Motor axonopathies in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by deleterious mutations in the DMD gene which encodes the dystrophin protein. Skeletal muscle weakness and eventual muscle degradation due to loss of dystrophin are well-documented pathological hallmarks of DMD. In contrast, the neuropathology of this disease remains understudied despite the emerging evidence of neurological abnormalities induced by dystrophin loss. Using quantitative morphological analysis of nerve sections, we characterize axonopathies in the phrenic and hypoglossal (XII) nerves of mdx mice. We observe dysfunction in these nerves – which innervate the diaphragm and genioglossus respectively – that we propose contributes to respiratory failure, the most common cause of death in DMD. These observations highlight the importance in the further characterization of the neuropathology of DMD. Additionally, these observations underscore the necessity in correcting both the nervous system pathology in addition to skeletal muscle deficits to ameliorate this disease.


Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by deleterious mutations
in the DMD gene which encodes the dystrophin protein. Skeletal muscle weakness and eventual muscle degradation due to loss of dystrophin are well-documented pathological hallmarks of DMD. in contrast, the neuropathology of this disease remains understudied despite the emerging evidence of neurological abnormalities induced by dystrophin loss. Using quantitative morphological analysis of nerve sections, we characterize axonopathies in the phrenic and hypoglossal (Xii) nerves of mdx mice. We observe dysfunction in these nerves -which innervate the diaphragm and genioglossus respectively -that we propose contributes to respiratory failure, the most common cause of death in DMD. these observations highlight the importance in the further characterization of the neuropathology of DMD. Additionally, these observations underscore the necessity in correcting both the nervous system pathology in addition to skeletal muscle deficits to ameliorate this disease.
Duchenne muscular dystrophy (DMD) is a severe, progressive muscle wasting disorder that results from loss-of-function mutations in the DMD gene 1 . The encoded protein, dystrophin, is a large structural protein that connects the cytoskeleton of muscle fibers to the extracellular matrix via the sarcolemma, thereby acting as an important stabilizer of muscle during movement 2 . When dystrophin is deficient or absent, muscles are damaged during contraction, resulting in chronic inflammation and fibrosis 2,3 . Eventually muscle regeneration is inhibited, and healthy muscle cells are replaced by fibrotic and adipose tissue 3 .
In the most common mouse model of DMD -the mdx mouse -a point mutation in exon 23 of the dystrophin gene confers loss-of-function of the full-length dystrophin protein 4 . While the function of dystrophin and pathophysiology of DMD in skeletal muscle is well-described in this model, recent evidence suggests that dystrophin may also play an important role in the nervous system 5 . Dystrophin deficiency in mdx mice alters neuron proliferation, survival, and/or differentiation as well as regulation of genes associated with axon development and synaptic organization 6,7 . In addition, considerable morphological alterations have been observed in the neuromuscular junction (NMJ) of mdx mice, and these alterations hinder NMJ transmission and motor endplate function 8 .
Respiratory failure is the most common cause of death in patients with DMD 9 . Characterization studies of respiratory pathology in DMD have solely investigated muscular insufficiency and have overlooked potential neuropathologies [9][10][11] . In this study, we demonstrate that mdx mice exhibit a motor neuron axonopathy in the phrenic and hypoglossal (XII) nerves, which implies that an underlying neuropathology may also contribute to the characteristic respiratory failure of DMD. We show that mdx mice exhibit substantial demyelination and loss of large-caliber axons. Furthermore, we find that mitochondria accumulate in the axoplasms of these nerves and exhibit morphological abnormalities. Altogether, we provide some of the first direct evidence that nervous system dysfunction may represent an important, but often overlooked, source of respiratory insufficiency in DMD. nerve processing and images. Phrenic and hypoglossal (XII) nerves were harvested from 12-month-old wildtype (n = 2) and mdx mice (n = 3). The nerves were placed in 2.5% glutaraldehyde and 0.1% sodium cacodylate. They were then processed, embedded in hard plastic, sectioned to 1 μm and consequently stained with 1% toluidine blue and 1% sodium borate by the Duke University Electron Microscopy Core. Semi-thin sections were imaged on a Keyence BZ-X710 All-in-One Fluorescence Microscope. Light micrographs were analyzed using a public downloadable ImageJ plugin 12 to examine the g-ratio, fiber diameter, axon diameter, and myelin thickness. The g-ratio is a highly reliable indicator of optimal myelination. Specifically, the g-ratio is the measure of the ratio of the axon diameter to the diameter of the axon plus myelin 13 . 100 randomly selected axons from each nerve were manually outlined for each animal.
