Allogenic umbilical cord-derived mesenchymal stromal cells improve motor function in prenatal surgical repair of myelomeningocele: An ovine model study.

OBJECTIVE: To investigate the effects of an adjuvant allogenic umbilical cord mesenchymal stromal cell (UC-MSC) patch applied during fetal surgery on motor and sphincter function in the ovine MMC model. DESIGN: MMC defects were surgically created at 75 days of gestation and repaired 14 days later. POPULATION: Ovine MMC model: fetal lambs. METHODS: We compared lambs that received a UC-MSC patch with a control group of lambs that received an acellular patch. MAIN OUTCOME MEASURES: Clinical neurological assessment was performed at 2 and 24 hours of life and included determination of the Sheep Locomotor Rating scale (SLR), which has been validated in the ovine MMC model. Electrophysical examinations, spine scans and histological analyses were also performed. RESULTS: Of the 13 operated lambs, nine were born alive: five had of these had received a UC-MSC patch and four an acellular patch. At 24 hours of life, lambs in the UC-MSC group had a significantly higher score (14 versus 5, P = 0.04). Amyotrophy was significantly more common in the control group (75% versus 0%, P = 0.02). All the lambs in the control group and none of those in the UC-MSC group were incontinent. No significant differences were observed between the UC-MSC and control groups in terms of the presence of spontaneous EMG activity, nerve conduction or spinal evoked potentials. In the microscopic examination, lambs in the UC-MSC group had less fibrosis between the spinal cord and the dermis (mean thickness, 453 versus 3921 µm, P = 0.03) and around the spinal cord (mean thickness, 47 versus 158 µm, P < 0.001). Examination of the spinal cord in the area of the MMC defect showed a higher large neuron density in the UC-MSC group (14.5 versus 5.6 neurons/mm2, P < 0.001). No tumours were observed. CONCLUSIONS: Fetal repair of MMC using UC-MSC patches improves motor and sphincter function as well as spinal preservation and reduction of fibrosis.

A PNPLA8 frameshift variant in Australian shepherd dogs with hereditary ataxia.

Hereditary ataxias are common among canine breeds with various molecular etiology. We identified a hereditary ataxia in young-adult Australian Shepherd dogs characterized by uncoordinated movements and spasticity, worsening progressively and leading to inability to walk. Pedigree analysis suggested an autosomal recessive transmission. By whole genome sequencing and variant filtering of an affected dog we identified a PNPLA8:c.1169_1170dupTT variant. This variant, located in PNPLA8 (Patatin Like Phospholipase Domain Containing 8), was predicted to induce a PNPLA8:p.(His391PhefsTer394) frameshift, leading to a premature stop codon in the protein. The truncated protein was predicted to lack the functional patatin catalytic domain of PNPLA8, a calcium-independent phospholipase. PNPLA8 is known to be essential for maintaining mitochondrial energy production through tailoring mitochondrial membrane lipid metabolism and composition. The Australian Shepherd ataxia shares molecular and clinical features with Weaver syndrome in cattle and the mitochondrial-related neurodegeneration associated with PNPLA8 loss-of-function variants in humans. By genotyping a cohort of 85 control Australian Shepherd dogs sampled in France, we found a 4.7% carrier frequency. The PNPLA8:c.[1169_1170dupTT] allele is easily detectable with a genetic test to avoid at-risk matings.

Magnetic resonance imaging of a giant frontal hemorrhagic mucocele with intracranial extension in a cat.

A 6-year-old domestic short-haired cat was presented with an acute onset of right cortical encephalopathy. Magnetic resonance imaging (MRI) performed 4 days after the onset of clinical signs revealed a lesion originating from the right frontal sinus with intracranial extension and compression of the right frontal lobe. The lesion was T1-weighted hypointense and T2-weighted and fluid-attenuated inversion recovery hyperintense. Signal voids within the lesion were observed on T2* images, consistent with hemorrhage. Peripheral ring enhancement was visible on postcontrast sequences. These features were consistent with a giant hemorrhagic mucocele. To the authors' knowledge, this is the first report of MRI characteristics of this lesion in a cat.

Altered myofilament structure and function in dogs with Duchenne muscular dystrophy cardiomyopathy.

