A dog model for centronuclear myopathy (CNM) carrying the most common DNM2 mutation

Mutations in DNM2 cause autosomal dominant centronuclear myopathy (ADCNM), a rare disease characterized by skeletal muscle weakness and structural anomalies of the myofibres including nuclear centralization and mitochondrial mispositioning. Following the clinical report of a Border Collie male with exercise intolerance and histopathological hallmarks of CNM on the muscle biopsy, we identified the c.1393C>T (R465W) mutation in DNM2, corresponding to the most common ADCNM mutation in humans. In order to establish a large animal model for longitudinal and preclinical studies on the muscle disorder, we collected sperm samples from the Border Collie male and generated a dog cohort for subsequent clinical, genetic, and histological investigations. Four of the five offspring carried the DNM2 mutation and showed muscle atrophy and a mildly impaired gait. Morphological examinations of transverse muscle sections revealed CNM-typical fibres with centralized nuclei and remodelling of the mitochondrial network. Overall, the DNM2-CNM dog represents a faithful animal model for the human disorder, allows the investigation of ADCNM disease progression, and constitutes a valuable complementary tool to validate innovative therapies established in mice.


INTRODUCTION
Cellular membranes undergo constant shape remodelling through curvature, tubulation, constriction, and fission to enable fundamental biological processes such as cytokinesis, migration, endocytosis, phagocytosis, signalling, intracellular trafficking, recycling, or compartmentalization (McMahon and Gallop, 2005). These dynamic events rely on the concerted interplay of lipids, proteins, and the cytoskeleton, and one of the key factors of membrane remodelling is dynamin 2 (DNM2) (Ferguson and De Camilli, 2012). This ubiquitously expressed mechanochemical enzyme is able to reorganize the microtubule and actin networks, and self-assembles into helical structures at the neck of nascent vesicles to induce vesicle release under GTP hydrolysis (Antonny et al., 2016;Chappie et al., 2009;Gu et al., 2010;Warnock et al., 1997). interactions (Faelber et al., 2011;Ferguson and De Camilli, 2012). Mutations in the DNM2 gene are associated with three distinct autosomal dominant neuromuscular disorders -Charcot-Marie-Tooth neuropathy (CMT1B, MIM# 606482), spastic paraplegia (Sambuughin et al., 2015) and centronuclear myopathy (ADCNM, MIM# 160150), the predominant CNM form in adult patients (Bitoun et al., 2005;Zuchner et al., 2005). ADCNM is characterized by generalized muscle weakness, ptosis, and ophthalmoplegia, and muscle biopsies from affected individuals typically show fibre size heterogeneity, abnormal nuclear centralization, mitochondrial mispositioning, and radial arrangement of sarcoplasmic strands (Bitoun et al., 2005). The age of onset and disease severity ranges from severe neonatal hypotonia and respiratory distress to mild adult-onset muscle weakness, and correlates with the position of the mutation. To date, more than 100 families and about 20 different ADCNM-related DNM2 mutations have been reported, essentially clustering in hot spot regions in exons 8, 11, 14, 15, and 16 (Abath Neto et al., 2015;Bohm et al., 2012;Werlauff et al., 2015). The most common mutation c.1393C>T resides in exon 11, affects approximately 25% of the cases, leads to the amino acid substitution p.ArgR465Trp (R465W) in the MID domain, and most often results in a moderate clinical presentation involving childhood-onset and slowly progressive distal muscle weakness (Bohm et al., 2012). In accordance with the clinical and histological presentation of the patients, the Dnm2 R465W/+ knock-in mouse model manifests reduced force associated with abnormal muscle structure and function (Durieux et al., 2010). Here we describe a spontaneous canine model harbouring the DNM2 R465W mutation and the generation of a dog cohort with clinical and histopathological characteristics paralleling the human disorder.

