibodies from WT mice. Scale bar: 40 μm. (E) Representative confocal image of flexor digitorum brevis (FDB) immunostained with indicated antibodies from 4‐month‐old WT mice. Scale bar: 45 μm (left panel), 45 μm (right panel). (F) Cdon and MBP (myelin marker) staining of transverse section of 4‐month‐old WT mice sciatic nerve. Scale bar: 10 μm.

mnKO mice exhibit age‐related lethality and impaired motor function due to motor neuron degeneration

Whole‐body Cdon knockout mice exhibit 80–85% embryonic lethality, and the surviving 15–20% knockout mice die within 4–12 weeks after birth exhibiting breathing difficulties and immobility. ¹⁸ , ¹⁹ The early death of Cdon‐knockout mice complicates a close examination of Cdon's role in motor neuron function. Thus, we generated mice lacking Cdon specifically in motor neurons by crossing two floxed‐Cdon alleles (Cdon^fl/fl, f/f) to f/f mice hemizygous for Hb9‐Cre expressing the Cre recombinase under the control of a motor neuron‐specific Hb9 promoter (Figure S1A). The resulting mice lacking Cdon in motor neurons (mnKO) and their f/f littermates were used for the phenotype analysis. Mice were born with an expected ratio, and no overt phenotype associated with developmental defects or growth deficits were observed in mnKO mice. Knockout of Cdon in motor neurons was confirmed by immunostaining and immunoblot of Cdon in Hb9‐positive motor neurons and sciatic nerve extract of mnKO mice, respectively (Figure 2A). Interestingly, mnKO mice exhibited lethality starting from 7 months of age while displaying tremor and breathing difficulties, and only about 60% of mnKO mice survived beyond 12 months of age (Figure 2B). In addition, mnKO mice frequently displayed upper spinal kyphosis, indicative of back muscle weakness (Figure 2C).

Figure 2.

Motor neuron‐specific Cdon deficient mice exhibit age‐related lethality and muscle weakness accompanied by NMJ abnormalities. (A) Confirmation of Cdon ablation in the motor neurons of mnKO mice via immunostaining of lumbar spinal cord (left) and immunoblot analysis of sciatic nerve (right). Scale bar: 10 μm. (B) Kaplan–Meier survival curve of f/f (n = 26) and mnKO (n = 24) mice. The data were analysed by log‐rank (Mantel–Cox) test. (C) Impaired motor function of mnKO mice demonstrated by Kyphosis phenotype of 10‐month‐old mnKO mice (left) and forelimb grip strength test (middle) and gait test (right) of 24‐month‐old f/f and mnKO mice (n = 6 per each group). The grip strength was recorded in grams from the digital force transducer. (D) Representative image of CMAP waveform from TA muscles of 24‐month‐old f/f and mnKO mice and bar graphs showing CMAP measurement (n = 6 per each group). (E) Representative semi‐thin section (500 nm) of 24‐month‐old f/f and mnKO mice sciatic nerve stained with toluidine blue (left), measurement of myelin thickness (middle), and number of degenerating axons (right) (n = 4 per each group). Examples of a large dilated axon (yellow asterisk) and a degenerating axon (red asterisk) are indicated. Scale bar: 10 μm. (F) Representative image of whole‐mount EDL muscle stained for α‐BTX and neurofilament + synaptophysin (left) and quantification of fully occupied, partially occupied, and denervated NMJs (right) (n = 3 per each group). Scale bar: 25 μm. The data are expressed as mean ± SD and analysed by Student's t‐test (C, D, and E) or two‐way ANOVA (F). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