AIM: Duchenne Muscular Dystrophy (DMD) is associated with progressive depressed left ventricular (LV) function. However, DMD effects on myofilament structure and function are poorly understood. Golden Retriever Muscular Dystrophy (GRMD) is a dog model of DMD recapitulating the human form of DMD. OBJECTIVE: The objective of this study is to evaluate myofilament structure and function alterations in GRMD model with spontaneous cardiac failure. METHODS AND RESULTS: We have employed synchrotron X-rays diffraction to evaluate myofilament lattice spacing at various sarcomere lengths (SL) on permeabilized LV myocardium. We found a negative correlation between SL and lattice spacing in both sub-epicardium (EPI) and sub-endocardium (ENDO) LV layers in control dog hearts. In the ENDO of GRMD hearts this correlation is steeper due to higher lattice spacing at short SL (1.9µm). Furthermore, cross-bridge cycling indexed by the kinetics of tension redevelopment (ktr) was faster in ENDO GRMD myofilaments at short SL. We measured post-translational modifications of key regulatory contractile proteins. S-glutathionylation of cardiac Myosin Binding Protein-C (cMyBP-C) was unchanged and PKA dependent phosphorylation of the cMyBP-C was significantly reduced in GRMD ENDO tissue and more modestly in EPI tissue. CONCLUSIONS: We found a gradient of contractility in control dogs' myocardium that spreads across the LV wall, negatively correlated with myofilament lattice spacing. Chronic stress induced by dystrophin deficiency leads to heart failure that is tightly associated with regional structural changes indexed by increased myofilament lattice spacing, reduced phosphorylation of regulatory proteins and altered myofilament contractile properties in GRMD dogs.

Progressive Structural Defects in Canine Centronuclear Myopathy Indicate a Role for HACD1 in Maintaining Skeletal Muscle Membrane Systems.

Mutations in HACD1/PTPLA cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids, and HACD1 has a role in early myogenesis, but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1 deficiency is histopathologically classified as a centronuclear myopathy (CNM). We investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We found progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Furthermore, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including T-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiologic link with these disorders, confirm that dogs deficient in HACD1 are relevant models, and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1 CNM. Consequently, alterations in membrane properties associated with HACD1 mutations should be investigated in humans with related phenotypes.

Prevalence of neurological disorders in French bulldog: a retrospective study of 343 cases (2002-2016).

BACKGROUND: French Bulldog (FB) has significantly gained in popularity over the last few years, and seems to be frequently affected by various neurological conditions. The purpose of this retrospective study was to report the prevalences of neurological diseases in a large population of FB, presented with neurological signs between 2002 and 2016, and for which a definitive diagnosis was established. A secondary objective was to identify epidemiological characteristics regarding specific diseases in this singular breed. RESULTS: During the study period, 533 FBs were presented for neurological signs, representing 18.7% of all admitted FBs (N = 2846). In total, 343 FBs with definitive diagnosis were included in this descriptive epidemiological study. Hansen type I intervertebral disk herniation (IVDH) was by far the most common neurological disorder (45.5% of all cases). The IVDH location was cervical in 39.8%, and thoracolumbar in 60.2% of cases. The median ages for cervical and thoracolumbar IVDH were 4.2 and 4 years, respectively. C3-C4 was the most commonly affected disk (57.8% of cervical IDVH) all locations combined. Spinal arachnoid diverticulum (SAD) was detected in 25 FBs, representing the second most common myelopathy (11.3%). A concurrent spinal abnormality was identified in 64.0% of SAD cases. Brain tumours represented 36.8% of encephalopathies, with glioma (confirmed or suspected) being the most common. Meningoencephalitis of unknown origin (MUO) represented 25.0% of brain disorders, females less than 5.5 years being more likely to be affected. Aside from central nervous system conditions, otitis interna associated with peripheral vestibular signs and bilateral congenital deafness (associated with white coat) were also common. CONCLUSIONS: The findings of this study suggest that FB seems to be prone to several neurological diseases. IVDH is clearly predominant in FB and cervical location seems more represented than in other breeds. FBs affected by IVDH tend to be younger than previously described, either for both cervical and thoracolumbar locations. Thoracic SAD was the second most common myelopathy, with a concurrent spinal anomaly identified in two thirds of the cases. MUO was more likely to affect young to middle-aged females. These findings could be of interest for owners, breeders, practicing veterinarians and insurance companies.