For electron microscopy, phrenic and hypoglossal (XII) nerves from wildtype (n = 2) and mdx (n = 2) mice were placed in 2.5% glutaraldehyde and 0.1% sodium cacodylate and then post-fixed in 1% osmium tetroxide. The nerves were placed in 1% uranyl acetate and then dehydrated with acetone. They were then processed in epoxy resin (EPON), cut into 60 nm ultrathin sections on a Reichert Ultracut E ultramicrotome, and stained with 2% uranyl acetate and SATO's Lead stain. The nerves were imaged on a Philips CM12 electron microscope. The area of the axoplasm and diameter of mitochondria was measured using ImageJ 1.48 v.
Motor neuron counting. 12-month-old mdx mice (n = 3) and age-matched wildtype mice (n = 3) were anesthetized and harvested for their spinal cords, which were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). The spinal cords were placed in 30% sucrose at 4 °C. The spinal cord was separated into sections: medulla, cervical spinal cord, thoracic and lumbar. The sections were embedded in OCT and frozen at −80 °C before cryosectioning. Medulla and cervical spinal cords were cut into 40 μm cross sections using a Leica CM3050 S Cryostat. The sections were preserved in a 96-well plate in 2% PFA. Every third cervical spinal cord cross-section, and every other section of the medulla were transferred to a master 96-well plate with 1x PBS for histochemical analysis.
The mounted tissues were then stained using Cresyl Violet. The Cresyl Violet solution was prepared using 0.1% Cresyl Violet Acetate in distilled H 2 O and stirred overnight. The slides were sequentially submerged in the following solutions: distilled H 2 O, Cresyl Violet, distilled H 2 O, 50% EtOH, 75% EtOH, 95% EtOH, 100% EtOH, Xylene, and then re-submerged in Xylene. Sections were then cover slipped and imaged using a Leica DMRA2 for brightfield microscopy. The images were analyzed and counted by two different individuals, and their results were compared with each other. Statistical analysis. Differences between wildtype and mdx mice were evaluated using either an unpaired, two-tailed Two Sample t-test or a Mann-Whitney U test. The Two Sample t-test was employed when the data were normally distributed, and the nonparametric Mann-Whitney U test was used when the data were not normally distributed. Normality for each group was tested using the Shapiro-Wilk test, with alpha set at 0.05. Significance was set at p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****). Values in figure legends are reported as the mean ± standard error of mean (SEM). Computations were completed using RStudio, an integrated development editor of R. Plots were also generated in RStudio, using the ggplot2 package.

Results and Discussion
Demyelination of large-caliber axons in mdx mice. Myelin ensheathment of axons is a vital process that allows for rapid impulse propagation along the nerve, promoting efficient nervous system function 14 . Myelination deficiency leads to axon degeneration and is a pathological hallmark of numerous neurological disorders 15 . Myelin sheath degradation -demyelination -is a well-documented phenotype in multiple sclerosis 16 , while myelin decompaction is a characteristic of ALS 17 . To investigate abnormal myelination, we quantified the g-ratio of axons with intact myelin sheaths in both nerves. Myelination is naturally optimized to achieve maximum efficiency in impulse propagation, and an abnormal g-ratio may suggest impaired nerve conduction 13 . For intact axons, mdx mice exhibit smaller g-ratio values in comparison to wildtype mice in both the phrenic and XII nerves, representing an axonopathy (Fig. 1a). Furthermore, axons that were not analyzed in mdx mice had severely collapsed and deformed myelin sheaths, exhibiting severe pathology (Fig. 1f,g). Together, these observations reflect a significant nerve pathology present in mdx mice.