To examine the effect of Cdon depletion on motor function, 24‐month‐old mnKO mice and their f/f littermates were subjected to forelimb grip strength and gait tests. mnKO mice displayed a decrease in grip strength and sway and stance distance (Figure 2C), indicative of muscle weakness and declined motor function. The measurement of compound muscle action potentials (CMAPs) of tibialis anterior (TA) muscles showed that the distal duration was about 170% longer in mnKO muscles than f/f muscles, suggestive of defective nerve transmission (Figure 2D). Because nerve demyelination is a key factor causing slower nerve conduction, we next examined the sciatic nerves by toluidine blue staining. Sciatic nerves of mnKO mice had reduced myelin sheath thickness and more degenerating axons relative to those of f/f mice (Figure 2E). Furthermore, NMJs in mnKO EDL muscles were frequently fragmented and significantly denervated, while f/f muscles exhibited well occupied NMJs (Figure 2F). Finally, mnKO mice exhibited signs of muscle atrophy by reduced cross‐sectional area of myofibres and elevated expression of atrophy markers (Atrogin‐1 and MuRF1) in the TA muscle compared with that of f/f mice (Figure S1B–D). These data indicate that Cdon deficiency in motor neurons causes age‐related impairment in motor function due to motor neuron degeneration.

Cdon ablation exacerbates ALS‐related phenotypes of SOD1^G93A mutant mice

ALS is an age‐dependent neurodegenerative disease linked with motor neuron degeneration, muscle atrophy, and paralysis. About 20% of inherited forms of ALS are caused by mutations in Cu/Zn superoxide dismutase (SOD1). ²⁰ Consistently, mice expressing SOD1^G93A mutant exhibit ALS‐like phenotypes with motor neuron degeneration, NMJ abnormalities, muscle atrophy, and lethality. ²¹ Thus, we investigated the role of Cdon in ALS‐like phenotypes of SOD1^G93A mutant mice. The mnKO::SOD1^G93A mice displayed Kyphosis and hind limb clasping phenotype at 16 weeks of age (Figure 3A) and died around 15 to 16 weeks of age while f/f::SOD1^G93A littermates died around 18 to 20 weeks of age (Figure 3B). mnKO::SOD1^G93A mice exhibited rapid decrease in body weight starting around 14 weeks of age, while f/f::SOD1^G93A mice maintained their body weight at this stage (Figure 3B). While no statistically significant differences were observed in grip strength and gait tests between mnKO::SOD1^G93A and f/f::SOD1^G93A mice (Figure 3C,D), mnKO::SOD1^G93A mice displayed drastic decrease in the latency from CMAP measurement (Figure 3E). The endplate immunostaining revealed that NF‐positive axons in EDL muscles of mnKO::SOD1^G93A were thinner, while the NMJ occupancy was not significantly altered compared with those of f/f::SOD1^G93A mice (Figure 3F). In addition, mnKO::SOD1^G93A mice showed reduced gastrocnemius (GAS) and TA muscle mass and smaller cross‐sectional area of myofibres compared with those of f/f::SOD1^G93A mice (Figure S2 A,B). Quantitative RT‐PCR (qRT‐PCR) analysis revealed that mnKO::SOD1^G93A TA muscles express greatly elevated levels of denervation markers (MyoG, AchRa1 and AchRb1) and atrophy markers (Atrogin‐1 and MuRF1) compared with f/f and f/f::SOD1^G93A muscles (Figure S2 C). On the other hand, expression of the satellite cell marker (Pax7) significantly decreased in TA muscles of mnKO::SOD1^G93A than that of f/f::SOD1^G93A mice (Figure S2 D). These data suggest that Cdon depletion in motor neurons exacerbates ALS‐related phenotypes of SOD^G93A mice.

Figure 3.