Gait characterization in golden retriever muscular dystrophy dogs using linear discriminant analysis.

BACKGROUND: Accelerometric analysis of gait abnormalities in golden retriever muscular dystrophy (GRMD) dogs is of limited sensitivity, and produces highly complex data. The use of discriminant analysis may enable simpler and more sensitive evaluation of treatment benefits in this important preclinical model. METHODS: Accelerometry was performed twice monthly between the ages of 2 and 12 months on 8 healthy and 20 GRMD dogs. Seven accelerometric parameters were analysed using linear discriminant analysis (LDA). Manipulation of the dependent and independent variables produced three distinct models. The ability of each model to detect gait alterations and their pattern change with age was tested using a leave-one-out cross-validation approach. RESULTS: Selecting genotype (healthy or GRMD) as the dependent variable resulted in a model (Model 1) allowing a good discrimination between the gait phenotype of GRMD and healthy dogs. However, this model was not sufficiently representative of the disease progression. In Model 2, age in months was added as a supplementary dependent variable (GRMD_2 to GRMD_12 and Healthy_2 to Healthy_9.5), resulting in a high overall misclassification rate (83.2%). To improve accuracy, a third model (Model 3) was created in which age was also included as an explanatory variable. This resulted in an overall misclassification rate lower than 12%. Model 3 was evaluated using blinded data pertaining to 81 healthy and GRMD dogs. In all but one case, the model correctly matched gait phenotype to the actual genotype. Finally, we used Model 3 to reanalyse data from a previous study regarding the effects of immunosuppressive treatments on muscular dystrophy in GRMD dogs. Our model identified significant effect of immunosuppressive treatments on gait quality, corroborating the original findings, with the added advantages of direct statistical analysis with greater sensitivity and more comprehensible data representation. CONCLUSIONS: Gait analysis using LDA allows for improved analysis of accelerometry data by applying a decision-making analysis approach to the evaluation of preclinical treatment benefits in GRMD dogs.

C8 cross transfer for the treatment of caudal brachial plexus avulsion in three dogs.

OBJECTIVE: To evaluate the cervical nerve 8 cross-transfer technique (C8CT) as a part of surgical treatment of caudal brachial plexus avulsion (BPA) in the dog. STUDY DESIGN: Case series. ANIMALS: Client-owned dogs suspected to have caudal BPA based on neurological examination and electrophysiological testing (n = 3). METHODS: The distal stump of the surgically transected contralateral C8 ventral branch (donor) was bridged to the proximal stump of the avulsed C8 ventral branch (recipient) and secured with 9-0 polypropylene suture under an operating microscope. A carpal panarthrodesis was performed on the injured limb after C8CT. RESULTS: Surgical exploration confirmed avulsion of nerve roots C7, C8, and T1 in all cases. There was no evidence of an iatrogenic effect on the donor forelimb. Gradual improvement in function of the affected forelimb occurred in all dogs, with eventual recovery of voluntary elbow extension. Reinnervation was evident in EMG recordings 6 months postoperatively in all three dogs. Stimulation of the donor C8 ventral branch led to motor evoked potentials in the avulsed side triceps brachialis and radial carpus extensor muscles. Variable functional outcome was observed in the 3 dogs during clinical evaluation 3-4 years after surgery. Digital abrasion wounds, distal interphalangeal infectious arthritis, and self-mutilation necessitated distal phalanx amputation of digits 3 and 4 in 2 dogs. CONCLUSION: C8CT provided partial reconnection of the donor C8 ventral branch to the avulsed brachial plexus in the 3 dogs of this series. Reinnervation resulted in active elbow extension and promoted functional recovery in the affected limb.

