A lower motor neuron disease in takahe (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease.

AIMS: To investigate the pathogenesis of a disease in takahe (Porphyrio hochstetteri) with intracytoplasmic inclusion bodies in lower motor neurons. METHODS: Four birds aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the spinal cord from the bird with spinal dysfunction were examined by electron microscopy. RESULTS: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D, so were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method.A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. CONCLUSIONS: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Animal medical genetics: a historical perspective on more than 50 years of research into genetic disorders of animals at Massey University.

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.

Intracisternal enzyme replacement therapy in lysosomal storage diseases: dispersal pathways, regional enzyme concentrations and the effect of posttreatment posture.

AIMS: To investigate routes of dispersal of enzyme, its regional uptake and the effect of posture when replacement enzyme is administered directly into the cerebrospinal fluid (CSF). METHODS: Dispersal pathways of particles and solutes were investigated using intracisternal injections of india ink with visual assessment, and a contrast medium (Iohexol) with computer tomography (CT). Replacement enzyme was measured at 46 loci within the central nervous system (CNS) in four groups of dogs subjected to different post-injection postural changes. RESULTS: India ink and CT studies showed dispersal pathways for CSF to be mainly via cisterns and sulci. Replacement enzyme reached all areas of the CNS tested, although mean concentrations varied 49-fold over different areas of the brain. Posttreatment posture had only modest effects on enzyme uptake in limited anatomical sites. CONCLUSIONS: Dispersal of solutes after injection is rapid and initially enhanced by the injection process. Preferential pathways for CSF flow in the subarachnoid spaces of the brain involve cisterns and sulci. The splenial and suprasplenial sulci in particular appear important conduits for dispersal to more dorsal and rostral areas of the brain. Expansion and contraction of these sulci during brain pulsation is considered important to the forward flow of solutes in CSF through these compartments. Following intracisternal enzyme replacement therapy, enzyme reached all areas of the brain, but there was considerable disparity of enzyme uptake with some areas recording much higher levels than others. Posttreatment posture made only modest differences to enzyme uptake.

Intracisternal enzyme replacement therapy in lysosomal storage diseases: routes of absorption into brain.

AIMS: The research concerns enzyme replacement therapy in lysosomal storage diseases with central nervous system involvement. The principle aim was to understand the routes of entry of enzyme into the brain when delivered directly into the cerebrospinal fluid (CSF) via the cerebellomedullary cistern. METHODS: Pathways for absorption of replacement enzyme were investigated in dogs with mucopolysaccharidosis IIIA (MPSIIIA) following intracisternal injections of human recombinant N-sulphoglucosamine sulphohydrolase (rhSGSH, EC3.10.1.1) by light and confocal microscopy using chromogenic and fluorescent immune probes. RESULTS: Enzyme entered the brain superficially by penetration of the pia/glia limitans interface, but the main route was perivascular along large veins, arteries and arterioles extending onto capillaries. It further dispersed into surrounding neuropil to be taken up by neurones, macrophages, astrocytes and oligodendroglia. Enzyme also entered the lateral ventricles adjacent to the choroid plexus, probably also by the tela choroidea and medullary velum, with further spread throughout the ventricular system and spinal canal. There was secondary spread back across the ependyma into nervous tissue of brain and spinal cord. CONCLUSIONS: Enzyme mainly enters the brain by a perivascular route involving both arteries and veins with subsequent spread within the neuropil from where it is taken up by a proportion of neurones and other cells. Penetration of enzyme through the pia/glia limitans is minor and superficial.

Familial episodic ataxia in lambs is potentially associated with a mutation in the fibroblast growth factor 14 (FGF14) gene.