Cdo ablation exacerbates ALS‐like phenotype of SOD1^G93A mice. (A) Kyphosis phenotype of 4‐month‐old mnKO::SOD1^G93A mice. (B) Kaplan–Meier survival curve (left) and changes in body weight (right) during the first 18 weeks of f/f, mnKO, f/f::SOD1^G93A, and mnKO::SOD1^G93A mice. The survival curve data were analysed by log‐rank (Mantel–Cox) test. (C) Forelimb grip strength tests of 4‐month‐old f/f, f/f::SOD1^G93A, and mnKO::SOD1^G93A mice. The grip strength was recorded in grams from the digital force transducer (n = 3 per each group). (D) Gait test of 4‐month‐old f/f, f/f::SOD1^G93A, and mnKO::SOD1^G93A littermates (n = 3 per each group). (E) Latency from CMAP measurement of 4‐month‐old f/f, f/f::SOD1^G93A, and mnKO::SOD1^G93A mice (n = 3 per each group). (F) Representative images of whole‐mount FDB muscle stained for α‐BTX and neurofilament + synaptophysin (left), quantification of fully occupied, partially occupied, and denervated NMJs (top right), and measurement of axon branch diameter (bottom right) (n = 3 per each group). Scale bar: 50 μm and 50 μm (inset). The data are expressed as mean ± SD and analysed by one‐way (C, D, E, and F; bottom right) or two‐way ANOVA (F; top right). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

mnKO mice exhibit impaired nerve regeneration and remyelination after sciatic nerve crush injury

Next, we investigated the role of Cdon in peripheral nerve repair via sciatic nerve crush injury (Figure 4A). Both f/f and mnKO mice exhibited reduced hindlimb muscle weights compared with sham‐treated f/f mice 4 weeks post‐injury, suggestive for the incomplete recovery (Figure S3 A). Compared with f/f mice, mnKO mice exhibited mild but statistically significant reduction in stance and stride distances from gait test (Figure 4B). Also, the duration of nerve transmission was significantly increased in mnKO mice compared with f/f mice (Figure 4C). To further define the defects in nerve repair of mnKO mice, the distal segments of sciatic nerves from both f/f and mnKO mice 35 days post‐injury were harvested and subjected to toluidine blue staining. The axon diameter of mnKO sciatic nerves was substantially reduced than that of f/f sciatic nerves (Figure 4D). Furthermore, the g‐ratio calculated as axon diameter/fibre diameter to assess motor axonal myelination revealed a dramatic increase in mnKO mice than that of f/f mice, suggesting remyelination defect (Figure 4D). The transmission electron microscopic images of f/f and mnKO sciatic nerves further demonstrated impaired remyelination of mnKO mice via significantly reduced myelin thickness (Figure 4E). The NMJ morphology in the flexor digitorum brevis muscles of f/f mice showed mostly well recovered and fully occupied NMJs with thick axons, while mnKO muscles had considerably higher proportion of denervated NMJs and thinner axons (Figure 4F). qRT‐PCR analysis of mnKO muscles revealed a specifically elevated myofibre type I transcript and significant alterations in the expression of muscle denervation and atrophy markers compared with those of f/f mice (Figure S3 B–D). Taken together, these data suggest that motor neuron‐specific Cdon ablation attenuates motor neuron's capacity to regenerate and remyelinate after crush injury.

Figure 4.

Cdo deficiency in motor neurons causes impaired reinnervation and remyelination in sciatic nerve crush injury model. (A) Timeline of sciatic nerve crush injury and analysis. (B) Parameters measured from gait test 28 days after sciatic nerve injury (n = 16 per each group). (C) Representative image of CMAP waveform from TA muscles of f/f and mnKO mice 30 days post‐injury and bar graphs showing CMAP measurements (n = 8 per each group). (D) Representative semi‐thin section (500 nm) at the distal site of f/f and mnKO mice sciatic nerve post‐injury stained with toluidine blue (left) and measurement of axon diameter and g‐ratio (right) (n = 4 per each group). Scale bar: 20 μm. (E) Representative transmission electron microscope images for sciatic nerve ultra‐thin section at the distal site (left) and measurement of myelin thickness (right) (n = 2 per each group). Scale bar: 10 μm. (F) Representative confocal images of FDB muscles of f/f and mnKO mice post‐injury immunostained for α‐BTX and neurofilament + synaptophysin (left), quantification of fully occupied, partially occupied, and denervated NMJs (middle), and measurement of axon branch diameter (right) (n = 3 per each group). Scale bar: 50 and 25 μm (inset). The data are expressed as mean ± SD and analysed by Student's t‐test (B, C, D, E, and F; right) or two‐way ANOVA (F; middle). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Genes related to neurogenesis, inflammation and apoptosis are majorly altered in regenerating mnKO lumbar spinal cords after injury

