The CLN9 Protein, a Regulator of Dihydroceramide Synthase

Abstract

A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients. A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients. The neuronal ceroid lipofuscinoses (NCL) 2The abbreviations used are: NCL, neuronal ceroid lipofuscinosis/dhC16-Cer; 4-HPR, N-(4-hydroxyphenyl)retinamide; Cer, ceramide; SM, sphingomyelin; β4GalT, β-1,4-galactosyltransferase; GB3/CD77, globoside synthase; SMS1 and SMS2, sphingomyelin synthase; TRAM, translocating chain-associated membrane protein; PI, propidium iodide; JC-1, 5,50-60-tetrachloro-1,1,3,3- tetraethylbenzimidazolylcarbocyanine iodide; FB1, fumonisin B1; PDMP, phenyl-2-decanoulamino-3-morpholino-1-propranolol; DGK, diacylglycerol kinase; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). are a group of autosomal, recessively inherited storage diseases. They represent the most common cause of neurodegeneration in childhood. Neuronal death is caused by apoptosis (1Lane S.C. Jolly R.D. Schmechel D.E. Alroy J. Boustany R.M. J. Neurochem. 1996; 67: 677-683Crossref PubMed Scopus (90) Google Scholar). Clinical features of these diseases include visual loss, mental and motor deterioration, seizures, and early death (2Goebel H.H. Mole S.E. Lake B.D. Goebel H.H. Mole S.E. Lake B.D. The Neuronal Ceroid Lipofuscinoses (Batten Disease). Vol. 33. IOS Press, Amsterdam, the Netherlands1999: 1-211Google Scholar, 3Haltia M. J. Neuropathol. Exp. Neurol. 2003; 62: 1-13Crossref PubMed Scopus (219) Google Scholar). To date nine forms of NCL have been described. Six genes are known. CLN1 and CLN2 encode for the lysosomal enzymes palmitoyl protein thioesterase 1 and tripeptidylpeptidase 1, respectively (4Vesa J. Hellsten E. Verkruyse L.A. Camp L.A. Santavuori P. Hofman S.L. Peltonen L. Nature. 1995; 376: 584-587Crossref PubMed Scopus (628) Google Scholar, 5Sleat D.E. Donnelly R.J. Lackland H. Liu C.G. Sohar I. Pullarkat R.K. Lobel P. Science. 1997; 277: 1802-1805Crossref PubMed Scopus (507) Google Scholar). CLN5p has been described as a soluble glycoprotein (6Savukoski M. Klockars T. Holmberg V. Santavuori P. Lander E.S. Peltonen L. Nat. Genet. 1998; 19: 286-288Crossref PubMed Scopus (244) Google Scholar, 7Isosomppi J. Vesa J. Jalanko A. Peltonen L. Hum. Mol. Genet. 2002; 11: 885-891Crossref PubMed Scopus (101) Google Scholar). CLN3, CLN6, and CLN8 genes code for novel transmembrane proteins (8International Batten Disease ConsortiumCell. 1995; 82: 949-957Abstract Full Text PDF PubMed Scopus (561) Google Scholar, 9Gao H. Boustany R.M. Espinola J.A. Cotman S.L. Srinidhi L. Antonellis K.A. Gillis T. Qin X. Liu S. Donahue L.R. Bronson R.T. Faust J.R. Stout D. Haines J.L. Lerner T.J. MacDonald M.E. Am. J. Hum. Genet. 2002; 70: 324-335Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 10Wheeler R.B. Sharp J.D. Schultz R.A. Joslin J.M. Williams R.E. Mole S.E. Am. J. Hum. Genet. 2002; 70: 537-542Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 11Ranta S. Zhang Y. Ross B. Lonka L. Takkunen E. Messer A. Sharp J. Wheeler R. Kusumi K. Mole S. Liu W. Soares M.B. Bonaldo M.F. Hirvasniemi A. de la Chapelle A. Gilliam T.C. Lehesjoki A.E. Nat. Genet. 1999; 23: 233-236Crossref PubMed Scopus (255) Google Scholar). Increased apoptosis has been documented in CLN1-3-, 6-, and -8-deficient variants (1Lane S.C. Jolly R.D. Schmechel D.E. Alroy J. Boustany R.M. J. Neurochem. 1996; 67: 677-683Crossref PubMed Scopus (90) Google Scholar, 12Cho S. Dawson P.E. Dawson G. Eur. J. Neurol. 2001; 5: 53-55Abstract Full Text PDF Scopus (20) Google Scholar). Dysregulated sphingolipid metabolism and increased apoptosis have been described in the late infantile CLN2-, juvenile CLN3-, variant late infantile CLN6-, and the Northern Epilepsy with Mental Retardation CLN8-deficient variants. CLN3 is a negative modulator of ceramide synthesis resulting in high ceramide levels in CLN3-deficient patient fibroblasts (14Persaud-Sawin D.A. McNamara II J.O. Rylova S. Vandongen A. Boustany R.M. Pediatr. Res. 2004; 56: 449-463Crossref PubMed Scopus (54) Google Scholar). High ceramide levels, caspase-8 activation, and engagement of both the extrinsic and intrinsic apoptotic pathway as well as caspase-dependent autophagy explain the increased apoptosis and slow growth observed in CLN3-deficient cells. Also, CLN3 protein harbors a galactosylceramide binding motif that is involved in CLN3 trafficking from Golgi to lipid rafts via recycling endosomes (14Persaud-Sawin D.A. McNamara II J.O. Rylova S. Vandongen A. Boustany R.M. Pediatr. Res. 2004; 56: 449-463Crossref PubMed Scopus (54) Google Scholar). The ninth form of NCL, CLN9, has recently been described in four patients; two German brothers and two Serbian sisters (15Schulz A. Dhar S. Rylova S. Dbaibo G. Alroy J. Hagel C. Artacho I. Kohlschütter A. Lin S. Boustany R.M. Ann. Neurol. 