Spinal cord pathology of ovine CLN5 and CLN6 neuronal ceroid lipofuscinoses (Batten disease) : A thesis submitted in partial fulfilment of the requirements for the Degree of Master at Lincoln University

Abstract

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.