Basal Autophagy is altered in Lagotto Romagnolo Dogs with an ATG4D mutation

A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.Peer reviewe

Sensory Ataxic Neuropathy in Golden Retriever Dogs Is Caused by a Deletion in the Mitochondrial tRNATyr Gene

Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNATyr gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0–11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNATyr had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNATyr gene is the causative mutation for SAN

A Deletion in the N-Myc Downstream Regulated Gene 1 (NDRG1) Gene in Greyhounds with Polyneuropathy

The polyneuropathy of juvenile Greyhound show dogs shows clinical similarities to the genetically heterogeneous Charcot-Marie-Tooth (CMT) disease in humans. The pedigrees containing affected dogs suggest monogenic autosomal recessive inheritance and all affected dogs trace back to a single male. Here, we studied the neuropathology of this disease and identified a candidate causative mutation. Peripheral nerve biopsies from affected dogs were examined using semi-thin histology, nerve fibre teasing and electron microscopy. A severe chronic progressive mixed polyneuropathy was observed. Seven affected and 17 related control dogs were genotyped on the 50k canine SNP chip. This allowed us to localize the causative mutation to a 19.5 Mb interval on chromosome 13 by homozygosity mapping. The NDRG1 gene is located within this interval and NDRG1 mutations have been shown to cause hereditary motor and sensory neuropathy-Lom in humans (CMT4D). Therefore, we considered NDRG1 a positional and functional candidate gene and performed mutation analysis in affected and control Greyhounds. A 10 bp deletion in canine NDRG1 exon 15 (c.1080_1089delTCGCCTGGAC) was perfectly associated with the polyneuropathy phenotype of Greyhound show dogs. The deletion causes a frame shift (p.Arg361SerfsX60) which alters several amino acids before a stop codon is encountered. A reduced level of NDRG1 transcript could be detected by RT-PCR. Western blot analysis demonstrated an absence of NDRG1 protein in peripheral nerve biopsy of an affected Greyhound. We thus have identified a candidate causative mutation for polyneuropathy in Greyhounds and identified the first genetically characterized canine CMT model which offers an opportunity to gain further insights into the pathobiology and therapy of human NDRG1 associated CMT disease. Selection against this mutation can now be used to eliminate polyneuropathy from Greyhound show dogs

Mitochondrially inherited sensory ataxic neuropathy in golden retriever dogs

A novel neurological syndrome, mitochondrially inherited sensory ataxic neuropathy (SAN), was discovered in Golden Retriever dogs in Sweden. The purpose of the work described in the present thesis was to investigate the phenotype, clinical course and genotype of this syndrome by clinical, neurological and pathological examination of affected dogs, as well as to determine the mode of inheritance and identify the causative mutation. Mitochondrially inherited SAN in Golden Retriever dogs has an insidious onset during puppyhood. Affected dogs develop ataxia and dysmetria, with abnormal postural reactions and depressed spinal reflexes. The disease has a chronic, slowly progressive clinical course. Of the affected dogs investigated within the scope of this thesis, none became non-ambulatory or died spontaneously during the study period. However, about half of the affected dogs were euthanized because of neurological impairment before attaining 4 years of age. Post mortem examinations of affected dogs revealed degenerative changes in both the central and the peripheral nervous system. A chronic active central–peripheral axonopathy, neuroaxonal dystrophy-like alterations in spinal cord and brainstem, and a neuron-sparing encephalopathy with spongiosis in the basal nuclei were the most prominent findings. A maternal mode of inheritance was concluded from pedigree analysis, indicating a causative mutation in the mitochondrial DNA. Laboratory data confirmed that affected dogs had malfunctioning mitochondria. A single base-pair deletion in the mitochondrial tRNATyr gene was found and proven to be pathogenic. In summary, canine SAN is a slowly progressive neurodegenerative disease with onset in puppyhood. The disease is maternally inherited and is caused by a mutation in the mitochondrial tRNATyr gene

Re-emergence of hereditary polyneuropathy in Scandinavian Alaskan malamute dogs - old enemy or new entity? A case series

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Granulomatous interstitial polymyositis and intramuscular neuritis in a dog

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Thoracolumbar meningeal fibrosis in pugs

Background Thoracolumbar myelopathies associated with spinal cord and vertebral column lesions, with a similar clinical phenotype, but different underlying etiologies, occur in pugs. Objectives To further characterize the clinical and neuropathological characteristics of pugs with longstanding thoracolumbar myelopathy. Animals Thirty client‐owned pure‐bred pugs with a history of more than a month of ataxia and paresis of the pelvic limbs, suggesting a myelopathy localized to the thoracolumbar spinal cord, were included in the study. Methods Prospective clinicopathological study. Included pugs underwent a complete neurological examination and gross and histopathologic postmortem studies with focus on the spinal cord. Computed tomography (n = 18), magnetic resonance imaging (n = 17), and cerebrospinal fluid analysis (n = 27) were performed before or immediately after death. Results Twenty male and 10 female pugs had a median age at clinical onset of 84 months (interquartile range, 66‐96). Affected pugs presented with a progressive clinical course and 80% were incontinent. There was circumferential meningeal fibrosis with concomitant focal, malacic, destruction of the neuroparenchyma in the thoracolumbar spinal cord in 24/30 pugs. Vertebral lesions accompanied the focal spinal cord lesion, and there was lympho‐histiocytic inflammation associated or not to the parenchymal lesion in 43% of the pugs. Conclusions and Clinical Importance Meningeal fibrosis with associated focal spinal cord destruction and neighboring vertebral column lesions were common findings in pugs with long‐standing thoracolumbar myelopathy

Thoracolumbar meningeal fibrosis in pugs

Background Thoracolumbar myelopathies associated with spinal cord and vertebral column lesions, with a similar clinical phenotype, but different underlying etiologies, occur in pugs. Objectives To further characterize the clinical and neuropathological characteristics of pugs with longstanding thoracolumbar myelopathy. Animals Thirty client-owned pure-bred pugs with a history of more than a month of ataxia and paresis of the pelvic limbs, suggesting a myelopathy localized to the thoracolumbar spinal cord, were included in the study. Methods Prospective clinicopathological study. Included pugs underwent a complete neurological examination and gross and histopathologic postmortem studies with focus on the spinal cord. Computed tomography (n = 18), magnetic resonance imaging (n = 17), and cerebrospinal fluid analysis (n = 27) were performed before or immediately after death. Results Twenty male and 10 female pugs had a median age at clinical onset of 84 months (interquartile range, 66-96). Affected pugs presented with a progressive clinical course and 80% were incontinent. There was circumferential meningeal fibrosis with concomitant focal, malacic, destruction of the neuroparenchyma in the thoracolumbar spinal cord in 24/30 pugs. Vertebral lesions accompanied the focal spinal cord lesion, and there was lympho-histiocytic inflammation associated or not to the parenchymal lesion in 43% of the pugs. Conclusions and Clinical Importance Meningeal fibrosis with associated focal spinal cord destruction and neighboring vertebral column lesions were common findings in pugs with long-standing thoracolumbar myelopathy