Familial episodic ataxia of lambs is a congenital transient autosomal dominant disorder of newborn lambs, with varying expressivity. Affected lambs show episodes of an asymmetric ataxic gait, base-wide extensor hypertonia of the thoracic limbs and flexor hypertonia of the pelvic limbs. The aim of the study was to determine the genetic variant causing familial episodic ataxia in lambs. Using whole genome sequencing of two half-sib affected lambs, their sire, and their two normal dams, a heterozygous C>T transition at OAR10:77593415 (Oar_v3.1) in exon 1 of the fibroblast growth factor 14 (FGF14) gene (c.46C>T) was identified. The c.46C>T transition resulted in a premature stop codon at position 16 of the 247 amino acid FGF14 protein (p.Q16*). PCR and Sanger sequencing was used to genotype an additional 20 clinically affected animals, demonstrating all lambs carried the c.46C>T variant but 1 clinically more severely affected inbred lamb was homozygous (TT). A further 11 unrelated normal ewes were positionally sequenced, none of which had the variant, while in 18 lambs of unknown status born over 2 years of breeding trials six lambs were found to have the c.46C>T variant, likely clinically unidentified heterozygotes due to the variable expressivity, while 12 did not. In conclusion, familial episodic ataxia of lambs is potentially associated with a c.46C>T variant in the FGF14 gene. Further research is required into the mechanism behind the apparent recovery of lambs.

Animal medical genetics: a perspective on the epidemiology and control of inherited disorders.

This perspective considers genetic disorders of domestic animal populations, in particular their epidemiology and control. Inherited disorders of animals share the same basic molecular biology as those of human beings, but they differ in their epidemiology due largely to the breed structure of the various species, human control of breeding and a greater influence of the founder effect, particularly due to extensive use of a limited number of sires, and inbreeding. Control of genetic disorders in animals is also more practical through extensive screening for disease, or heterozygous animals within defined breed populations, followed by exclusion of affected or carrier animals from breeding. This is assisted by the fact that, within a breed, many inherited monogenic disorders are associated with a single mutation. However some of the more important disorders may be inherited in a non-Mendelian manner, being influenced by multiple genes as well as environmental factors. These aspects are discussed and contrasted with similar aspects in human medical genetics.

Phospholipid fatty acids in brains of normal sheep and sheep with ceroid-lipofuscinosis.

The ceroid-lipofuscinoses are a group of inherited diseases of humans and animals characterised by brain atrophy and the storage of a fluorescent lipopigment. Brain grey matter phospholipid fatty acids of diseased sheep are compared with those of normal sheep. Phosphatidylethanolamine of diseased sheep contains more 18:1(n-9) and less 22:6(n-3) than normal and their phosphatidylcholine less 16:0. Other differences are minor. All differences are in the same direction as those reported for the infantile form of human ceroid-lipofuscinosis, but are smaller. Normal sheep grey matter phosphatidylinositol contains 8.5% 20:4(n-6) and 24.6% 22:6(n-3), in contrast to 28.5 and 4.6%, respectively, in humans. The other sheep phospholipids have similar fatty acid profiles to those from humans. Apart from low levels of 20:3(n-9) and 22:3(n-9) they contain no additional non-essential fatty acid derived species. No sign of essential fatty acid deficiency occurs in either diseased or normal sheep. It is concluded that sheep must conserve their restricted essential fatty acid supply for structural functions, and that an abnormality in fatty acid metabolism is not primarily involved in the pathogenesis of ceroid-lipofuscinosis. The results also call into question the primary role of peroxidation of polyunsaturated fatty acids in lipopigment formation in this disease.

Analysis of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis.

Ovine ceroid-lipofuscinosis is an inherited neurodegenerative disorder characterised by the accumulation of storage cytosomes in brain and visceral organs. Phosphorylated dolichol-containing compounds, largely in the form of dolichyl pyrophosphoryl oligosaccharides, have been shown to constitute 1-2% of the dry weight of storage cytosomes isolated from brain and pancreas, and 0.5 and 0.1% respectively of storage cytosomes isolated from liver and kidney. The carbohydrate portion of these glyconjugates in storage cytosomes isolated from brain, pancreas and liver consisted of a series of oligosaccharides of composition Man2-9GlcNAc2, with Man5-8GlcNAc2 predominating. The concentrations of dolichyl pyrophosphoryl oligosaccharides in storage cytosomes from ovine ceroid-lipofuscinosis are much higher than has been reported for endoplasmic reticulum, their normal functional location.

