Over the last 50 years, there have been major advances in knowledge and technology regarding genetic diseases, and the subsequent ability to control them in a cost-effective manner. This review traces these advances through research into genetic diseases of animals at Massey University (Palmerston North, NZ), and briefly discusses the disorders investigated during that time, with additional detail for disorders of major importance such as bovine α-mannosidosis, ovine ceroid-lipofuscinosis, canine mucopolysaccharidosis IIIA and feline hyperchylomicronaemia. The overall research has made a significant contribution to veterinary medicine, has provided new biological knowledge and advanced our understanding of similar disorders in human patients, including testing various specific therapies prior to human clinical trials.

1 Department of Biochemistry, Children’s Hospital, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 2 Department of Biochemistry 2, Georg-August-University Göttingen, 37073 Göttingen, Germany 3 UMR CNRS/USTL 8576, Unité de Glycobiologie Structurale et Fonctionnelle, IFR 147, Bat C9, Université de Lille 1, 59655 Villeneuve d’Ascq, France 4 ZMNH, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 5 Department of Biochemistry I, Faculty of Chemistry, University of Bielefeld, 33501 Bielefeld, Germany 6 Department of Biosciences, P.O. Box 65, FI-00014 University of Helsinki, Helsinki, Finland 7 Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated 'knock-in' mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as alpha-L-fucosidase, beta-hexosaminidase, alpha-mannosidase or Niemann-Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero) phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autopha-golysosomes and prevent neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease.

Vitamin E (α-tocopherol) is the major lipid-soluble antioxidant in many species. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by mutations in the NPC1 or NPC2 gene, which regulates lipid transport through the endocytic pathway. NPC disease is characterized by massive intracellular accumulation of unesterified cholesterol and other lipids in lysosomal vesicles. We examined the roles that NPC1/2 proteins play in the intracellular trafficking of tocopherol. Reduction of NPC1 or NPC2 expression or function in cultured cells caused a marked lysosomal accumulation of vitamin E in cultured cells. In vivo, tocopherol significantly accumulated in murine Npc1-null and Npc2-null livers, Npc2-null cerebella, and Npc1-null cerebral cortices. Plasma tocopherol levels were within the normal range in Npc1-null and Npc2-null mice, and in plasma samples from human NPC patients. The binding affinity of tocopherol to the purified sterol-binding domain of NPC1 and to purified NPC2 was significantly weaker than that of cholesterol (measurements kindly performed by R. Infante, University of Texas Southwestern Medical Center, Dallas, TX). Taken together, our observations indicate that functionality of NPC1/2 proteins is necessary for proper bioavailability of vitamin E and that the NPC pathology might involve tissue-specific perturbations of vitamin E status.

Dogs with mucopolysaccharidosis (MPS) IIIA were bred within an experimental colony. As part of characterizing them as a model for testing therapeutic strategies for the analogous disease of children, a pathologic study was undertaken. By histology, there were variably stained storage cytosomes within neurons, including many that stained for gangliosides. On ultrastructure examination, these cytosomes contained either moderately dense granular material, tentatively interpreted as precipitated glycosaminoglycan; a variety of multilaminar bodies, interpreted as being associated with secondary accumulation of gangliosides; or a mixture of both types. In the liver, storage vesicles also contained excess glycogen as a secondary storage product. In various tissues, there were large foamy macrophages. In the brain, many of these were in juxtaposition with neurons, and, on ultrastructure examination, they contained storage cytosomes similar to those in neurons. However, the neuron in association with such a macrophage frequently showed little such material.

Alterations in neuronal geometry in a feline model of sphingomyelin lipidosis were evaluated using Golgi staining. Neurons in cerebral cortex, basal ganglia, amygdala, thalamus, and cerebellum were impregnated and many were found to possess conspicuous enlargements at the axon hillock-initial segment region (meganeurites) and/or to sprout secondary neuritic processes from this same area. The latter were sometimes well developed and resembled small dendrites. These changes were cell type specific with distribution limited to certain types of neurons in select brain regions, while others remained normal, or underwent only simple somatic enlargement or generalized degenerative changes. Occasional cortical pyramidal neurons also displayed thinning of dendrites and extensive loss of dendritic spines.

Feline Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disease which shares many of the clinical, biochemical and pathological features of the corresponding human disorder. Cytopathological alterations in distinct neuronal cell populations were investigated in this animal model to gain a better understanding of the pathogenesis of brain dysfunction. Golgi and immunocytochemical methods were employed to characterize the cell architectural changes occurring in neuronal somata, dendrites and axons at different stages of disease progression. Cortical pyramidal neurons in laminae II, III, and V exhibited various degrees of meganeurite and/or swollen axon hillock formation with or without ectopic dendritogenesis. Enlarged axon hillock regions with neuritic processes and spines were recognized early in the progression of feline NPC but were less prevalent in mid to late stages of the disease. Glutamic acid decarboxylase (GAD) immunocytochemistry demonstrated immunoreactive spheroids in numerous GABAergic axons in neocortex, subcortical areas, and cerebellum. Parvalbumin-immunoreactive axonal spheroid distribution in brain closely mirrored results from the GAD studies, whereas calbindin D-28k-immunoreactive spheroids were conspicuously absent in most cortical and subcortical areas examined. Purkinje cell axonal spheroid formation progressed in a distal to proximal direction, with eventual involvement of recurrent axon collaterals. Purkinje cell death and a concomitant decrease in the numbers of spheroids in the cerebellum were observed late in the disease course. Clinical neurological signs in feline NPC occur in parallel with neuronal structural alterations and suggest that GABAergic neuroaxonal dystrophy is a contributor to brain dysfunction in this disease.

