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2012 CLN5 Boustany

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The CLN5 and CLN8 proteins as Ceramide Synthases or Their Modulators

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2012 CLN5 Boustany

  1. 1. 3-Complementation studies in patient cell lines 1- CLN5 sequencing Exon 3 2-Western blot showing the presence of a truncated CLN5 protein in patient cell lysates. 4- Measurement of Ceramide leves 7 5-Perturbation of sphingolipid metabolism in mouse mnd fibroblasts. KEY PROJECTS 6-Ceramide and dhCeramide changes in CLN5 and CLN8-deficient Cells WHAT THIS MEANS FOR THERAPY Conclusion CLN5 Neurogenetics lab:Neurogenetics lab: The CLN5 (aka CLN9) and CLN8 proteins as Ceramide Synthases orThe CLN5 (aka CLN9) and CLN8 proteins as Ceramide Synthases or their Modulatorstheir Modulators Boustany R-MBoustany R-M11 , Saria El Haddad, Saria El Haddad11 , Marwan Khoury, Marwan Khoury11 ,Mohammad Daoud,Mohammad Daoud11 , Talal Mousallem, Talal Mousallem22 , Oscar Alzate, Oscar Alzate22 .. 11 Neurogenetics Program and Division ofNeurogenetics Program and Division of Pediatric Neurology, Departments of Pediatrics and Biochemistry, American University of Beirut, Beirut, Lebanon.Pediatric Neurology, Departments of Pediatrics and Biochemistry, American University of Beirut, Beirut, Lebanon. 22 Duke University Medical Center, Departments ofDuke University Medical Center, Departments of Pediatrics and Neurobiology, Durham, NC, USA. rb50@aub.edu.lbPediatrics and Neurobiology, Durham, NC, USA. rb50@aub.edu.lb NEUROGENETICSPROGRAM DEPARTMENTOFPEDIATRICS NEUROGENETICSPROGRAM DEPARTMENTOFPEDIATRICS INTRODUCTION (Laboratory Objectives) Four patients with variant CLN9 NCL are reclassified as CLN5 disease. CLN5-/- fibroblasts demonstrate adhesion defects, increased growth, apoptosis and ↓ ceramide (Cer), sphingomyelin, and glycosphingolipids. CLN8p corrects growth/apoptosis in CLN5-/- cells. This study highlights role of CLN5/8 as activators of (dihydro)ceramide synthase (CerS). Homozygositymapping led to identifying the CLN5 gene. CerS activity and Cer are ↓ in CLN8-/- / CLN5-/-cells. Normal vs. CLN5-/- CerS1-bound proteins by immunoprecipitation, differential gel electrophoresis, and mass spectrometry reveal absence of γ-actin. γ-actin gene sequence is normal in CLN5-/-. γ-actin-bound proteins vimentin and histone proteins H2Afz/H3F3A/Hist1H4 were absent from the γ-actin protein complex. Interpretation: functions of these proteins and failure to bind γ-actin explain the CLN5 phenotype. We explore the role of CLN5/ CLN8 in sphingolipid biosynthesis and suggest that CLN5/ CLN8 are closely related. Probands Mother FatherNormal Dendrogram of a stretch of nucleotides in Exon 3 revealing the presence of a substitution mutation (red arrows) replacing cytosine by thymine in DNA of probands. In the parents, there is a clear super-position of a red a blue peak reflecting presence of cytosine and thymine denoting that both parents are carriers of the mutated allele. A. No band is seen using anti-CLN5 against the C- terminus while. B. a smaller band is detected with the N- terminus anti-CLN5 antibody. NFB= normal fibroblasts, CLN5= CLN5-deficient fibroblasts. Apoptosis rate of CLN5-deficient fibroblasts transfected with emptypCMV(control) vs CLN5 pCMV (CLN5) 0 10 20 30 40 50 60 70 80 0 1 2 3 4 Time (days) Apoptosisrate(%) Control CLN5 Growth curve of CLN5-deficient fibroblasts transfected with empty pCMV (control) vs CLN5 pCMV (CLN5) 0 500000 1000000 1500000 2000000 0 1 2 3 4 Time (days) Numberofcells/ml Control CLN5 Measurement of de novo C17 ceramide levels generated over 4 hours reflecting CerS activity in mnd cells in top panel. Ceramide levels were measured by tandem mass spectrometry (values from single measures are shown). Ceramide species levels in mnd cells are decreased compared to normal mouse fibroblasts (lower panel). This is mostly seen in C16, C20 and C 24:1 carbon length of ceramide species. Ceramide (A), glucosylceramide (B), gallceramide (C), lactosylcer (D), ceramide trihexoside (E), globoside (F) levels , are decreased in mnd cells compared to normal fibro-blasts measured by 14 C-palmitate (A) γ- actin NF CLN5 Merge NFCLN5 Merge A B C A. 1DGel of proteins from Normal (NF) and CLN5 deficient (CLN5) fibroblasts after immunoprecipitation with LASS1 antibody. Overlay samples display a strong red band present in NF sample but absent in CLN5.This band identified as γ- actin by mass spec. The gene ACTG1 coding for γ- actin was normal in CLN5-/- Fibroblasts. B. 1DGel of NF and CLN5 protein samples after immunoprecipitation with ACTG1 antibody. It is clear that there is a difference in the bands between the 2 cell lines. C. Gel bands present in NF, absent in CLN5-/- were cut and analyzed by Mass Spec. 8 Ceramide and Dihydroceramide species in CLN5- deficient cells Sphingolipid abnormalities Ceramide species in CLN5 Overexpression of CLN8 in normal Fibroblasts Overexpression of CLN8 in normal Fibroblasts: effect on Ceramide Species Sphingolipid abnormalities Ceramide species in CLN5 Overexpression of CLN8 in normal Fibroblasts Sample (Band/ Spot On gel) Protein Name Protein Function Correlation with CLN9 phenotype 1 No Significant Hits     3 Glycerol-3- phosphate dehydrogenase Ubiquitous protein involved in respiration and metabolism Unknown 2 and 4 Vimentin Imparts Resilience to cell Body. Maintains cell shape. Binds Globoside Adhesion defect Rounded morphology Low Globoside level 5 No Significant Hits     6 Histone H2AFZ DNA binding Gene expression DNA repair Mitosis Aneuploidy after several culture passages accelerated growth and apoptosis rates Histone H3F3A 7  Histone1H4H Histone H2A type 2-C Metaphase spread: passage #22 CLN5-/- fibroblasts demonstrating aneuploidy. This was shown in 2 different cell lines from two unrelated families. Acknowledgements: 1. Patient families for their support. 2. The Tarailo family for their funding and support (American Serbian Orthodx church). 3. The Batten Disease Research and Support Foundation (BDRSA). 4. The MPP /URB AUBMC fund. This study highlights the close interaction between CLN5/CLN8 proteins, and their role in sphingolipid metabolism. Our findings suggest that CLN5p/CLN8p are positive modulators of CerS. CLN5/CLN8p defects affect metabolism of C24/C24:1 ceramide species and CLN8p corrects CLN5p defects. There is a strong possibility that these proteins are functionally related. CLN8p and NCL proteins cross-correcting the CLN8-/- phenotype, such as CLN3p and CLN6p, could form a dynamic protein complex, that could include CLN5p, modulating CerS. Phosphorylation is a major post-translational modification that affects CerS function Similarly, CLN5p and CLN8p could impact CerS function through a mechanism still to be determined. Vimentin and several histone proteins, crucial to cell cycle regulation and cellular morphology, may fail to function due to CLN5p defects, in part explaining the CLN5 cellular phenotype.

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