To determine the underlying mechanisms leading to impaired motor function of mnKO mice, the lumbar spinal cord from f/f and mnKO mice at 35 days post‐nerve crush injury was collected, and the transcriptome was assessed by the next‐generation sequencing. The obtained transcriptomic profile was applied to the Gene Set Enrichment Analysis (GSEA; Broad Institute), a computational method that identifies statistically enriched gene sets. mnKO spinal cords exhibited alterations in genes related to neurogenesis, inflammation, cell death, metabolism and synaptic organization compared with the control (Figure 5A–D). Furthermore, the expression of genes altered in mnKO spinal cords correlated with one another, implying that the altered gene expression is a systematic consequence of Cdon ablation in the motor neuron (Figure 5E,F). Taken together, the data suggest that impaired nerve repair post‐crush injury due to Cdon ablation in motor neurons is associated with the dysregulation of diverse pathways including neurogenesis, inflammation and cell death.

Figure 5.

Alteration of gene expression profiles in the spinal cord of mnKO mice. (A) A volcano plot representing significantly altered gene groups (fold change > ± 1.3; nominal p value) between f/f and mnKO mice post‐sciatic nerve injury from gene set enrichment analysis (GSEA). (B) The bubble plots present the nominal P values (purple) and false discovery rates (FDR, q values) of the top correlated gene set. The depth of the shading in each bubble indicates the magnitude of P or q values. (C) The GSEA revealed enrichment gene set which were significantly differentiated in f/f mice versus mnKO mice. (D) Heatmap shows the expression of representative genes in those gene sets presented in panel (A). (E) Correlogram matrices display Spearman's rho of two genes facing each side of the square (matrix). The shading intensity of the correlation matrices displays Spearman's rho displayed in the scale (left‐hand side of the correlogram). (F) Gene network shows co‐expression of genes involved in top correlated gene sets. The Spearman's rho between two nodes (genes) generates the colour and depth of each edge. The colour (green, purple and orange) of each node indicates cell death, inflammation and neurogenesis‐related gene sets, respectively. The size of a node indicates the number of connected (correlated) nodes (|Spearman's rho| > 0.5).

Cdon depletion perturbs neuronal survival with impaired neurotrophin signalling

In line with the transcriptome analysis, Cdon depletion in NSC34 cells augmented cellular stress response evident by the increased expression of pro‐apoptotic gene, decreased expression of anti‐apoptotic genes, and higher level of cleaved caspase 3 and γ‐H2AX (Figure 6A,B). In addition, Cdon ablation in SOD1^G93A mice increased cellular stress response in motor neurons compared with f/f and SOD1^G93A mice (Figure  S4 ). Overexpression of Cdon in NSC34 cells prevented the increase of cleaved Caspase 3 and γ‐H2AX levels induced by tumour necrosis factor alpha, further supporting the role of Cdon in the protection of motor neurons against cellular stress (Figure 6C).

Figure 6.

