Investigation of the Batten disease protein CLN

The neuronal ceroid lipofuscinoses (NCLs, Batten Disease) are a group of lysosomal storage disorders caused by mutations in known and unknown proteins with different cellular locations. Mutations in the CLN6 gene cause a type of variant late infantile NCL (vLINCL). The CLN6 protein is located in the ER and how mutations in CLN6 cause accumulation of storage material in the lysosome is unclear. The aims of this thesis were to establish the tools and techniques necessary to analyse the CLN6 protein and to utilise these to investigate its subcellular location, to identify proteins that interact with CLN6 and to elucidate the early cellular responses to loss of functional vLINCL proteins, CLN5, CLN6 and CLN8. It was shown that CLN6 can be detected by Western blotting, indirect immunofluorescence and immunoprecipitation. A construct of CLN6 fused to horseradish peroxidase was cloned for localisation studies by indirect immunofluorescence and electron microscopy. CLN6 protein and mRNA levels could be depleted using RNA interference and this depletion assessed by Western blotting, indirect immunofluorescence and Q-PCR. It was not possible to establish whether CLN6 is located within a sub-domain of the ER using indirect immunofluorescence or electron microscopy. There was no effect on the localisation or stabilisation of wild-type or mutant CLN6 when proteasomal or lysosomal inhibitors were used, indicating that CLN6 does not traffic to the lysosome and that wild-type and mutant CLN6 were not degraded by ER-associated degradation. Endogenous CLN6 was identified in coimmunoprecipitation experiments, confirming that it may bind to itself. No other proteins were identified that bind to CLN6. Depletion of CLN5, CLN6 and CLN8 resulted in an increase in the size and a redistribution to perinuclear areas of late endosomes and lysosomes. An increase in long cis-Golgi structures was also observed in response to siRNA against CLN6 and CLN8 but not CLN5.