Altered Basal Autophagy Affects Extracellular Vesicle Release in Cells of Lagotto Romagnolo Dogs With a Variant ATG4D.

Lagotto Romagnolo breed dogs develop a progressive neurological disease with intracellular vacuolar storage when homozygous for a variant in the autophagy-related gene 4D (ATG4D). A lysosomal enzyme deficiency has not been proven in this disease, despite its overlapping morphology with lysosomal storage diseases. Instead, basal autophagy was altered in fibroblasts from affected dogs. The aim of this study was to clarify the origin of the limiting membrane of the accumulating vacuoles and determine whether altered basal autophagy affects the extracellular release of vesicles in cells from diseased dogs. When assessed by immunoelectron microscopy, the membrane of the cytoplasmic vacuoles in affected tissues contained ATG4D, markers for autolysosomes (microtubule-associated protein 1A/B light chain 3 and lysosome-associated membrane protein 2) and for recycling endosomes (transferrin receptor 2), indicating that the vacuoles are hybrid organelles between endocytic and autophagic pathways. Ultracentrifugation, nanoparticle tracking analysis, and mass spectrometry were used to analyze the vesicles released from cultured fibroblasts of affected and control dogs. The amount of extracellular vesicles (EVs) released from affected fibroblasts was significantly increased during basal conditions in comparison to controls. This difference disappeared during starvation. The basal EV proteome of affected cells was enriched with cytosolic, endoplasmic reticulum, and mitochondrial proteins. Heat shock proteins and chaperones, some of which are known substrates of basal autophagy, were identified among the proteins unique to EVs of affected cells. An increased release of extracellular vesicles may serve as a compensatory mechanism in disposal of intracellular proteins during dysfunctional basal autophagy in this spontaneous disease.

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.

Lisosomal storage disease caused by ingestion of Astragalus spp in llamas: an emergent concern.

Lysosomal storage diseases are inherited or acquired disorders characterized by dysfunctional lysosomes that lead to intracytoplasmic accumulation of undegraded substrates, causing impaired cellular function and death. Many acquired lysosomal storage diseases are produced by toxic plants, which have indolizidine alkaloids, including swainsonine, that inhibits lysosomal α-mannosidase and Golgi α-mannosidase II. Swainsonine-induced nervous disease associated with various plants has been reported, including species of the genus Astragalus, Sida, Oxitropis, Swainsona, and Ipomoea. Two species of Astragalus (i.e. Astragalus garbancillo and Astragalus punae) have been found to cause neurologic disease in llamas. In addition, A. garbancillo was also associated with malformations in the offspring, and possibly abortions and neonatal mortality in llamas. The diagnosis of Astragalus spp. intoxication is established based on clinical signs, microscopic and ultrastructural findings, lectin histochemistry, abundance of these plants in the grazing area and determination of swainsonine in plant specimens.

A Homozygous MAN2B1 Missense Mutation in a Doberman Pinscher Dog with Neurodegeneration, Cytoplasmic Vacuoles, Autofluorescent Storage Granules, and an α-Mannosidase Deficiency.

A 7-month-old Doberman Pinscher dog presented with progressive neurological signs and brain atrophy suggestive of a hereditary neurodegenerative disorder. The dog was euthanized due to the progression of disease signs. Microscopic examination of tissues collected at the time of euthanasia revealed massive accumulations of vacuolar inclusions in cells throughout the central nervous system, suggestive of a lysosomal storage disorder. A whole genome sequence generated with DNA from the affected dog contained a likely causal, homozygous missense variant in MAN2B1 that predicted an Asp104Gly amino acid substitution that was unique among whole genome sequences from over 4000 dogs. A lack of detectable α-mannosidase enzyme activity confirmed a diagnosis of a-mannosidosis. In addition to the vacuolar inclusions characteristic of α-mannosidosis, the dog exhibited accumulations of autofluorescent intracellular inclusions in some of the same tissues. The autofluorescence was similar to that which occurs in a group of lysosomal storage disorders called neuronal ceroid lipofuscinoses (NCLs). As in many of the NCLs, some of the storage bodies immunostained strongly for mitochondrial ATP synthase subunit c protein. This protein is not a substrate for α-mannosidase, so its accumulation and the development of storage body autofluorescence were likely due to a generalized impairment of lysosomal function secondary to the accumulation of α-mannosidase substrates. Thus, it appears that storage body autofluorescence and subunit c accumulation are not unique to the NCLs. Consistent with generalized lysosomal impairment, the affected dog exhibited accumulations of intracellular inclusions with varied and complex ultrastructural features characteristic of autophagolysosomes. Impaired autophagic flux may be a general feature of this class of disorders that contributes to disease pathology and could be a target for therapeutic intervention. In addition to storage body accumulation, glial activation indicative of neuroinflammation was observed in the brain and spinal cord of the proband.