Serum profiling identifies novel muscle miRNA and cardiomyopathy-related miRNA biomarkers in Golden Retriever muscular dystrophy dogs and Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked neuromuscular disease that affects 1 boy in 3500 to 5000 boys. The golden retriever muscular dystrophy dog is the best clinically relevant DMD animal model. Here, we used a high-thoughput miRNA sequencing screening for identification of candidate serum miRNA biomarkers in golden retriever muscular dystrophy dogs. We confirmed the dysregulation of the previously described muscle miRNAs, miR-1, miR-133, miR-206, and miR-378, and identified a new candidate muscle miRNA, miR-95. We identified two other classes of dysregulated serum miRNAs in muscular dystrophy: miRNAs belonging to the largest known miRNA cluster that resides in the imprinting DLK1-DIO3 genomic region and miRNAs associated with cardiac disease, including miR-208a, miR-208b, and miR-499. No simple correlation was identified between serum levels of cardiac miRNAs and cardiac functional parameters in golden retriever muscular dystrophy dogs. Finally, we confirmed a dysregulation of miR-95, miR-208a, miR-208b, miR-499, and miR-539 in a small cohort of DMD patients. Given the interspecies conservation of miRNAs and preliminary data in DMD patients, these newly identified dysregulated miRNAs are strong candidate biomarkers for DMD patients.

Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy.

Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy-dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here, we show in mice, that 4-month pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling downregulates porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phosphorus Magnetic Resonance Spectroscopy ((31)P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparß, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.

Nematode dermatitis due to Angiostrongylus vasorum infection in a dog.

BACKGROUND: Angiostrongylus vasorum is a nematode that primarily infects Canidae. The adult parasites are found in the pulmonary arterial circulation and the right side of the heart. The most common clinical sign is respiratory dysfunction. Bleeding, neurological, ocular, cardiovascular and gastrointestinal disorders are also reported. Skin lesions are very unusual. HYPOTHESIS/OBJECTIVES: This report describes a nematode dermatitis due to A. vasorum infection. To the best of the authors' knowledge, this is the first case of a dog infected with this parasite that initially presented with skin lesions only. ANIMAL: A 3-year-old female Weimaraner dog presented with a crusted papular dermatitis on the bridge of the nose and on the pinnae, and an erythematous pododermatitis with erosions and perionyxis of one digit of 1 week's duration. Two weeks later the dog developed respiratory distress. METHODS AND RESULTS: Skin scrapings and fungal culture were negative for parasites and dermatophytes. Histopathological examination showed dermal granulomas and pyogranulomas with eosinophils centred around parasitic elements compatible with nematode larvae. Angiostrongylus vasorum DNA was demonstrated in skin biopsies. Chest radiographs were compatible with verminous pneumonia and a Baermann test revealed A. vasorum larvae. The dog was treated orally with fenbendazole, with rapid improvement and complete cure after 3 months. CONCLUSIONS AND CLINICAL IMPORTANCE: Angiostrongylus vasorum should be considered in dogs presented with skin lesions and respiratory signs. Skin biopsy, chest radiographs and Baermann test should be included in the diagnostic investigation.

Acute idiopathic polyneuritis with spontaneous remission in an Abyssinian cat.

An Abyssinian kitten was presented after a sudden onset of neurological disorders consistent with a polyneuropathy. Electrophysiological and histological investigations revealed an inflammatory polyneuropathy. No infectious agents were detected. Spontaneous recovery occurred rapidly without relapse (2 years follow-up). This is the first description of a histologically confirmed self-limiting feline polyneuritis.

Polyneurite idiopathique aiguë avec rémission spontanée chez un chat Abyssinien. Un chaton Abyssinien a été présenté suite à l'apparition soudaine de troubles neurologiques conformes à une polyneuropathie. Les enquêtes électrophysiologiques et histologiques ont révélé une polyneuropathie inflammatoire. Aucun agent infectieux n'a été détecté. Le rétablissement spontané s'est produit rapidement sans rechute (suivi de 2 ans). Il s'agit de la première description d'une polyneurite féline auto-limitée confirmée par histologie.(Traduit par Isabelle Vallières).

Use of video-electroencephalography as a first-line examination in veterinary neurology: development and standardization of electroencephalography in unsedated dogs and cats.