alpha-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal alpha-mannosidase. Feline alpha-mannosidosis is a well-characterized animal model used for studying pathological and therapeutic aspects of lysosomal storage disorders. We here report the purification of feline liver lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consisted of three polypeptides, with molecular masses of 72, 41 and 12 kDa, joined by non-covalent forces. The cDNA sequence of feline lysosomal alpha-mannosidase was determined from reverse transcriptase PCR products obtained from skin fibroblast mRNA. The deduced amino acid sequence contained the N-terminal sequences of the 72 and 41 kDa peptides. This indicated that the enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the three polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1 and 83.2% identical with the human and bovine lysosomal alpha-mannosidases sequences respectively. A 4 bp deletion was identified in an alpha-mannosidosis-affected Persian cat by DNA sequencing of reverse transcriptase PCR products. The deletion resulted in a frame shift from codon 583 and premature termination at codon 645. No lysosomal alpha-mannosidase activity could be detected in the liver of this cat. A domestic long-haired cat expressing a milder alpha-mannosidosis phenotype than the Persian cat had a lysosomal alpha-mannosidase activity of 2% of normal. This domestic long-haired cat did not possess the 4 bp deletion, proving molecular heterogeneity for feline alpha-mannosidosis.

Journal of Inherited Metabolic DiseaseVolume 16, Issue 2 p. 284-287 NCL Symposium — Pathogenetic Mechanisms Biology of neuronal dysfunction in storage disorders S. U. Walkley, S. U. Walkley Department of Neuroscience, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY, 10461 USASearch for more papers by this authorP. A. March, P. A. March Department of Neuroscience, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY, 10461 USASearch for more papers by this author S. U. Walkley, S. U. Walkley Department of Neuroscience, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY, 10461 USASearch for more papers by this authorP. A. March, P. A. March Department of Neuroscience, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, NY, 10461 USASearch for more papers by this author First published: 01 March 1993 https://doi.org/10.1007/BF00710266Citations: 17AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume16, Issue2March 1993Pages 284-287 RelatedInformation

Three Domestic Long-haired cats from a litter of five afflicted with alpha-mannosidosis (alpha-mannosidosis) were studied clinically and pathologically. Many of these findings contrasted with those made previously in kittens with deficiency of alpha-mannosidase. In these cats, the clinical signs were generally milder, more slowly progressive, and did not include the prominent skeletal deformities, ocular abnormalities, or hepatomegaly that were reported in prior studies of Persian and Domestic Short-haired kittens. While the Domestic Long-haired cats were spared the central nervous system (CNS) myelin deficiency, which was severe in the Persian but mild in the Domestic Short-haired cats, the extensive loss of Purkinje cells in their cerebellar cortices was without precedent. Additionally, ultrastructural study of the neuronal cytosomes showed a diversity not recorded in the earlier cases. The observed phenotypic heterogeneity was sufficient enough to consider separating feline alpha-mannosidosis into severe, acute and milder, chronic forms in a manner analogous to the Type I and Type II distinctions made in infants and juveniles.

Morphological changes in neurons with inborn defects of the lysosomal hydrolase, α-l-iduronidase, and with concomitant storage of glycosaminoglycans, were evaluated by Golgi staining in two animal models and compared to a similar study of a child with the same disease. Cortical pyramidal neurons in feline mucopolysaccharidosis type I often displayed axon hillock enlargements (meganeurites) and/or ectopic, secondary neuritic processes sprouting from this same region of the cell. The latter structures were prominent and often appeared longer than similar neurites reported in other neuronal storage discases. Although most meganeurites were aspiny, a few were observed which possessed spine-like processes or neurites. Other than these morphological changes in cortical pyramidal neurons, few other cell types displayed abnormalities demonstrable by Golgi impregnation. In the canine model of this disorder, abnormal Golgi-impregnated cortical neurons resembled more closely those seen in human mucopolysaccharidosis. That is, they possessed meganeurites which typically were aspiny in appearance. Ectopic neurite growth was not observed on any Golgi-impregnated neurons in the cases of canine or human mucopolysaccharidosis used in this study. The latter finding given the advanced ages of these cases, is consistent with the view that ectopic neuritogenesis seen in neuronal storage diseases may be subject to a developmental window, albeit one open well beyond the period of early postnatal maturation.