Identification of the DNM2 c. 1393C>T (R465W) mutation in a Border Collie
A 2-year-old Border Collie male presented with a 1-year history of exercise-induced pelvic limb collapse, short-strided and stiff gait, and morphological analysis of a muscle biopsy uncovered histopathological anomalies suggestive of CNM (Eminaga et al., 2012). We therefore Sanger-sequenced the known canine CNM genes HACD1 (previously named PTPLA), MTM1, and BIN1. An insertion of the SINE retrotransposon in HACD1 exon 2 is associated with CNM in Labrador retrievers (Pele et al., 2005), and missense and splice site mutations in MTM1 and BIN1 were respectively shown to cause CNM in Labrador retrievers,

Generation of a dog cohort -clinical and histopathological characterization
To confirm the pathogenicity of the identified DNM2 mutation in dogs and to establish a large and relevant animal model for long-term studies on disease development and the evaluation of innovative therapeutic approaches, sperm was collected from the Border Collie male to inseminate a Beagle female. The resulting litter of two female and three male pups were genotyped and underwent thorough clinical follow-up over 12 months. Biopsies from the tibialis cranialis and biceps femoris muscles, both easily accessible and extensively studied in centronuclear myopathies, were taken at one year of age and used for protein extraction and histological investigations. confirming that the identified missense mutation does not impair mRNA or protein stability.
At 12 months of age, all DNM2 R465W/+ dogs showed general muscle atrophy particularly affecting the masticatory and paraspinal muscles (Fig. 1C). Transcutaneous ultrasound confirmed atrophy of the biceps femoris and sartorius cranialis muscles, and revealed enhanced echo intensities suggesting an alteration of the muscle texture ( Fig. 1D-E). The affected dogs had increasing difficulties with jumping and standing on the pelvic limbs ( Fig. S1), and gait analysis evidenced subtle anomalies and notably a reduced craniocaudal power pointing to a decreased forward propulsion (Fig. 1F). Complete blood counts and routine biochemistry profiles were within the reference ranges, and serum creatine kinase (CK) levels were normal (85±18 U.I/l compared with 87 U.I./l in the healthy littermate).

Disease Models & Mechanisms • DMM • Accepted manuscript
Histological and histochemical examinations of transverse sections of tibialis cranialis and biceps femoris muscles disclosed fibre size variability, endomysial enlargement, and fibres with centralized nuclei in the DNM2 R465W/+ dogs ( Fig. 2A). We also observed major cytoplasmic rearrangements such as central and subsarcolemmal accumulations on H&E, oxidative staining, and COX assay, indicating a remodelled mitochondrial network in 19% to 57% of the myofibers ( Fig. 2A-C). In addition, the fibre diameter was significantly reduced in the DNM2 R465W/+ dogs compared with the control littermate (Fig. 2D). Overall and based on the cumulative anomalies on the muscle sections, the histopathology index was increased by a factor of 3 to 5 in DNM2 R465W/+ dogs (Fig. 2B).

DISCUSSION
The present study describes the first canine model for ADCNM, and the affected dogs carrying the DNM2 c.1393C>T (R465W) mutation showed muscle weakness and ADCNMtypical morphological anomalies on muscle sections. Hence, we propose that DNM2 R465W/+ dogs can be alternatively named DNM2-CNM dogs. Spontaneous canine MTM1 and BIN1 models were previously reported, and all recapitulated the human disorders at the clinical and histopathological level with severe muscle atrophy, swallowing difficulties, and a rapidly progressive tetraparesis. These major functional deficits required daily veterinary support and most often necessitated compassionate euthanasia between three and six months of age, preventing longitudinal studies (Beggs et al., 2010;Bohm et al., 2013;Olby et al., 2020;Shelton et al., 2015). Of note, none of the affected Labrador Retrievers, Rottweilers, and Boykin Spaniels carried mutations found in patients.