Objective: To assess the feasibility and validate the use of video-electroencephalography (EEG) in conscious dogs and cats and to propose guidelines of routine EEG in veterinary clinical practice. Design: Prospective clinical study. Data: One hundred and fifty EEG recordings were carried out to validate the clinical adding-value, reproducibility, and guidelines on 140 owned animals. One hundred and one EEGs were performed on dogs and 49 on cats. Procedures: We compared recordings performed with 8 EEG unwired stud Ag/AgCl electrodes held by elastic straps and 8 EEG wired cup Ag electrodes held by a tailor-made manufactured headset combined with a wired video-EEG device. Electrodes placement was determined according to previously published animal EEG protocols. Physiological sensors, such as electrocardiography, electromyography, and respiratory sensors were added. Stimulation protocols were tested. Quality and interpretability were evaluated. Results: Headsets and recording procedures appeared suitable for all skull shapes and sizes. Video-EEG recordings were successfully performed without tranquilization or anesthesia except for 9 animals. Median EEG recordings time was 40 min. Impedance remained below 20 kΩ in 99% of dog EEGs and 98% of cat EEGs. Isosynchrony was reported in 6% of the channels. Seventy-five percent of dog EEGs and 83% of cat EEGs were readable for more than 50% (to 100%) of their duration. Successful discrimination of vigilance states from rhythm analysis (wakefulness, drowsiness, and sleepiness) was possible in 99% of dog EEGs and 91% of cat EEGs. Photic driving responses during photic stimulations were observed in 11% of dog EEGs and 85% of cat EEGs. Electroencephalography recordings were directly informative in 32% of the examinations: in 25% EEG abnormalities were associated with clinical signs and 7% concerned EEG abnormalities without clinical symptoms during recording. Thirteen percent of dogs subjected to photic stimulation exhibited epileptic anomalies. Among 9 EEGs with other history-based stimulations, three displayed epileptic graphoelements. Conclusions: We have developed a standardized unanesthetized video-EEG procedure easily performed and reproducible in dogs and cats. Qualitative and quantitative technical and medical criteria were evaluated and were in accordance with human EEG recommendations. Moreover, we have demonstrated its relevance and accuracy for diagnostic purposes, providing further arguments for the use of EEG as a first-line neurological functional exploration test.

Ambulatory electrocardiographic longitudinal monitoring in a canine model for Duchenne muscular dystrophy identifies decreased very low frequency power as a hallmark of impaired heart rate variability.

Duchenne muscular dystrophy (DMD) patients exhibit a late left ventricular systolic dysfunction preceded by an occult phase, during which myocardial fibrosis progresses and some early functional impairments can be detected. These latter include electrocardiographic (ECG) and heart rate variability (HRV) abnormalities. This longitudinal study aimed at describing the sequence of ECG and HRV abnormalities, using Holter ECG in the GRMD (Golden retriever muscular dystrophy) dog model, known to develop a DMD-like disease, including cardiomyopathy. Most of the known ECG abnormalities described in DMD patients were also found in GRMD dogs, including increased heart rate, prolonged QT and shortened PR intervals, ventricular arrhythmias, and several of them could be detected months before the decrease of fractional shortening. The HRV was impaired like in DMD patients, one of the earliest evidenced abnormalities being a decrease in the very low frequency (VLF) component of the power spectrum. This decrease was correlated with the further reduction of fractional shortening. Such decreased VLF probably reflects impaired autonomic function and abnormal vasomotor tone. This study provides new insights into the knowledge of the GRMD dog model and DMD cardiomyopathy and emphasizes the interest to monitor the VLF power in DMD patients, still unexplored in this disease, whilst it is highly predictive of deleterious clinical events in many other pathological conditions.

Clinical findings and outcome in feline tetanus: a multicentric retrospective study of 27 cases and review of the literature.

Tetanus is a toxigenic illness caused by the action of Clostridium tetani neurotoxin (TeNT), which results in partial or generalized muscle stiffness in infected mammals and birds. The disease is rarely reported in cats due to their innate resistance to the toxin. This multicentric retrospective study aimed to describe a significant population of cats with a diagnosis of tetanus and report their signalment, clinical and neurological signs, diagnostic findings, treatment, and outcome. A retrospective search through medical records from 11 referral centers in Europe resulted in the identification of 27 cases of feline tetanus from July 2005 to April 2023. These cases were further compared with previously reported cases in the veterinary literature. Young cats were more commonly represented than older cats, with a median age of 4 years. Clinical signs were initially characterized by a lame and/or stiff limb, near the primary injury site, in 17/26 (65%) cats. Signs were focal or multifocal in 21/27 (78%) cats of this study and one typical sign was the inability to flex the most severely affected limbs. Electrodiagnostic studies revealed characteristic changes, such as continuous spontaneous motor unit discharges in both agonist and antagonist muscles. Such studies are particularly useful in focal and multifocal cases and should be performed to further support the diagnosis. The toxin was successfully identified in one case using the mouse bioassay. Treatment included antibiotherapy (metronidazole) in most cases, muscle relaxants, appropriate nursing cares and handling of potential complications. Overall, the outcome appeared to be positive, with only 1/27 (3.7%) cats being euthanized due to financial restrains. 23/25 (92%) cats returned to an independent ambulatory capacity on all limbs within a median delay of 25 days. Mild to moderate long-term sequelae were reported in eight (30%) cats. This multicentric study is the first to bring together such a large number of cats affected with tetanus. Presentation of the disease in cats differs from that observed in humans and dogs, with most cats being locally affected. Compared to previous reports of tetanus, this series of cats had a better outcome overall, especially for cats affected with generalized tetanus.