Lysosomal storage disease associated with a CNP sequence variant in Dalmatian dogs.

A progressive neurological disorder was identified in purebred Dalmatian dogs. The disease is characterized by anxiety, pacing and circling, hypersensitivity, cognitive decline, sleep disturbance, loss of coordination, loss of control over urination and defecation, and visual impairment. Neurological signs first became apparent when the dogs were approximately 18 months of age and progressed slowly. Two affected littermates were euthanized at approximately 7 years, 5 months and 8 years, 2 months of age due to the severity of neurological impairment. The mother of the affected dogs and four other relatives exhibited milder, later-onset neurological signs. Pronounced accumulations of autofluorescent intracellular inclusions were found in cerebral cortex, cerebellum, optic nerve, and cardiac muscle of the affected dogs. These inclusions co-localized with immunolabeling of the lysosomal marker protein LAMP2 and bound antibodies to mitochondrial ATPase subunit c, indicating that the dogs suffered from a lysosomal storage disease with similarities to the neuronal ceroid lipofuscinoses. Ultrastructural analysis indicated that the storage bodies were surrounded by a single-layer membrane, but the storage granules were distinct from those reported for other lysosomal storage diseases. Whole genome sequences, generated with DNA from the two euthanized Dalmatians, both contained a rare, homozygous single-base deletion and reading-frame shift in CNP which encodes the enzyme CNPase (EC 3.1.4.37). The late-onset disease was exhibited by five of seven related Dalmatians that were heterozygous for the deletion allele and over 8 years of age, whereas none of 16 age-matched reference-allele homozygotes developed neurologic signs. No CNPase antigen could be detected with immunohistochemical labeling in tissues from the dogs with the earlier-onset disorder. Similar to the later-onset Dalmatians, autofluorescent storage granules were apparent in brain and cardiac tissue from transgenic mice that were nullizygous for Cnp. Based on the clinical signs, the histopathological, immunohistochemical, ultrastructural, and molecular-genetic findings, and the finding that nullizygous Cnp mice accumulate autofluorescent storage granules, we propose that the earlier-onset Dalmatian disorder is a novel lysosomal storage disease that results from a loss-of-function mutation in CNP and that shares features characteristic of the neuronal ceroid lipofuscinoses. That the later-onset disorder occurred only in dogs heterozygous for the CNP deletion variant suggests that this disorder is a result of the variant allele's presence.

Lysosomal storage disease in two presumed-related springboks (Antidorcas marsupialis).

In April 2007, two newborn springboks (Antidorcas marsupialis) from a zoo of southern France were found dead. Necropsy was performed on the two animals and revealed arthrogryposis, mild facial structural abnormalities, and bilateral enlargement of the kidneys with concurrent hydronephrosis in both newborns. Histopathologically, extensive cytoplasmic vacuolation of neurons in the central nervous system, thyroid follicular epithelium, renal tubular epithelium, enterocytes, hepatocytes, and ruminal epithelial cells was the most remarkable lesion in both animals. By electron microscopy, the vacuoles were membrane bound and contained scattered membranous and granular material within a primarily electron-lucent background. Hence, a diagnosis of lysosomal storage disease was established, with gross, histological, and ultrastructural features suggestive of an inherited form of mannosidosis. This report documents the first case of lysosomal storage disease in springboks.

Impact of gene therapy for canine monogenic diseases on the progress of preclinical studies.

