R. D. Jolly

Lysosomal Storage Diseases of Animals: An Essay in Comparative Pathology

A wide variety of inherited lysosomal hydrolase deficiencies have been reported in animals and are characterized by accumulation of sphingolipids, glycolipids, oligosaccharides, or mucopolysaccharides within lysosomes. Inhibitors of a lysosomal hydrolase, e.g., swainsonine, may also induce storage disease. Another group of lysosomal storage diseases, the ceroid-lipofuscinoses, involve the accumulation of hydrophobic proteins, but their pathogenesis is unclear. Some of these diseases are of veterinary importance, and those caused by a hydrolase deficiency can be controlled by detection of heterozygotes through the gene dosage phenomenon or by molecular genetic techniques. Other of these diseases are important to biomedical research either as models of the analogous human disease and/or through their ability to help elucidate specific aspects of cell biology. Some of these models have been used to explore possible therapeutic strategies and to define their limitations and expectations.

Apoptosis as the Mechanism of Neurodegeneration in Batten's Disease

Abstract: Battens disease is a genetic neurodegenerative disease of childhood. Its hallmarks are retinitis pigmentosa and neuronal degeneration. As some types of photoreceptor death in mice are mediated by apoptosis, we investigated whether apoptosis is responsible for retinal and neuronal degeneration in the late infantile and juvenile forms of Battens disease. Using the terminal dUDP nick end‐labeling (TUNEL) staining method, we detected apoptotic neuronal cells in brain from patients and a canine model and in brain and retina from an ovine model for Battens disease. We confirmed apoptosis by flow cytometry, electron microscopy, and DNA laddering. This is the first inherited neurodegenerative disease involving brain and retina in which apoptosis has been established as the mechanism of neuronal and photoreceptor cell death in both humans and animal models.

OVINE CEROID-LIPOFUSCINOSIS: A MODEL OF BATTEN'S DISEASE

Jolly R.D., Janmaat A., West D.M. & Morrison I. 1980 Neuropathology and Applied Neurobiology 6,195–209

Separation of some neutral lipids by normal-phase high-performance liquid chromatography on a cyanopropyl column: Ubiquinone, dolichol, and cholesterol levels in sheep liver

The normal-phase high-performance liquid chromatographic separation of neutral lipids into molecular classes was carried out on a cyanopropyl (CN) column eluted with isopropanol in hexane. Cholesteryl, retinyl, and dolichyl esters, triglycerides and vitamin E, ubiquinone, dolichol, phytol, and cholesterol eluted as separate peaks with 0.05% isopropanol in hexane. Cholesterol, retinol, diglyceride, and monoglyceride eluted as separate peaks with 0.75% isopropanol in hexane. These separations could not be achieved on a silica gel column. The method was used to assay sheep liver ubiquinone, dolichol, and cholesterol levels, that were determined as 77, 108 and 1864 micrograms/g wet wt, respectively.

CEROID–LIPOFUSCINOSIS (BATTEN'S DISEASE): PATHOGENESIS AND SEQUENTIAL NEUROPATHOLOGICAL CHANGES IN THE OVINE MODEL

A sequential morphological study of ovine ceroid–lipofuscinosis showed that brains of affected lambs were normal at birth, grew until four months of age but then atrophied. Laminar necrosis of cerebral cortex was noted at 10 weeks, occurring first in the parietal area, then spreading to involve frontal and occipital areas while the temporal lobe was least and last affected. With progression of the disease, the laminar pattern was lost. Neuronal necrosis was accompanied by a severe astrocytosis. The granular and multilamellar storage cytosomes increased in size with age. Their structure was interpreted as paracrystalline in which repeating molecules of the dominantly stored lipid binding subunit of mitochondrial ATP synthase interact with neutral lipids and phospholipids. Abnormal cytosomes in neurons of lamb fetuses and a neonate were interpreted as early lesions which contained whorls or stacks of bilayered membrane as well as the more complex multilamellar material. The underlying anomaly leading to the storage of the lipid binding subunit of mitochondrial ATPsynthase remains to be defined. However, it is noted that this disease should be regarded as a lipid binding protein proteinosis or alternatively as a proteolipid proteinosis.

