Abstract
Neuronal Ceroid Lipofuscinoses (NCL) are a group of debilitating and fatal neurodegenerative diseases of childhood resulting from progressive brain atrophy. The human and ovine variant late infantile CLN6 (ceroid lipofuscinosis protein) forms of NCL result from mutations encoding the endoplasmic reticulum transmembrane protein CLN6. Mutations encoding the soluble lysosomal protein CLN5 also cause an NCL in humans and sheep. The functions of these proteins are not understood, however previous research suggests a relationship between them (Lyly et al., 2009). In CLN6-/- ovine neural tissue, CLN5 protein expression was reduced (McIntyre, K.M., summer studentship unpublished observations, 2013/2014). The aim of this study was to investigate this potential interaction in the ovine model of CLN6 NCL.
In CLN6-/- and CLN6+/- control secondary ovine cultures derived from primary ovine neural cultures, concentrations of CLN5 mRNA were assessed by relative quantitative polymerase chain reaction (qPCR). Protein expression was assessed by 3, 3 diaminobenzidine (DAB) immunocytochemistry, immunofluorescence and western blotting techniques. To detect CLN5 protein, cultures were transduced with CLN5 lentivirus (pCDH.MND.CLN5 (VSVG)). Proteasome inhibitor MG132 was applied for 2 and 4 hours to determine whether the reduction in CLN5 was due to ERAD (Endoplasmic Reticulum Associated-Protein Degradation). Absence of GFAP (Glial Fibrillary Acidic Protein) and MAP2 (Microtubule-associated protein 2) immunofluorescence in cultures indicated a cell population devoid of astrocytes and neurons respectively. Relative to ATPase and RPLPO (Large Ribosomal Protein) (M-value 0.936), no statistical difference in CLN5 mRNA concentration was found between CLN6-/- and control cultures (p = 0.68, n = 3). DAB immunocytochemistry showed low CLN5 protein expression in non-transduced cultures; reduced CLN5 protein expression was not evident. In immunofluorescence studies, no significant difference in relative fluorescent intensity was seen in transduced cultures (p = 0.75, n = 3). Western blot analysis of overexpressed CLN5 protein between CLN6-/- and control cultures was inconclusive. In non-transduced cultures treated with MG132, CLN5 expression was not detectable by western blotting. Data from transduced cultures are currently inconclusive. These results suggest that in this cell population, CLN5 mRNA is unaltered in CLN6 NCL. These methods may subsequently be translated to primary cultures as a foundation for on-going investigations into NCL protein interactions in ovine cultures.