Abstract:
Four patients with juvenile neuronal ceroid lipofuscinoses, a childhood neurodegenerative disorder that was previously described as CLN9 variant, are reclassified as CLN5 disease. CLN5-deficient (CLN5---) fibroblasts demonstrate adhesion defects, increased growth, apoptosis, and decreased levels of ceramide, sphingomyelin, and glycosphingolipids. The CLN8 protein (CLN8p) corrects growth and apoptosis in CLN5--- cells. Related proteins containing a Lag1 motif (CerS1-2-4-5-6) partially corrected these deficits, with CerS1, which is primarily expressed in brain, providing the best complementation, suggesting CLN5p activates CerS1 and may co-immunoprecipitate with it. CLN8p complements CLN5-deficient cells, consolidating the interrelationship of CLN5p-CLN8p, whose potential roles are explored as activators of (dihydro)ceramide synthases. Homozygosity mapping using microarray technology led to identification of CLN5 as the culprit gene in previously classified CLN9-defective cases. Similar to CLN5---cells, ceramide synthase activity, C16-C18:0-C24:0-C24:1 ceramide species, measured by MS is decreased in CLN8--- cells. Comparison of normal versus CLN5--- cell CerS1-bound proteins by immunoprecipitation, differential gel electrophoresis, and MS revealed absence of γ-actin in CLN5--- cells. The γ-actin gene sequence is normal in CLN5--- derived DNA. The γ-actin-bound proteins, vimentin and histones H2Afz-H3F3A-Hist1H4, were absent from the γ-actin protein complex in CLN5--- cells. The function of CLN5p may require vimentin and the histone proteins to bind γ-actin. Defective binding could explain the CLN5--- cellular phenotype. We explore the role of the CLN5-CLN8 proteins in ceramide species specific sphingolipid de novo synthesis, and suggest that CLN5-CLN8 proteins are more closely related than previously believed. © 2012 WILEY-VCH Verlag GmbH andamp; Co. KGaA, Weinheim.