Rapid progress in knowledge of the organization of the dog genome has facilitated the identification of the mutations responsible for numerous monogenic diseases, which usually present a breed-specific distribution. The majority of these diseases have clinical and molecular counterparts in humans. The affected dogs have thus become valuable models for preclinical studies of gene therapy for problems such as eye diseases, immunodeficiency, lysosomal storage diseases, hemophilia, and muscular dystrophy. Successful gene therapies in dogs have significantly contributed to decisions to run clinical trials for several human diseases, such as Leber's congenital amaurosis 2-LCA2 (caused by a mutation of RPE65), X-linked retinitis pigmentosa-XLRP (caused by mutation RPGR), and achromatopsia (caused by mutation of CNGB3). Promising results were also obtained for canine as follows: hemophilia (A and B), mucopolysaccharidoses (MPS I, MPS IIIB, MPS VII), leukocyte adhesion deficiency (CLAD), and muscular dystrophy (a counterpart of human Duchenne dystrophy). Present knowledge on molecular background of canine monogenic diseases and their successful gene therapies prove that dogs have an important contribution to preclinical studies.

Neurodegenerative lysosomal storage disease in European Burmese cats with hexosaminidase beta-subunit deficiency.

GM2 gangliosidosis is a fatal, progressive neuronopathic lysosomal storage disease resulting from a deficiency of beta-N-acetylhexosaminidase (EC 3.2.1.52) activity. GM2 gangliosidosis occurs with varying degrees of severity in humans and in a variety of animals, including cats. In the current research, European Burmese cats presented with clinical neurological signs and histopathological features typical of a lysosomal storage disease. Thin layer chromatography revealed substantial storage of GM2 ganglioside in brain tissue of affected cats, and assays with a synthetic fluorogenic substrate confirmed the absence of hexosaminidase activity. When the hexosaminidase beta-subunit cDNA was sequenced from affected cats, a 91 base pair deletion constituting the entirety of exon 12 was documented. Subsequent sequencing of introns 11 and 12 revealed a 15 base pair deletion at the 3' end of intron 11 that included the preferred splice acceptor site, generating two minor transcripts from cryptic splice acceptor sites in affected Burmese cats. In the cerebral cortex of affected cats, hexosaminidase beta-subunit mRNA levels were approximately 1.5 times higher than normal (P<0.001), while beta-subunit protein levels were substantially reduced on Western blots.

Swainsonine-induced lysosomal storage disease in goats caused by the ingestion of Turbina cordata in Northeastern Brazil.

A disease of the central nervous system in goats was observed in the municipalities of Juazeiro, Casa Nova and Curaça, state of Bahia, and Petrolina, state of Pernambuco, Northeastern Brazil. The disease was produced experimentally in two goats by the administration of dry Turbina cordata mixed with grain. Clinical signs were observed after the ingestion of 62 and 106 g/kg body weight in 28 and 54 days, respectively. The concentration of swainsonine in the plant varied from less than 0.001% to 0.14% (dry weight). Clinical signs of natural and experimental cases included difficulties in standing, ataxia, hypermetria, wide-based stance, intention tremors, spastic paresis mainly in the hind legs, nystagmus, abnormal postural reactions, head tilting, and falling. Diffuse vacuolation of neurons, epithelial cells of pancreas, thyroids, and renal tubules were observed on the histology. From the electron microscopy of Purkinje cells the vacuoles represented dilated lysosomes. These findings demonstrated that T. cordata causes an acquired glycoprotein lysosomal storage disease. The intoxication occurs at least in an area of 27,000 km2 causing severe losses in goats, and some farmers report the disease also in cattle.

Presumptive Spontaneous Lysosomal Storage-Like Disease in a Crl:CD1(ICR) Mouse.

A clinically unremarkable 4.5-mo-old female Crl:CD1(ICR) VAF/Elite mouse was euthanized for scheduled sentinel processing. Gross necropsy findings included significant hepatosplenomegaly and visceral lymphadenomegaly, resulting in a preliminary gross diagnosis of lymphoma. Histology revealed florid accumulations of large, 'foamy' macrophages present in the bone marrow, small intestines, and viscera including liver, spleen, lymph nodes, thymus, uterus, and ovaries. The cytoplasm of these cells was abundant, stained pale blue with Wright-Giemsa and was periodic acid-Schiff positive. Given these characteristic gross and histologic findings, a spontaneous lysosomal storage-like disease was diagnosed in this mouse. Cholesterol ester storage disease is likely, because of the visceral involvement with sparing of the CNS, but could not be diagnosed definitively. To our knowledge, this report is the first to describe a case of spontaneous lysosomal storage disease in an outbred mouse of the CD1(ICR) background.

