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Neuronal ceroid lipofuscinosis (NCL or Batten disease) is a fatal neurodegenerative disorder, which occurs in approximately 1 in 12,500 children worldwide and currently has no cure. To date, there are thirteen known variants of NCL, caused by mutations in the genes CLN1-8 or CLN10-14. Two naturally occurring forms of CLN5 and CLN6 NCL exist in Borderdale and South Hampshire sheep breeds respectively. These large animal models recapitulate the clinicopathological features of the human disease and have been instrumental in understanding the human condition and in the development of gene therapies. Affected sheep have a well-characterised pathogenic cascade in the brain, which begins prenatally and involves neurodegeneration, neuroinflammation and lysosomal storage, but very little is known about their spinal cord pathology. This study examined anatomy and neurochemistry in the healthy sheep spinal cord and then compared that to pathology in the CLN5 and CLN6 affected spine over the disease course. Neurodegeneration was assessed by area measurements and neuron counts. This revealed that there was no grey matter loss in the spinal cord for either diseased genotype, but total cross-sectional area and white matter spinal cord areas declined rapidly at end-stage disease (18 months of age). There was also an equivalent late-stage reduction in motor neuron counts in the CLN5 affected sheep spinal cord. Fluorescent microscopy showed that lysosomal storage increased in the CLN6 affected sheep spinal cord from 3 to 18 months of age, whilst rates of accumulation were slower in CLN5 affected sheep until late in the disease course. Immunohistochemistry found that activated astrocytes were present in the CLN6 affected spinal cord at 18 months of age, but not in the ovine CLN5 model. Collectively, these results show that pathological changes in the spinal cord occurred late in the disease process, making them secondary to that occurring in the brain. This indicates that CLN5 and CLN6 affected sheep may have a “top-down” or “brain-first” propagation of disease. Despite subtle differences in the pathogenic cascade, the pathological endpoint in the spinal cord for CLN5 and CLN6 affected animals was very similar. This natural history study of spinal cord pathology in ovine NCL increases our knowledge about disease progression and informs subsequent decisions on optimal routes of administration for gene therapy in NCL.

Batten disease or neuronal ceroid lipofuscinoses (NCLs) is one of the most common reasons for childhood dementia, characterized by brain atrophy, blindness, mental decline and premature death. Although animal model neurological research is traditionally conducted using small rodents, large animal models are becoming more popular for several reasons. Longitudinal monitoring of disease progression and individual variations are essential in the development of translational medicine and whereas small rodents have relatively short life spans and different brain structures compared with humans, sheep provide an excellent model for long-term studies of diseases and their treatment. Three naturally occurring ovine models are currently being utilised to increase knowledge about different types of NCL and to aid in development of treatment strategies for translation to humans. These sheep models include a CLN5 Borderdale, a CLN6 South Hampshire and a CLN6 Merino model. This thesis contains investigations of techniques for longitudinal in vivo monitoring that can be correlated with the progression of neuropathological events in all three ovine NCL models, both in the natural progression of the diseases and in trials of potential therapeutic regimes. Previous studies have described the neuropathological development of disease in the CLN5 Borderdale and the CLN6 South Hampshire models. In the present study similar investigations were conducted with the CLN6 Merino model, and the three ovine models compared. The general development of neuropathology in CLN6 Merino sheep follows that of the other two models. Common themes are neuronal loss from the cerebral cortex that proceeds in a regional pattern, with neuroinflammation, neurogenesis and ubiquitous accumulation of storage material within the cells. Immunohistochemistry indicated possible cross-regulation between the CLN5 and CLN6 proteins. In vivo techniques are essential in the monitoring of progressive diseases such as the NCLs. Neuroimaging through computed tomography (CT) and magnetic resonance imaging (MRI) are suitable tools to describe neuropathological changes without the need to sacrifice animals. Here, current techniques and protocols for CT scanning in ovine NCL research were reviewed and improved. The congruence of brain size and intracranial volume (ICV) in ovine NCL was established and techniques for the monitoring of brain size in therapeutic trials were validated and correlated with neuropathological changes. Furthermore, 3-dimensional reconstruction was used to monitor regionality of the cortical changes and this could be correlated with findings from post examination. CT based surgery planning was used to improve accuracy of intracereboventricular gene therapy delivery and reduce duration of the surgical procedure. Pilot studies on the use of MRI as a longitudinal in vivo monitoring modality in ovine NCL research were conducted and these indicated that MRI will yield enhanced detail compared with CT and that MRI measurements can be closely correlated with the neuropathological changes observed at post mortem examination. However, some specific technical issues were identified and will need to be addressed in the future. These include image quality, fit of templates and an important need to establish ovine-specific image acquisition protocols. Blindness in ovine NCL results from atrophy of photoreceptor cells in the retina as well as changes in the central visual pathways. Treatment should aim not only to treat the atrophy of the brain, but also to prevent or halt the development of blindness. Previous trials have indicated the need for ocular delivery of therapy to achieve this. As for neurodegeneration, in vivo monitoring of retinal pathology is essential for the longitudinal assessment of treatment efficacy. Here, protocols were established for the use of repeated electroretinography (ERG) to monitor the development of blindness in the CLN5 Borderdale and the CLN6 South Hampshire models. The results show that ERG is an invaluable tool for monitoring the progression of retinal degeneration in ovine NCL. Furthermore, it was established that the course of development of retinal changes differs between the two ovine models that were investigated. The measurements enabled some degree of differentiation between the retinal and central components of the blindness in ovine NCL. Finally, it was shown that the in vivo monitoring tools established throughout the studies described in this thesis are invaluable for the assessment of treatment efficacy in gene therapy trials for ovine NCL. The measures evaluated here were shown to correspond with other clinical measures of treatment efficacy, such as behavioural observations, maze-testing and neuropathology. Key words: Neuronal ceroid lipofuscinosis, Batten disease, lysosomal storage disease, large animal models, sheep, neuroimaging, neurodegeneration, neuroinflammation, gene therapy, computed tomography, magnetic resonance imaging, electroretinography, in vivo monitoring.

Neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of genetically distinct neurodegenerative diseases of children and young adults, leading to premature death and characterised by the intracellular accumulation of fluorescent lipofuscin-like (ceroid) storage bodies within lysosomes of neurons and other cell types. NCLs are the most common neurogenetic storage disease in children world-wide. Typical clinical symptoms of the disease include retinopathy leading to blindness, sleep problems, motor abnormalities, epilepsia, dementia and eventually premature death. However, mechanisms underlying these neuropathies are not fully understood. In NCLs, glial activation precedes neurodegeneration that marked by the gross atrophy, cortical thinning and neuronal loss. The neurodegeneration observed in CLN 6 defective cells and tissues could be associated with up-regulated expression of the radical scavenger MnSOD in CLN6 defective cells and tissues. A significant increase in MnSOD activity has been detected in fibroblasts and brain extracts from CLN6 sheep. The increased MnSOD levels, elevated ROS and glial inflammation have been suggested to be associated with molecular pathways in NCL pathology. The aim of this study was to investigate the regionality and cell-type selection in the change in MnSOD expression in the CLN6 and CLNS affected sheep brains. Immunohistochemical staining was used to localize MnSOD expression and distribution in the affected sheep brains. Microscopy and computational image analysis were used to examine the MnSOD stained brain region and cell morphology. The MnSOD immunoreactivity parallels the neuronal distribution in 5&6 brains. The enhanced expression of MnSOD was confined to the layer V of OCL5 and OCL6 affected sheep cerebral cortices, including the frontal, parietal and occipital lobes. Other brain regions examined did not show any marked difference in MnSOD immunoreactivity between the affected brains and the controls. .

The Neuronal Ceroid Lipofuscinosis (NCL, Batten disease) are a group of related hereditary neurodegenerative diseases that afflict children and animals. They are characterised by brain atrophy and the accumulation of a hydrophobic protein within lysosome-derived cytosomes. Biochemical studies on a well characterised ovine model of ceroid lipofuscinosis (OCL) established the nature of the storage material. Subunit c of mitochondrial ATP synthase specifically accumulates in OCL and the human diseases except for the infantile onset form. The gene defective in the ovine disease is different to the recently identified gene for a juvenile form of NCL, CLN3. This gene has been sequenced but nothing is known about the CLN3 protein or its function. We studied CLN3 expression in normal and OCL affected sheep to test whether the two genes are cross-regulated. The sheep cDNA sequence was used to generate a probe for Northern blotting and to verify that the amino acid sequence corresponds to that of synthetic peptides used to raise antibodies. CLN3 Northern blot analysis did not reveal a significant difference in the amount of message being present in brain and liver from control and OCL affected animals. No large differences were apparent on Western blots of brain and liver homogenates. Bands of 40, 50 and 90 kD in liver, or 50 and 62 kD in brain were detected in both control and affected animals. No definite band could be assigned to CLN3 which appears to be a differentially spliced gene. In brain, antiserum raised to a C-terminal peptide detects a 50 kD band that is specifically quenched by the peptide used to raise the antibody. Increased 50 kD brain and 90 kD liver immunostaining to this antiserum in three OCL affected sheep compared to normal is the only indication of cross-regulation between the OCL gene and CLN3. The increased potential CLN3 protein activity may reflect an attempt to compensate for the OCL gene defect. Western blotting of subcellular fractions indicated that the CLN3 protein may reside in a soluble compartment.