Cdon ablation in motor neurons alters neurotrophin signalling. (A) mRNA expression levels of anti‐apoptotic markers (Bcl2, bid) and a pro‐apoptotic marker (Bax) in NSC34 cells transfected with control vector or Cdon shRNA. (B) Immunoblot analysis showing C‐Caspase3 and γ‐H2AX levels in NSC34 cells transfected with control vector or Cdon shRNA. β‐Tubulin is shown as a loading control. (C) Immunoblot analysis showing C‐Caspase3 and γ‐H2AX levels in NSC34 cells transfected with either control or Cdon expression vector following tumour necrosis factor alpha treatment. β‐Tubulin is shown as a loading control. (D) RNA analysis of lumbar spinal cords of f/f and mnKO littermates post‐sciatic nerve injury. A heat map of neurotrophin signalling related genes (left) and GSEA showing enrichment of genes related to MAPK signalling pathway (middle) reveal alterations in neurotrophin signalling in mnKO mice, which is confirmed by qRT‐PCR analysis of ErbB4 and Fgfr1 expression (right). (E) qRT‐PCR analysis of ErbB4 and Fgfr1 expression in NSC34 cells transfected with control vector or Cdon shRNA. (F) Activation of AKT in NSC34 cells in response to neuregulin‐1 (NRG1; 10 ng/mL). The cells were transfected with either control vector or Cdon shRNA and changes phosphorylated AKT levels over time were analysed via immunoblot. HSP90 is shown as a loading control. The data are expressed as mean ± SEM and analysed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Despite Cdon being a coreceptor of Shh signalling, the induction of Shh and its coreceptors' expression following nerve injury was not impaired in the spinal cord and motor neurons of mnKO mice, suggesting that other signalling pathways are involved in the neuronal death of mnKO mice (Figure  S5 ). Of note, Gas1 expression was significantly increased in mnKO spinal cords at 72 h post‐injury, which may be complementing for the loss of Cdon.

A closer examination of the transcriptome analysis revealed that several components of neurotrophin signalling were altered in the spinal cords of mnKO mice (Figure 6D). Notable downregulated genes were Erb‐B2 receptor tyrosine kinase 4 (ErbB4) and fibroblast growth factor receptor 1 (FgfR1), which play multiple roles in neurogenesis, neuronal regeneration and remyelination. ²² , ²³ Genes related to mitogen‐activated protein kinase signalling pathway, which is downstream of ErbB4 and Fgf signalling, were also downregulated in the mnKO mice compared with the WT mice (Figure 6D). qRT‐PCR analysis confirmed a significant decrease in ErbB4 and FgfR1 expression in the lumbar spinal cords of mnKO mice (Figure 6D). To verify the role of Cdon in neurotrophin signalling, NSC34 cells were transfected with control or Cdon shRNA expression vectors. Cdon‐depleted NSC34 cells also exhibited greatly decreased expression of ErbB4 and FgfR1 compared with the control NSC34 cells (Figure 6E). To examine the changes in responsiveness to neuregulin‐1 (NRG1), a ligand for ErbB4, control and Cdon‐depleted NSC34 cells were treated with NRG1 for 0, 5, 10 and 30 min in serum starved condition and the lysates were subjected to immunoblotting. Following 10 min of NRG1 treatment, the active, phosphorylated form of AKT was elevated in the control cells while Cdon depletion attenuated this increase in response to NRG1 treatment (Figure 6F). These data suggest that Cdon depletion causes alteration in neurotrophin signalling, leading to motor neuron degeneration and neuromuscular dysfunction.

Discussion

Sciatic nerve injury elicits multiple cellular events, such as the removal of debris, axon sprouting and guidance, and formation of synapses with muscle to ensure rapid synapse reinnervation and muscle recovery. ²⁴ During the repair process, the axon pathfinding and synapse formation require distinct cell to cell or cell to matrix interaction mediated through cell adhesion molecules (CAMs), Ig superfamily of cell adhesion molecules (IgCAMs), Cadherins, and Integrins. ²⁵ As a result, these CAMs play important roles in a variety of nerve regeneration processes. ²⁶ IgCAMs, such as N‐CAM, Nr‐CAM and L1, have been implicated in the control of NMJ function through synaptic structure maintenance, vesicle release and recycling, and neurotrophin signalling regulation. ²⁷ , ²⁸ Cdon has been shown to function in a complex with N‐cadherin or other IgCAMs to mediate cell adhesion‐mediated signalling in promotion of myoblast differentiation. ¹⁵ , ²⁹ Thus, it can be speculated that Cdon and other IgCAMs and Cadherins cooperate to modulate the specific cell‐to cell interaction required for nerve regrowth, synapse formation, and remyelination.