Ceroid-lipofuscinosis (Batten's disease). Sequential electrophysiologic and pathologic changes in the retina of the ovine model.

The sequential electrophysiologic and pathologic changes in the retina in ceroid-lipofuscinosis were recorded in a time-course study in the ovine model. Over a relatively short period in the course of the disease, a severe reduction in both rod and cone b-wave amplitudes developed with rod b-wave changes preceeding those of cones. These changes paralleled a similar loss of rod and cone photoreceptor cells. In affected retinas, outer segments appeared shorter than normal. By 84 weeks of age, the outer nuclear layer was reduced to the width of a single nucleus. In addition to these changes, electronmicroscopy showed the formation of abnormal dystrophic rod and cone outer segments in photoreceptor cells. Most cells in the retina showed the accumulation of a fluorescent lipopigment, this being most prominent in ganglion cells. Ultrastructural studies showed them to be made of electron dense granular material and a variety of membranous and tubular arrays.

Membrane interlocking domains in the lens.

"Ball and socket"-like membrane processes interlock fiber cells in the sheep lens cortex, but appear reduced deeper in the lens. Wheat germ agglutinin (WGA) binds preferentially to these ball and socket structures, and more weakly to other membrane regions. On protein blots, 125I WGA binds to glycoproteins with 140,000 and 32,000 apparent molecular weight, the smaller protein also binding 125I fibronectin. In two animal cataract models, the intense WGA labeling of globular bodies replaces the spotty WGA staining pattern associated with the ball and sockets in the normal lens.

Comparative biology of the neuronal ceroid-lipofuscinoses (NCL): an overview.

Multiple forms of ceroid-lipofuscinosis occur in human beings and animals. They are characterized by brain and retinal atrophy associated with selective necrosis of neurons. This neurodegenerative disease appears associated with the disease process rather than storage of fluorescent lipopigment per se, and there is now growing evidence that pathogenesis may involve mitochondria rather than a primary defect of lysosomal catabolism. Of the forms of ceroid-lipofuscinosis studied, most but not all reflect accumulation of subunit c of mitochondrial ATP synthase. If there is a common denominator between all forms other than the presence of fluorescent lipopigment, then it may be the accumulation of hydrophobic protein. Analogous diseases in animals can be expected to reflect the same spectrum of biochemical changes, and they warrant in-depth study to help understand the pathogenesis and heterogeneity of the group.

Inborn errors of lysosomal catabolism--principles of heterozygote detection.

Carriers of an inborn error of lysosomal catabolism can be recognized, as they have enzyme levels approximately half those of normal individuals. Of the various tissues readily available for assay, plasma and leukocytes and, in some situations, tears are preferred. Although mixed leukocytes have proved satisfactory in Tay-Sachs screening programs, purified preparations of granulocytes or lymphocytes will allow better discrimination in most situations. Enzymes are assayed relative to some other reference parameter which must be a constant or highly correlated with test enzyme activity. In the two mass screening programs in operation, beta-hexosaminidase A and alpha-mannosidase have both been assayed relative to total beta-hexosaminidase activity. Carrier detection is particularly important in X-linked diseases. The techniques used mostly involve hair roots or fibroblasts and depend on random inactivation of the X chromosome. In the mucolipidoses II and III, in which there are a number of deficient enzymes in cells, carriers may be identified on the basis of the ratio of beta-hexosaminidase I1 and I2 to total hexosaminidase.

Sheep and other animals with ceroid-lipofuscinoses: their relevance to Batten disease.

Distinct pathological and histopathological changes distinguish the ceroid-lipofuscinoses from other storage diseases of humans and animals. These various disease entities likely reflect a variety of mutations of the same gene, or mutations of different genes associated with metabolism of the same or similar substrates. The disease in sheep most closely resembles the juvenile human disease. In it 50% of the lipopigment consists of subunit c of mitochondrial ATP synthase while the remaining constituents are considered normal for a lysosomal derived cytosome. The same subunit c has been shown to be also stored in affected English Setter, Border Collie, and Tibetan Terrier dogs, the Devon cow, and in the late infantile and juvenile human forms of disease but not in the infantile form. Thus it gives a chemical unity to at least some members of the group and allows a major conceptual change in regard to further directions of research.