Lectin-histochemistry: glycogenosis in cattle.

Ten out of 47 calves that were born in a small Brahman herd from southern Brazil developed progressive muscular weakness and tremors, lethargy and poor body condition. Necropsy was performed on three affected animals. The only gross lesion detected was paleness of the muscles of the trunk and limbs. Multiple cytoplasm vacuoles located in different tissues were the principal microscopic lesions. Vacuoles were particularly evident in skeletal muscles and myocardium. PAS-positive granules were numerous in skeletal muscle fibres and Purkinje fibres of the myocardium, but were also observed in the neurons of the brain and spinal cord, and in the vascular smooth muscle fibres from all the examined tissues. Pretreatment with diastase completely abolished the PAS reactivity. The vacuoles reacted strongly to Griffonia simplicifolia II and Concanavalia ensiformes lectins, whose biding pattern has been reported as useful for demonstration of glycogen. Examination of the electron micrographs revealed that glycogen was free within the cytoplasm or accumulated in membrane-bound granules of several tissues, especially in striated muscle, liver and neurons of the CNS. These findings were consistent with generalized glycogenosis.

A variant form of neuronal ceroid lipofuscinosis in American bulldogs.

Neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases characterized by accumulations of autofluorescent lipopigments within cells of the nervous system. Nine related American Bulldogs demonstrated dysmetria in all limbs and paraparesis. Nonambulatory tetraparesis was observed only in the later stages of the disease. The clinical signs developed between 1 and 3 years of age and were slowly progressive over several years, which is inconsistent with most reports in other breeds. Results from blood tests for 8 different lysosomal storage diseases on 4 affected and 6 related but unaffected dogs were negative. Four affected dogs were euthanized and histopathologic examinations showed diffuse accumulations of periodic acid-Schiff-positive inclusions in neurons and axonal spheroids along the entire neuraxis and retinae. The most severe lesions were in the brainstem proprioceptive nuclei and spinal cord, consistent with clinical signs. The storage material was autofluorescent and immunohistochemically positive for products of lipid peroxidation. Ultrastructural analysis was consistent with NCL. Pedigree analysis supports an autosomal-recessive mode of inheritance. NCL has not been previously reported in the American Bulldog and these findings suggest a variant form of the canine disease.

Suspected natural lysosomal storage disease from ingestion of pink morning glory (Ipomoea carnea) in goats in northern Argentina.

This study describes an occurrence of pink morning glory (Ipomoea carnea) intoxication in goats in northern Argentina. The clinical signs displayed by the affected animals were ataxia, lethargy, emaciation, hypertonia of the neck muscles, spastic paresis in the hind legs, abnormal postural reactions and death. The clinico-pathologic examination revealed that the affected animals were anemic and their serum level of aspartate aminotransferase was significantly increased. Cytoplasmic vacuolation in the Purkinje cells and pancreatic acinar cells was observed by histological examination. The neuronal lectin binding pattern showed a strong positive reaction to WGA (Triticum vulgaris), sWGA (succinylated T. vulgaris) and LCA (Lens culinaris). Although I. carnea is common in tropical regions, this is the first report of spontaneous poisoning in goats in Argentina.

A lysosomal storage disease induced by Ipomoea carnea in goats in Mozambique.

A novel plant-induced lysosomal storage disease was observed in goats from a village in Mozambique. Affected animals were ataxic, with head tremors and nystagmus. Because of a lack of suitable feed, the animals consumed an exotic hedge plant growing in the village that was identified as Ipomoea carnea (shrubby morning glory, Convolvulaceae). The toxicosis was reproduced by feeding I. carnea plant material to goats. In acute cases, histologic changes in the brain and spinal cord comprised widespread cytoplasmic vacuolation of neurons and glial cells in association with axonal spheroid formation. Ultrastructurally, cytoplasmic storage vacuoles in neurons were membrane bound and consistent with lysosomes. Cytoplasmic vacuolation was also found in neurons in the submucosal and mesenteric plexuses in the small intestine, in renal tubular epithelial cells, and in macrophage-phagocytic cells in the spleen and lymph nodes in acute cases. Residual alterations in the brain in chronic cases revealed predominantly cerebellar lesions characterized by loss of Purkinje neurons and gliosis of the Purkinje cell layer. Analysis of I. carnea plant material by gas chromatography-mass spectrometry established the presence of the mannosidase inhibitor swainsonine and 2 glycosidase inhibitors, calystegine B2 and calystegine C1, consistent with a plant-induced alpha-mannosidosis in the goats. The described storage disorder is analogous to the lysosomal storage diseases induced by ingestion of locoweeds (Astragalus and Oxytropis) and poison peas (Swainsona).