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.

The Pathology of Bovine Mannosidosis

Mannosidosis of Angus calves is an inherited lethal disease associated with a deficiency of the lysosomal enzyme α-mannosidase. It is characterised by vacuolation of neurons, macrophages, fixed reticuloendothelial cells, exocrine epithelial cells and to a lesser extent a wide variety of other cells. Vacuoles are shown to be membrane-bound vesicles and are considered to be secondary lysosomes in which are stored mannose-containing oligosaccharides that result from incomplete degradation of glycoproteins. Such glycoproteins may enter the lysosomal system by heterophagy, autophagy or crinophagy. The presence in the pancreas of zymogen granules in storage vesicles indicates that incomplete degradation of secretory granules might help explain the widespread vacuolation of other exocrine cells. It is suggested that such granules may enter the lysosomal system by crinophagy.

CEROID‐LIPOFUSCINOSIS (BATTEN'S DISEASE): PATHOGENESIS OF BLINDNESS IN THE OVINE MODEL

Mayhew I. G., Jolly R. D., Pickett B. T. & Slack P. M. (1985)

Effect of cisternal sulfamidase delivery in MPS IIIA Huntaway dogs - a proof of principle study.

Mucopolysaccharidosis type IIIA (MPS IIIA) results from lack of functional sulfamidase (SGSH), a lysosomal enzyme. Its substrate, heparan sulfate, and other secondarily-stored compounds subsequently accumulate primarily within the central nervous system (CNS), resulting in progressive mental deterioration and early death. Presently there is no treatment. As a potential therapeutic strategy, recombinant human sulfamidase (rhSGSH) was administered into the CSF (via the cerebellomedullary cistern) of three adult MPS IIIA dogs either twice with a 4 day interval, or weekly for up to 4 weeks. The dogs were euthanased 24 h post-injection along with one untreated unaffected and two MPS IIIA controls. We have examined the three dimensional pattern of distribution of enzyme in the CNS and its ability to reduce primary substrate storage. High concentrations of rhSGSH protein, with up to 39-fold normal enzyme activity levels were detected within widespread areas of the CNS. RhSGSH protein was also detectable by immunohistochemistry in neurons and glia in all three enzyme-treated dogs. In both weekly-treated dogs, relative levels of a heparan sulfate-derived disaccharide, measured using tandem mass spectrometry, were lower in many brain regions when compared to untreated MPS IIIA controls. A moderately severe meningitis was also present as well as antibodies to rhSGSH in CSF/plasma. These findings demonstrate proof of principle that MPS IIIA can be treated by intracisternal enzyme replacement warranting further experiments in animals tolerant to rhSGSH. This enzyme delivery method may represent a means of treating neuropathology in MPS IIIA and other lysosomal storage disorders affecting the CNS.

A new large animal model of CLN5 neuronal ceroid lipofuscinosis in Borderdale sheep is caused by a nucleotide substitution at a consensus splice site (c.571 + 1G >>> A) leading to excision of exon 3

Batten disease (neuronal ceroid lipofuscinoses, NCLs) are a group of inherited childhood diseases that result in severe brain atrophy, blindness and seizures, leading to premature death. To date, eight different genes have been identified, each associated with a different form. Linkage analysis indicated a CLN5 form in a colony of affected New Zealand Borderdale sheep. Sequencing studies established the disease-causing mutation to be a substitution at a consensus splice site (c.571+1G>A), leading to the excision of exon 3 and a truncated putative protein. A molecular diagnostic test has been developed based on the excision of exon 3. Sequence alignments support the gene product being a soluble lysosomal protein. Western blotting of isolated storage bodies indicates the specific storage of subunit c of mitochondrial ATP synthase. This flock is being expanded as a large animal model for mechanistic studies and trial therapies.