Characterization of Neuronal Ceroid Lipofuscinosis Phenotype in Three Different Cell Lines Treated by RNA interference

Neuronal ceroid lipofuscinoses (NCL) are a group of genetically inherited encephalopathies, characterized by accumulation of autofluorescent lipopigment in neurons and several other cell types. These diseases are metabolic lysosomal storage disorders affecting mainly children, although some rare adult forms have also been discovered. They are clinically identified by mental and motor deterioration, epileptic seizures, loss of vision and premature death. The disease genotype of NCLs is mainly inherited in an autosomal recessive manner. As an exception, one of the two adult forms (Parry disease) displays dominant inheritance. The different forms of human NCLs are caused by mutations in at least ten genes (CLN1-10), eight of which have been identified (CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8 and CLN10). Proteins encoded by the NCL genes are mainly located in the lysosomes, late endosomes or the endoplasmic reticulum (ER). These NCL proteins have both soluble and transmembrane forms and thus have various different functions, some of which have been established but most of which are still under research. CLN3 (juvenile NCL, Batten disease) is the most common form of NCL and is enriched especially in the Finnish population. The CLN3 gene encodes a hydrophobic protein of lysosomal/endosomal membranes, the function of which is still unknown. The aim of this study was to find an optimal cell line for NCL-targeted RNA interference (RNAi) studies with small interfering RNA (siRNA) transfections, which would show easily detectable markers characteristic of NCL phenotype. In these studies, CLN3 was chosen as target gene for RNAi and autofluorescent lipopigment accumulation as a NCL marker to be detected. Three cell lines were used for RNAi-experiments and the down-regulation was detected with quantitative real-time PCR. Relatively effective down-regulation of CLN3 with RNAi was achieved in HEK293 cells, with 70-75% knock-down. The results obtained from HUH7 and SH-SY5Y cells were somewhat controversial, showing unexpected changes also in control-siRNA samples. Accurate down-regulation of CLN3 was thus not obtained from any of these cell lines, giving a ground for further studies in other cell lines or with different methods. These studies were aiming at laying basis for a larger project, to set up a method that utilizes genome-wide RNAi technology combined with phenotypic screening to detect pathways and genes behind NCL diseases. When succesful, this technology would also create a method for functional studies in human cells, bypassing the use of knock-out mouse models.