Mice lacking Cdon in motor neurons (mnKO) showed multiple defects in NMJ morphology, remyelination, and neurotrophin signalling that led to middle age onset lethality and premature muscle weakness associated with neuromuscular dysfunction and motor neuron degeneration. In addition, Cdon ablation in motor neurons exacerbated disease phenotypes of ALS mice model and caused impaired peripheral nerve repair after crush injury. The transcriptome data obtained from lumbar spinal cords of mnKO mice post‐nerve injury indicated alterations in genes related to cell death, inflammation, and neurogenesis pathways. In line with the transcriptome analysis, Cdon depletion in NSC34 cells triggered elevated c‐Caspase 3 and γ‐H2AX levels, supporting for a neuroprotective role of Cdon. These data imply that Cdon is a critical factor for the maintenance of motor neuron function during aging and nerve repair post‐injury.

The importance of Shh signalling in the neuroprotection of peripheral nerve injury models has been well documented. Shh is re‐expressed in regenerating axons following a peripheral nerve injury and facilitates neuronal survival and regeneration. ¹³ , ³⁰ Consistent with the previous studies, the expression of Shh and its coreceptors, Cdon, Gas1 and Boc, was transiently increased in the lumbar spinal cords of wild‐type mice at 24 h post‐injury. Thus, we initially postulated that impaired motor neuron regeneration in mnKO post‐nerve injury is due to defects in Shh activation in the early stage of nerve injury. However, the induction of Shh expression was not greatly altered in mnKO spinal cords as well as in the sciatic nerves, suggesting for the dispensable role of Cdon in Shh signalling induction during early stage of nerve injury. Gas1 expression was greatly elevated in mnKO spinal cords at 72 h post nerve injury, suggesting for a potential compensatory mechanism to induce Shh signalling.

ErbB4 is a receptor tyrosine kinase expressed in various tissues including motor neurons, NMJs, and skeletal muscles. Upon activation by neuregulins (NRGs), ErbB4 forms a homodimer or a heterodimer with ErbB2 or ErbB3 to activate downstream signalling pathways such as phosphatidyinositide 3‐kinase‐Akt and Ras‐mitogen‐activated protein kinase pathways. ³¹ The NRG1/ErbB4 signalling axis has been found to have a critical role in motor neuron survival, NMJ formation, and axon myelination. ³² , ³³ Mice lacking ErbB4 exhibit embryonic lethality with defects in both motor neuron axon guidance and cardiac development, ³⁴ and heterozygous‐null mice display delayed motor development. ³⁵ NRG‐1 ablation in mice is also embryonic lethal with respiratory failure and loss of Schwann cells in the peripheral axons leading to degeneration of motor neurons. ³⁶ In line with the functional studies, mutation in ErbB4 is associated with familial amyotrophic lateral sclerosis. ³⁷ Also, a study on the sporadic ALS patients revealed a reduction in NRG1 level and ErbB4 expression in the motor neurons, further supporting the role of the NRG1/ErbB4 signalling in the pathophysiology of motor neuron diseases. ³⁸

In mnKO spinal cords, the expression of several neurotrophic signalling components, including ErbB4, was altered compared with that of f/f spinal cords. Similar to mnKO spinal cords, Cdon depletion in NSC34 cells caused decline in ErbB4 expression and NRG1‐mediated Akt activation. Considering the significance of NRG1/EbrB4 signalling in neurodegenerative diseases, decreased levels of ErbB4 in motor neurons caused by Cdon deficiency may be the underlying mechanism of motor neuron degeneration and impaired nerve repair in mnKO mice. While further studies are required to fully understand how Cdon regulates the expression of ErbB4, the current study proposes a novel link between Cdon and regulation of neurotrophin signalling in motor neurons.