Recognition and diagnosis of lysosomal storage diseases in the cat and dog.

Lysosomal storage diseases are rare, inherited disorders caused by the deficiency of 1 or more enzymes within the lysosomes of cells or by the deficiency of an activating protein or cofactor necessary for enzyme activity. The enzyme deficiency leads to a catabolic blockade and subsequent accumulation of storage material, and this in turn leads, albeit indirectly, to a wide array of clinical signs. Many features of storage diseases make them difficult to recognize and diagnose. In this review, we summarize the clinical features of these diseases and outline the steps required to confirm a diagnosis.

The effect of lysosomal storage diseases on secretory cells: an ultrastructural study of pancreas as an example.

The lysosome is the disposal and recycling center of eukaryotic cells. The pancreas is an exocrine and endocrine organ. It contains different cell types secreting enzymes, hormones, ions and fluid. The purpose of this study was to demonstrate that the manifestation of the disease in individual cells depends on the expression of the defected gene within the function of the cells. We have thus compared the ultrastructural features of normal pancreas to pancreas affected by eleven different lysosomal storage diseases in human, cats, dogs and mice. The diseases studied include GM1- and GM2- gangliosidosis, galactosialidosis, alpha-mannosidosis, mucopolysaccharidosis-I (MPS-I), mucopolysaccharidosis-VI (MPS-VI), mucopolysaccharidosis-VII (MPS-VII), mucolipidosis-IV (ML-IV), Niemann-Pick C, neuronal ceroid-lipofuscinosis, and an undefined glycolipid storage disease. We demonstrated that morphological changes are seen in some pancreatic cell types but not in others, even though the genetic defect occurs in all cell types. Furthermore, we identified and characterized primary and secondary changes in affected cells. The primary changes include presence of numerous and/or enlarged secondary lysosomes laden with partially degraded substrates. The secondary changes were associated with an abnormal formation and maturation of zymogen or secretory granules. The mechanisms contributing to the secondary changes are thought to be associated with altered synthesis of various constituents of these granules.

Lysosomal storage diseases in livestock.

Lysosomal storage diseases may be inherited or acquired. The former are usually inherited as autosomal traits, and heterozygotes may be identified by having enzyme activity levels approximately midway between normal and diseased values. Founder effect, enhanced by widespread acceptance of artificial breeding technologies, has been important in the spread of these genetic diseases in cattle. Acquired storage diseases of importance to livestock most frequently are plant intoxications.

Detection of swainsonine and calystegines in Convolvulaceae species from the semiarid region of Pernambuco / Detecção de swainsonina e calisteginas em espécies de Convolvulaceae da região semiárida de Pernambuco

Numerous plant species worldwide including some Ipomoea (Convolvulaceae) and Sida (Malvaceae) species in Brazil cause lysosomal storage disease in herbivores and are known to contain swainsonine and calystegines as the main toxic compounds. The aim of this work was to determine swainsonine and calystegines concentrations in species of Convolvulaceae from the semiarid region of Pernambuco. Seven municipalities in the Moxotó region were visited and nine species were collected and screened for the presence of swainsonine and calystegines using an HPLC-APCI-MS method. The presence and concentration of these alkaloids within the same and in different species were very variable. Seven species are newly reported here containing swainsonine and/or calystegines. Ipomoea subincana contained just swainsonine. Ipomoea megapotamica, I. rosea and Jacquemontia corymbulosa contained swainsonine and calystegines. Ipomoea sericosepala, I. brasiliana, I. nil, I. bahiensis and I. incarnata contained just calystegines. The discovery of six Ipomoea species and one Jacquemontia species containing toxic polyhydroxy alkaloids reinforces the importance of this group of poisonous plants to ruminants and horses in the semiarid region of Pernambuco. Epidemiological surveys should be conducted to investigate the occurrence of lysosomal storage disease associated to these new species.(AU)

