Prenatal Synaptic Pathology and Correction in CLN6 Ovine Neuronal Ceroid Lipofuscinosis

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of severe autosomal recessive and incurable childhood lysosomal storage disorders (LSD). They have a combined incidence of 1 in 100,000 live births, affecting males and females equally. There are at least ten distinct forms of NCL, each differing in terms of age of onset; one congenital, seven childhood, and two adult. The NCLs are the most common neurodegenerative disorders of childhood; characterised by severe cortical atrophy, visual impairment, seizures, personality and behavioural changes, dementia, and regression in communication and motor skills, culminating in premature death. Naturally occurring forms of Batten disease in three breeds of sheep have been extensively studied: including CLN6 in New Zealand South Hampshire and Australian Merino. Established flocks provide excellent large animal models to study pathology and potential treatment strategies. Lysosomal acidity is required for maintenance of neural function. We have previously shown defective lysosomal acidity in CLN6-/- primary ovine neural cultures, leading us to investigate the functionality and integrity of CLN6-/- synapses. Primary neural cell cultures from fetal South Hampshire sheep at mid-gestation (65 days, CLN6-/- n = 6 and CLN6+/- control n = 6) were studied to investigate the hypothesis of CLNprotein interaction networks, and to identify pathological features at the neuronal synapse. As NCL disease in the ovine model typically presents at 7-9 months of age the investigated pathologies represent pre-clinical disease features. It is widely hypothesised that the distinct NCL genes must participate in a shared signalling or trafficking pathway since mutations in the genes cause extremely similar pathological features. The potential role of CLN6 in lysosomal NCL-associated protein trafficking was investigated using CLN1, CLN2, and CLN10 enzyme assays. The trafficking of CLN5 was also investigated. Other than a small decrease in CLN10 enzymatic activity, no major defects in the trafficking of any of these select NCL enzymes was demonstrated, suggesting that CLN6 is not a vital component in the correct trafficking of these proteins to their lysosomal destination. A number of synaptic pathologies were investigated, including excitation-induced endocytosis through the use of fluorescent tetramethylrhodamine dextrans, and synaptic protein expression with immunocytochemistry, and autophagic flux with an autophagy monitoring fluorescent dye. Excitation-induced synaptic endocytosis of both 10 and 40 kDa dextrans was significantly impaired. The proportion of astrocytes was significantly increased in the CLN6-/- cultures, indicating early-onset gliosis. Expression of the key pre-synaptic vesicle protein, synaptophysin, was significantly reduced in CLN6-/- neurons and not trafficked to the synapse. Constitutive autophagy, a pathway required for the maintenance of healthy neurons, was significantly reduced in CLN6-/- neural cultures. CLN6-/- cultures were examined at the ultrastructural level with transmission electron microscopy, revealing a significant reduction in organelle density and significant cytoplasmic vacuolisation as compared to control primary ovine neural cell cultures. All pathologies were corrected via lentiviral-mediated overexpression of wild-type CLN6 into cultures or via treatment of cultures with a novel polyphenol drug compound. Together, these pathologies suggest major consequences for neuronal function in affected sheep, and indicate possible target sites for therapeutic correction via either gene therapy, drug treatment, or a combination approach