In summary, Cdon appears to regulate NRG1/ErbB4/Akt activation involved in neuroprotection, axon regrowth, and remyelination in motor neurons. Therefore, Cdon represents a promising target to intervene neuromuscular diseases and related muscle wasting.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Supporting information

Figure S1 Muscle phenotype of aged mnKO mice. (A) Schematic illustration of mnKO mice derivation. (B) Total hindlimb muscle mass of f/f and mnKO mice. (C) Representative image of f/f and mnKO mice TA muscle sections stained with laminin (left) and measurement of cross‐sectional areas of myofibers (right). Scale bar: 100 μm. (D) Relative mRNA expression of muscle atrophy markers (Atrogin1, Murf1) in TA muscles of f/f and mnKO mice. The data are expressed as mean ± SEM and analysed by Student's t‐test. *p < 0.05.

Figure S2 Muscle phenotype of f/f, f/f::SOD1, and mnKO::SOD1 mice. (A) Muscle mass of gastrocnemius (GAS), soleus (SOL), tibialis anterior (TA), and extensor digitorum longus (EDL) was averaged between left and right limbs. (B) Representative image of 4‐month‐old f/f::SOD1 and mnKO::SOD1 mice TA muscle sections stained with laminin (left) and measurement of cross‐sectional areas of myofibers (right). Scale bar: 100 μm. (C) Relative mRNA expression of denervation markers (MyoG, AchRα1, AchRβ1, HDAC4, HDAC5) and muscle atrophy markers (Atrogin1, Murf1) in f/f, f/f::SOD1, and mnKO::SOD1 mice TA muscles. (D) Relative mRNA expression of a denervation marker (AchRγ) and a satellite cell marker (Pax7) in f/f::SOD1, and mnKO::SOD1 mice TA muscles. The data are expressed as mean ± SEM and analysed by two‐way ANOVA. *p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001.

Figure S3 Muscle phenotype 35 days post‐sciatic nerve injury. (A) Relative muscle mass of GAS, SOL, TA, and EDL averaged between left and right limbs and normalized by body weight. (B‐D) Relative mRNA expression level of muscle type (B), atrophy (C), and denervation markers (D) in f/f and mnKO TA muscle 35 days post‐sciatic nerve crush injury. The data are expressed as mean ± SEM and analysed by two‐way ANOVA. *p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001.

Figure S4 Cdon depletion further increases cellular stress in motor neurons of SOD1^G93A mice. (A) Immunostaining for ChAT and γ‐H2AX in motor neurons of 4‐month‐old f/f, SOD1, and mnKO; SOD1 mice. Scale bar: 10 μm. (B) Quantification of the number of γH2AX foci per motor neuron from panel A. The data are expressed as mean ± SD and analysed by one‐way ANOVA. ^** p < 0.01 and ^**** p < 0.0001.

Figure S5 No overt changes in Shh signalling in mnKO mice post‐sciatic nerve injury. (A) Relative mRNA expression of Shh and Shh coreceptors in lumbar spinal cord of wile‐type mice post‐sciatic nerve injury (B) Relative mRNA expression of Shh and Shh coreceptors in lumbar spinal cord of f/f and mnKO littermates post‐sciatic nerve injury. (C) Relative mRNA expression of Shh in sciatic nerve of f/f and mnKO littermates post‐sciatic nerve injury. The data are expressed as mean ± SEM and analysed by two‐way ANOVA. *p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001.

Table S1. List of primary antibodies

Table S2. List of qRT‐PCR primers

Kim S., An S., Lee J., Jeong Y., You C.‐L., Kim H., et al (2023) Cdon ablation in motor neurons causes age‐related motor neuron degeneration and impaired sciatic nerve repair, Journal of Cachexia, Sarcopenia and Muscle, 14, 2239–2252, 10.1002/jcsm.13308