Ectopic dendrite growth and new synapse formation are known to occur on select kinds of neurons in a wide variety of neuronal storage diseases. As these changes in connectivity occur just proximal to the axonal initial segment, it has been hypothesized that they underlie the generation of abnormal neuronal function in these diseases. We have studied certain aspects of this phenomenon through the use of a plant-derived indolizadine alkaloid, swainsonine, which specifically inhibits the lysosomal hydrolase, alpha-mannosidase. These studies fully document the close morphological similarity between swainsonine-induced and inherited feline alpha-mannosidosis. This includes the presence of clear and floccule-filled storage vacuoles, as seen with routine EM, and axon hillock neurite growth on select cell types, as seen with Golgi staining. The latter was found only on cortical pyramidal neurons and multipolar cells of amygdala, and these same cell types are known to be involved in ectopic neuritogenesis in other storage diseases. Combined Golgi-electron-microscopic studies demonstrated the presence of normal-appearing synapses on these aberrant neuritic processes and also unusual, membranous inclusions specifically within the neurite-bearing pyramidal cells. The latter may be indicative of unique metabolic changes in these neurons and is consistent with the hypothesis that storage of gangliosides or other glycolipids underlies the recapitulation of dendritic growth features in these diseases. Experimental manipulation of the disease process using the swainsonine model indicated that induction of cortical pyramidal neuron neurite growth could be influenced by both age of onset and intensity of intraneuronal storage. Although Golgi studies clearly demonstrated neuritic sprouting in animals with disease onset as late as at 1 year, cortical pyramidal cells of older, adult animals appeared to undergo significant storage without a similar induction of neurite growth. These studies support the view that induced neuritogenesis in neuronal storage disease is associated with changes in metabolism, specifically within the neurite-bearing cells, that this change possibly involves gangliosides, and that the neuritogenic response may be limited to pre-adult stages of brain maturation.

Systematic Golgi studies have been performed on major subcortical, diencephalic, brain stem and spinal cord regions from cats with the inherited neuronal storage disease, GM1 gangliosidosis. Resulting data have been compared with other Golgi studies of neuronal storage disorders in man and animals, including an earlier, more limited examination of this same disease model. These previous studies have shown that in human and feline gangliosidoses cortical pyramidal neurons undergo remarkable changes in soma-dendritic geometry. The latter include the formation of conspicuous cellular enlargements between somata and axonal initial segments (meganeurites) and the sprouting of secondary neuritic processes from this same region of the cell. Further, ultrastructural studies have revealed normal appearing synapses on the surface of this ectopically placed dendritic-like membrane.

An experimentally induced and reversible model of a neuronal storage disease, swainsonine-induced feline alpha-mannosidosis, has been used to study the modifiability of ectopic, axon hillock-associated neurites and their new synaptic contacts. Earlier studies have fully documented that a variety of neuronal storage disorders are characterized by such changes in neuronal geometry and connectivity. Swainsonine administration was ended after 6 months of continuous treatment which had resulted in characteristic signs of alpha-mannosidosis. Studies of this animal 6 months after reversal showed that even though neuronal vacuolation and other CNS changes essentially normalized, ectopic neurites and their synaptic connections were still present and appeared similar to those of another animal which had been treated with swainsonine for the entire 12-month period. These results suggest that once initiated during the disease process, ectopic axon hillock-associated dendrites become an integral part of the soma-dendritic domain of affected neurons and may not be reversible. These findings may have relevance for current attempts to devise therapies involving enzyme replacement for individuals with inherited neuronal storage disease.

Ferric ion-ferrocyanide staining and safranin-0-counterstaining of neocortical tissue from cats with GM1 gangliosidosis have established that pyramidal neuron meganeurites occur proximal to axonal initial segments and that they are distinct from axonal spheroids. The latter, which were found to be widely distributed throughout cerebral cortex, were located distal to axonal initial segments and could be differentiated from meganeurites at both light and electron microscopic levels. This report confirms an earlier electron microscopic study which suggested that meganeurites are of axon hillock origin, and illustrates the striking distinction between abnormalities in the soma-dendritic and axonal domains of neurons in a lysosomal storage disease.

Postsynaptic potentials evoked by ventrolateral thalamic stimulation were recorded intracellularly from neurons in the precruciate cortex of GM1 mutants with HRP- or LY-loaded microelectrodes. Ganglioside-laden pyramidal neurons exhibiting somal distention and/or meganeurite formation were found to respond to thalamic stimulation with short duration IPSPs. Evoked EPSPs were recorded from two morphologically characterized large basket intrinsic neurons which deployed extensive intracortical axonal arborizations. These findings point to the preservation of intracortical inhibitory networks in the feline model of GM1 gangliosidosis, and to the possibility of abnormal integration of somadendritic inputs in ganglioside-laden pyramidal neurons.

A storage disease in a 2-year-old Japanese Spaniel resembled a GM2 gangliosidosis previously identified in a now extinct line of German Shorthaired Pointers. Despite a later appearance of signs in the Japanese Spaniel, the distribution, staining, and ultrastructure of the stored material were similar in the two breeds. Golgi studies of cerebral cortical neurons revealed the formation of spiny and aspiny enlargements at the axon hillock region (meganeurites) and the growth of secondary neurites from this region. As in the German Shorthaired Pointer model, there was massive storage of GM2 ganglioside as well as a seemingly paradoxical increase in total β-hexosaminidase activity measured in vitro.