R465W in humans, mice, and dogs
By contrast, the dogs described in the present study harbour the most common ADCNM mutation diagnosed in 25% of the patients, and the slowly progressive disease course enables a large panel of molecular investigations at different time points to decipher the etiopathology and implicated pathways, and to identify relevant therapeutic targets for the prevention or reversal of the muscle phenotype. Moreover, dog models served to establish exon skipping, genome editing, and minigene expression strategies for Duchenne muscular dystrophy (DMD) (Amoasii et al., 2018;Koo et al., 2011;Vulin et al., 2012), and have also been used for preclinical proofs-of-concept of disorders affecting other tissues and organs.

The importance of dogs for preclinical trials
In conclusion, the DNM2-CNM dog is a faithful model for the human disorder, allows longitudinal investigations to decipher the sequence of events leading to the muscle dysfunction, and represents an optimal complementary system to assess the safety and efficacy of therapeutic approaches before the translation to humans.

DNA analysis
Genomic DNA from the Border Collie male was prepared from peripheral blood by routine procedures, and Sanger-sequenced for HACD1 exon 2 and all coding exons and the adjacent splice elements of MTM1, BIN1, and DNM2. The five offspring were Sanger-sequenced for DNM2 exon 11 for genotyping using forward (TGCTTGTCTCCCAGCTGCAG) and reverse (TGGTACCTTGACTGAGGTG) primers. The identified DNM2 mutation was numbered according to GenBank XM_005632882.3 and XP_005632939.1.

Ethics, animals, and establishment of a colony
The establishment of the dog colony and animal experimentation were in accordance with the European Community Standards and were performed with approval of the ethics committee of EnvA, the University of Paris Est, and the Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES,

Muscle ultrasonography
The biceps femoris and sartorius cranialis muscles were longitudinally imaged using a linear 12.5-5 MHz transducer. Imaging depth was set at 4 cm, and muscle thickness was assessed with the internal measurement tool of the ultrasound Philips HD7 scanner (Philips, Amsterdam, The Netherlands). Echo intensity was determined in Image J as the mean grey level on the histogram of the muscle after drawing a region of interest. Three images were acquired per muscle and per dog, and the mean values were used for analysis.

Accelerometry
Gait analysis was performed using 3D-accelerometry as previously described (Barthelemy et al., 2009). Briefly, a device containing three orthogonally positioned Locometrix® accelerometers (Centaure Metrix, Evry, France) was inserted into a belt tightened around the thorax of the dogs. All dogs were evaluated at a trot. Accelerometric curves were acquired at 50 Hz along the cranio-caudal, dorso-ventral and medio-lateral gait axes, and data were analysed using the Equimetrix® software (Centaure Metrix) to calculate following parameters: stride length, stride frequency, speed, regularity, generated power for each axis, and total power. Generated power of each axis was expressed as percentage of the total power.
The DNM2/calnexin ratio of the integrated grey level density was calculated for each sample and expressed as the percentage of the average ratio in control dogs. The individual ratio values were plotted using Prism 9 for MacOS, version 9.3.1.

Muscle morphology
Biceps femoris and tibialis cranialis muscle biopsies were taken at 12 months of age.
Transverse sections (10 µm) were stained with Hematoxylin & Eosin (H&E), Sirius Red, modified Gomori trichrome, NADH tetrazolium reductase (NADH-TR), and COX, and assessed for fibre morphology, accumulation/infiltrations, and oxidative activity. The myofiber diameter was determined using an Image J-developed macro script (Reyes-Fernandez et al., 2019) and is defined as the minimum Feret on sections immune-stained with a rabbit anti-Caveolin antibody (ab173575; Abcam, Cambridge, UK). Quantitative analyses were performed on H&E staining. Entire muscle sections were analysed using the Visilog 7.0 software (Noesis, Orsay, France). A grid was superimposed onto the image, and muscle morphology was assessed at each of the intercepts and manually annotated (1000 annotations per section) as previously described (Spencer et al., 2001). The histopathology index corresponds to the percentage of pathological features not corresponding to normal myofibers.