Numerosas espécies de plantas em todo o mundo, incluindo algumas espécies de Ipomoea (Convolvulaceae) e Sida (Malvaceae) no Brasil, causam doença de armazenamento lisossomal em herbívoros e são conhecidas por conterem swainsonina e calisteginas como princípios tóxicos. O objetivo deste trabalho foi determinar a concentração de swainsonina e calisteginas em espécies de Convolvulaceae da região semiárida de Pernambuco. Sete municípios na região do Sertão do Moxotó foram visitados, onde foram coletadas amostras das folhas de nove espécies de Convolvulaceae para avaliação da presença de swainsonina e calisteginas utilizando-se cromatografia líquida com espectrometria de massa. A presença e concentração destes alcaloides nas folhas de plantas da mesma espécie e dentre as espécies foram muito variáveis. Seis novas espécies de Ipomoea e uma espécie de Jacquemontia contendo swainsonina e/ou calisteginas são relatadas neste estudo. Ipomoea subincana continha apenas swainsonina. Ipomoea megapotamica, I. rosea e Jacquemontia corymbulosa continham swainsonina e calisteginas. Ipomoea sericosepala, I. brasiliana, I. nil, I. bahiensis e I. incarnata continham apenas calisteginas. A descoberta de novas espécies de Ipomoea e Jacquemontia contendo alcaloides polihidroxílicos tóxicos reforçam a importância deste grupo de plantas tóxicas para ruminantes e equinos na região semiárida de Pernambuco. Pesquisas epidemiológicas devem ser realizadas para investigar a ocorrência de doença de depósito lisossomal associada a essas novas espécies.(AU)

Magnetic resonance findings of the corpus callosum in canine and feline lysosomal storage diseases.

Several reports have described magnetic resonance (MR) findings in canine and feline lysosomal storage diseases such as gangliosidoses and neuronal ceroid lipofuscinosis. Although most of those studies described the signal intensities of white matter in the cerebrum, findings of the corpus callosum were not described in detail. A retrospective study was conducted on MR findings of the corpus callosum as well as the rostral commissure and the fornix in 18 cases of canine and feline lysosomal storage diseases. This included 6 Shiba Inu dogs and 2 domestic shorthair cats with GM1 gangliosidosis; 2 domestic shorthair cats, 2 familial toy poodles, and a golden retriever with GM2 gangliosidosis; and 2 border collies and 3 chihuahuas with neuronal ceroid lipofuscinoses, to determine whether changes of the corpus callosum is an imaging indicator of those diseases. The corpus callosum and the rostral commissure were difficult to recognize in all cases of juvenile-onset gangliosidoses (GM1 gangliosidosis in Shiba Inu dogs and domestic shorthair cats and GM2 gangliosidosis in domestic shorthair cats) and GM2 gangliosidosis in toy poodles with late juvenile-onset. In contrast, the corpus callosum and the rostral commissure were confirmed in cases of GM2 gangliosidosis in a golden retriever and canine neuronal ceroid lipofuscinoses with late juvenile- to early adult-onset, but were extremely thin. Abnormal findings of the corpus callosum on midline sagittal images may be a useful imaging indicator for suspecting lysosomal storage diseases, especially hypoplasia (underdevelopment) of the corpus callosum in juvenile-onset gangliosidoses.

Vacuolar myopathy in an adult Warmblood horse.

Histopathological interpretation of semimembranosus muscle samples from an adult Warmblood mare with clinical signs suggestive of exertional rhabdomyolysis and intermittent mild elevations in muscle enzyme activities revealed abundant sarcoplasmic vacuoles in all fibre-types containing fine, apparently proteinaceous debris. Vacuolar contents stained lightly with PAS, but did not appear to contain amylopectate, lipid or acid phosphatase and their periphery was unstained with dystrophin immunohistochemistry. Electron microscopy revealed that vacuoles were not membrane bound. No vacuoles were detected in muscle samples evaluated at post mortem following 4 months of rest. To our knowledge, this is the first report of a presumed primary vacuolar myopathy in a horse.