Human galectin 3 binding protein interacts with recombinant adeno-associated virus type 6.

Recombinant adeno-associated viruses (rAAVs) hold enormous potential for human gene therapy. Despite the well-established safety and efficacy of rAAVs for in vivo gene transfer, there is still little information concerning the fate of vectors in blood following systemic delivery. We screened for serum proteins interacting with different AAV serotypes in humans, macaques, dogs, and mice. We report that serotypes rAAV-1, -5, and -6 but not serotypes rAAV-2, -7, -8, -9, and -10 interact in human sera with galectin 3 binding protein (hu-G3BP), a soluble scavenger receptor. Among the three serotypes, rAAV-6 has the most important capacities for binding to G3BP. rAAV-6 also bound G3BP in dog sera but not in macaque and mouse sera. In mice, rAAV-6 interacted with another protein of the innate immune system, C-reactive protein (CRP). Furthermore, interaction of hu-G3BP with rAAV-6 led to the formation of aggregates and hampered transduction when the two were codelivered into the mouse. Based on these data, we propose that species-specific interactions of AAVs with blood proteins may differentially impact vector distribution and efficacy in different animal models.

Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.

A canine Arylsulfatase G (ARSG) mutation leading to a sulfatase deficiency is associated with neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) represent the most common group of inherited progressive encephalopathies in children. They are characterized by progressive loss of vision, mental and motor deterioration, epileptic seizures, and premature death. Rare adult forms of NCL with late onset are known as Kufs' disease. Loci underlying these adult forms remain unknown due to the small number of patients and genetic heterogeneity. Here we confirm that a late-onset form of NCL recessively segregates in US and French pedigrees of American Staffordshire Terrier (AST) dogs. Through combined association, linkage, and haplotype analyses, we mapped the disease locus to a single region of canine chromosome 9. We eventually identified a worldwide breed-specific variant in exon 2 of the Arylsulfatase G (ARSG) gene, which causes a p.R99H substitution in the vicinity of the catalytic domain of the enzyme. In transfected cells or leukocytes from affected dogs, the missense change leads to a 75% decrease in sulfatase activity, providing a functional confirmation that the variant might be the NCL-causing mutation. Our results uncover a protein involved in neuronal homeostasis, identify a family of candidate genes to be screened in patients with Kufs' disease, and suggest that a deficiency in sulfatase is part of the NCL pathogenesis.

Replenishing NAD+ content reduces aspects of striated muscle disease in a dog model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene and loss of the protein dystrophin, which ultimately leads to myofiber membrane fragility and necrosis, with eventual muscle atrophy and contractures. Affected boys typically die in their second or third decade due to either respiratory failure or cardiomyopathy. Among the developed therapeutic strategies for DMD, gene therapy approaches partially restore micro-dystrophin or quasi-dystrophin expression. However, despite extensive attempts to develop definitive therapies for DMD, the standard of care remains corticosteroid, which has only palliative benefits. Animal models have played a key role in studies of DMD pathogenesis and treatment development. The golden retriever muscular dystrophy (GRMD) dog displays a phenotype aligning with the progressive course of DMD. Therefore, canine studies may translate better to humans. Recent studies suggested that nicotinamide adenine dinucleotide (NAD+) cellular content could be a critical determinant for striated muscle function. We showed here that NAD+ content was decreased in the striated muscles of GRMD, leading to an alteration of one of NAD+ co-substrate enzymes, PARP-1. Moreover, we showed that boosting NAD+ content using nicotinamide (NAM), a natural NAD+ precursor, modestly reduces aspects of striated muscle disease. Collectively, our results provide mechanistic insights into DMD.