Immunocytochemical studies in the ceroid-lipofuscinoses (Batten disease) using antibodies to subunit c of mitochondrial ATP synthase.

Immunocytochemistry, using antibodies against subunit c of mitochondrial ATP synthase, has been carried out in the ovine, canine, late infantile, and adult forms of ceroid-lipofuscinosis. Intensity of staining varied depending on the particular disease, species, fixation regime, and the antibody used. Differential staining of storage cytosomes in neurons of affected sheep and those in the late infantile patient suggested exposure of different epitopes. This was supported by the variable staining using two different antibodies in ovine, late infantile, and adult onset (Kufs) diseases. Immunostaining of muscle in the late infantile, and muscle and ear cartilage in affected sheep can assist diagnosis but positive results may depend on the age of the patient, at least in the latter species. In these tissues there was immunostaining of structures not identified by histochemical or fluorescence microscopy in addition to storage cytosomes that could be identified by these means. Poor or no immunostaining occurred with canine tissues. At the ultrastructural level, storage cytosomes but not other organelles stained with the immunogold method.

Pathogenesis of brain dysfunction in Batten disease.

Animal models of Batten disease and other neuronal storage disorders offer important opportunities to study the pathogenesis of brain dysfunction in this family of diseases. Although all of these conditions exhibit progressive intraneuronal storage, we have found that other aspects of the cellular pathology of Batten disease differ markedly from those of storage disorders caused by lysosomal hydrolase deficiencies. Likewise, lysosomal of cerebral cortex and other select brain regions, a prominent characteristic of Batten disease, does not occur in most other storage disorders. Our studies indicate that Batten disease has findings in common with human neurodegenerative diseases and that neuron death may be caused by excitotoxicity occurring secondary to the combined effects of suboptimal mitochondrial function and GABAergic (inhibitory) cell loss.

Variant proteins in ovine ceroid-lipofuscinosis.

Two-dimensional polyacrylamide gel electrophoresis has been used to search for disease-related protein variation in South Hampshire sheep with ovine ceroid-lipofuscinosis. Several hundred proteins in homogenates and subcellular fractions from livers have been examined, using isoelectric focusing as the first dimension separation, and SDS PAGE in the second dimension. Under these circumstances it was not possible to detect subunit c of the Fo region of ATP synthase, as this protein did not enter the isoelectric focusing gels. However, our studies emphasize the selective nature of misprocessing of subunit c, as we have not been able to detect any other consistent variation between affected and control animals for over 200 mitochondrial fraction proteins. Comparison of the presence or absence, and abundance, of proteins from isolated storage bodies with their counterparts in subcellular fractions from normal liver indicated that storage bodies contained a small subset of mitochondrial proteins, in addition to subunit c, with possible minor contributions from lysosomal, microsomal, and soluble proteins. Analysis of extramitochondrial proteins showed greater than 10-20-fold accumulation of ferritin light chains in microsomes, and partial loss of a putatively lysosomal protein, in ovine ceroid-lipofuscinosis. In addition, senescence marker protein was more abundant in the cytosolic fraction of controls, compared with affected individuals. We are currently investigating the basis and significance of these differences.

Hematopoietic cell transplantation in fetal lambs with ceroid-lipofuscinosis.

Hematopoietic cells from the liver of normal 45-48-day-old fetal lambs (Hb type AA) were transplanted intraperitoneally into 58-60-day-old recipient fetuses (Hb type BB). The recipient fetuses resulted from mating homozygous ceroid-lipofuscinosis affected males with heterozygous, phenotypically normal, females. The sex of the donor fetus was also recorded. At age 2 1/2 months the recipient lambs with ceroid-lipofuscinosis were diagnosed by histopathology of brain biopsies. Monitoring of blood and bone marrow cells showed that an average of 9% of blood cells in ceroid-lipofuscinosis affected recipients were of donor origin. No differences were evident in the clinical course of disease, brain weight, or histopathology of organs between transplanted and non-transplanted lambs with ceroid-lipofuscinosis. Under the conditions of this experiment, transplantation of fetal hematopoietic cells was not beneficial.

Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease).

The ceroid-lipofuscinoses (Batten disease) are neurodegenerative inherited lysosomal storage diseases of children and animals. A common finding is the occurrence of fluorescent storage bodies (lipopigment) in cells. These have been isolated from tissues of affected sheep. Direct protein sequencing established that the major component is identical to the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mitochondrial ATP synthase and that this protein accounts for at least 50% of the storage body mass. No other mitochondrial components are stored. Direct sequencing of storage bodies isolated from tissues of children with juvenile and late infantile ceroid-lipofuscinosis established that they also contain large amounts of complete and normal subunit c. It is also stored in the disease in cattle and dogs but is not present in storage bodies from the human infantile form. Subunit c is normally found as part of the mitochondrial ATP synthase complex and accounts for 2-4% of the inner mitochondrial membrane protein. Mitochondria from affected sheep contain normal amounts of this protein. The P1 and P2 genes that code for it are normal as are mRNA levels. Oxidative phosphorylation is also normal. These findings suggest that ovine ceroid-lipofuscinosis is caused by a specific failure in the degradation of subunit c after its normal inclusion into mitochondria, and its consequent abnormal accumulation in lysosomes. This implies a unique pathway for subunit c degradation. It is probable that the human late infantile and juvenile diseases and the disease in cattle and dogs involve lesions in the same pathway.

Mitochondrial dysfunction in the neuronal ceroid-lipofuscinoses (Batten disease).

There are at least eight genetic entities known as the ceroid-lipofuscinoses in humans which share clinical and pathological features that have caused them to be grouped together under the eponym of Batten disease. They present pathologically as lysosomal storage diseases but are also characterised by severe neurodegeneration. Although the biochemical defects appear primarily centred on lysosomes and defects in proteolysis, the link between this and pathogenesis of neuronal death is poorly understood. The pathogenesis of neurodegeneration has been studied particularly in two animal models these being the English setter dog and the New Zealand Southhampshire sheep (OCL6). In these, and some of the human entities, there is evidence of mitochondrial dysfunction. This includes the accumulation of subunit c of ATP synthase as a component of storage material in at least six of eight genetic forms of the disease; structural abnormalities of mitochondria and selective loss of neurons in areas of the brain that are particularly metabolically active. Direct evidence of dysfunction comes from mitochondrial function tests in fibroblasts and, in animal models, isolated liver mitochondria. Supporting evidence of mitochondrial dysfunction was shown by disturbances in proportions of energy-rich phosphates in fibroblasts in some of these diseases. If these various defects were reflected in neurons, then it would support the hypothesis that neuron death was associated with energy-linked excitotoxicity.

ATP synthase activity in ovine ceroid lipofuscinosis [OCL6].

We measured ATP synthase activities in mitochondria isolated from livers of lambs with ceroid lipofuscinosis (OCL6) and compared them with those from similar isolations from obligate heterozygous and control lambs. Addition of excess Ca2+ to the incubation mixture resulted in an up-regulation of activity in mitochondria from control lambs but down-regulation in those from OCL6 affected lambs. The mean change in activity with Ca2+ for heterozygous animals was midway between those from control and affected groups being significantly different from control but not from affected. The change in ATP synthase activity to added Ca2+ was also measured in isolated mitochondria from affected and control lambs from 3 days to 25 months of age. As above, there was down-regulation to the addition of Ca2+ in affected lambs. There was a fall in percentage change to Ca2+ with age in both affected and control lambs. This was not significantly different in affected lambs indicating it was not associated with the stage of disease. The above in vitro results, if extrapolated to neurons in vivo, imply a potential dysfunction of mitochondria in OCL6 lambs that could lead to calcium mediated neurotoxicity and neuron death due to production of free radicals as implicit in the energy-linked excitotoxic hypothesis.