Demyelination in mdx mice contributes to these observed g-ratio differences, as mdx mice exhibit significantly reduced myelin sheath thickness in both nerves (Fig. 1b). In addition, the size of the axons can impact the myelin thickness, as myelin thickness positively correlates with axon diameter. We find that larger-caliber axons (>5 μm diameter) 18 are selectively demyelinated in mdx mice, while smaller-caliber axons (<5 μm diameter) 18 are spared (Fig. 1c, Fig. S1). Because demyelination can lead to axon degeneration downstream 15 , we hypothesized that large-caliber axons are selectively degenerated in mdx mice. Furthermore, we considered that axon size may confound the g-ratio quantifications. To account for this potential confounder, we performed multivariate linear regression, regressing the g-ratio against both the axon diameter and myelin thickness (Fig. 1d,e). In both nerves, the models demonstrate that axon diameter is a better predictor of the g-ratio than myelin thickness. Therefore, we quantified the axon and fiber diameter (axon plus myelin diameter) and examined the distribution of axon caliber type. tively degraded, the axon and fiber diameters were noticeably reduced in mdx mice in both nerves (Fig. 2a,b). mdx mice clearly exhibited a skewed distribution of axonal diameter and area, favoring smaller axons and representing decreased heterogeneity of axon type (Fig. 2c-f). In contrast, wildtype mice show a more diverse composition of axon sizes, which is crucial for normal muscle function because large and small axons fulfill different roles. Small axons send high information signals to few muscle fibers, while large axons transmit low information signals to many muscle fibers 19 . Because large axons have a faster conduction time than small axons, they are vital for skeletal muscle function. Therefore, the selective loss of large-caliber axons in mdx phrenic and hypoglossal nerves likely hinders the proper function of the diaphragm and extrinsic tongue muscles, further exacerbating respiratory dysfunction in DMD. Figure 2g,h shows representative examples of the WT and mdx phrenic and XII nerves, respectively.
Altered mitochondrial morphology and accumulation in mdx mice. Mitochondrial dysfunction represents one of the earliest cellular deficits in mdx muscle 20 . Mitochondrial deficits impair muscle cells' ability to repair damaged membranes, contributing to persistent myofiber damage 20 . Therefore, we employed transmission electron microscopy (TEM) to investigate whether mitochondrial abnormalities are also present in the axoplasms of phrenic and XII nerves (Fig. 3a). The axoplasm -the cytoplasm of the axon -serves as an avenue for the www.nature.com/scientificreports www.nature.com/scientificreports/ transport of proteins, mRNA, lipids, and mitochondria in both the anterograde and retrograde directions 21 . We observe that the number of mitochondria in the axoplasmic space is increased in both nerves of mdx mice, and that there is an increase in mitochondrial diameter in only the XII nerve of mdx mice (Fig. 3b,c). Additionally, the density of mitochondria is increased in both nerves of mdx mice (Fig. 3d). The increase in mitochondrial diameter indicates swelling, which is a hallmark of dysfunction of these organelles 22 . Furthermore, the increased number and density of mitochondria suggests accumulation, which can represent either reduced axonal transport or impaired mitophagy 23 . Both of these features have been associated with other neuromuscular diseases, such as Charcot-Marie-Tooth type 2A 23 .
Proper myelination is required for maintenance of normal axon transport and long-term survival 24 , so the axonal demyelination we observe may have consequences on axonal transport leading to mitochondrial accumulation. Alternatively, axons may be transporting more mitochondria to the NMJ as a compensatory response to NMJ degradation in mdx mice 8 . Because axon transport deficits are linked with motor neuron death and dysfunction 21 , we quantified the number of phrenic and XII motor neurons (Fig. S2). There was no significant difference between the number of neurons between wildtype and mdx mice for both phrenic and XII neurons (p > 0.05), illustrating that motor neurons are not degraded in mdx mice. Therefore, the pathology at this time point seems to be constrained to the axons and does not affect the soma.
Taken together, our data suggest neuropathologies in the phrenic and XII nerves of mdx mice. However, because full-length dystrophin is not expressed in the spinal cord of healthy mice or humans 25 , we propose that these pathologies are secondary to the characteristic muscle-wasting. Specifically, we speculate that the nerve dysfunction may be a consequence of dystrophin deficiency at the NMJ. Dystrophin accumulates at the postsynaptic membrane in the form of the dystrophin-associated glycoprotein complex (DGC), and deficiency of the DGC has been shown to disrupt both the pre-and postsynaptic structures of the NMJ in the mdx model 26 . Therefore, we suspect that pathology starts at the NMJ and then acts in a retrograde manner later affecting the nerves, a process termed "dying-back" 27 .