2004; 56: 341-350Crossref Scopus (53) Google Scholar). These patients present with clinical features identical to those seen in juvenile or CLN3-deficient patients. The CLN9 gene is still unknown. Mutations in all known NCL genes, including CLN3, were ruled out in the CLN9-deficient variant. CLN9-deficient patient fibroblasts have a distinctive phenotype. They have small and rounded cell bodies, grow rapidly, are sensitive to apoptosis and manifest a cell adhesion defect. Their gene expression pattern is significantly different from that in other NCL forms. Genes involved in cell cycle, cell adhesion, and apoptosis are significantly dysregulated (15Schulz A. Dhar S. Rylova S. Dbaibo G. Alroy J. Hagel C. Artacho I. Kohlschütter A. Lin S. Boustany R.M. Ann. Neurol. 2004; 56: 341-350Crossref Scopus (53) Google Scholar). Sphingolipid metabolism, similarly to other NCL variants, is also perturbed in CLN9-deficient cells; ceramide, sphingomyelin (SM), lactosylceramide, gangliosides, ceramide trihexoside, and globoside, galactosylceramide, and glucosylceramide are diminished. The purpose of this study is to dissect out the specific biochemical defect in CLN9-deficient cells, with the ultimate goal of identifying the CLN9 gene and protein. Low levels of lactosylceramide and its metabolites including gangliosides, ceramide trihexoside, and globoside can implicate the seven genes for β-1,4-galactosyltransferase (β4GalT) and the two for GB3/CD77 synthase as possible candidate genes. Review of the gene chip data provided additional information; acid sphingomyelinase activity was normal, but mRNA expression of acid sphingomyelinase was 22-fold decreased in the setting of low baseline sphingomyelin levels. Defects in sphingomyelin synthase 1 and/or 2 (SMS1, SMS2) could account for this. A trafficking defect of ceramide from endoplasmic reticulum (ER) to Golgi due to defects in ceramide transporter protein, the ER-to-Golgi ceramide transporter, could also account for low SM (16Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (827) Google Scholar). Low ceramide levels also suggest a defect in de novo ceramide synthesis. Activity of serine palmitoyltransferase, the key enzyme of de novo ceramide synthesis, was 4-fold increased, expanding the list of candidate genes to those involved in de novo ceramide synthesis beyond the serine palmitoyltransferase step. Transfection with CLN8 corrected the CLN9 phenotype. The CLN8 protein contains a Lag1 motif similar to other TRAM-Lag1-CLN8 proteins (17Winter E. Ponting C.P. Trends Biochem. Sci. 2002; 27: 381-383Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Lag1p and Lac1p are homologous, multispanning transmembrane proteins that reside in the ER. Both contain a 52-amino acid-long Lag1 motif that is necessary for ceramide synthesis in yeast (18Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (220) Google Scholar, 19Schorling S. Vallee B. Barz W.P. Riezmann H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (221) Google Scholar). Lac1p, Lag1p, and Lip1p are components of the enzyme activity in yeast (20Vallee B. Riezmann H. EMBO J. 2005; 24: 730-741Crossref PubMed Scopus (116) Google Scholar). CLN8 sequencing results were normal in CLN9-deficient cells, suggesting human Lag1 homologs or activators of their proteins could be candidate genes for CLN9. Cell Lines—CLN9-deficient fibroblast lines were derived from skin biopsies of two CLN9 patients. Use of human cell lines in research is covered by an approved Duke institutional review board protocol. Tissue Culture Studies—Fibroblasts were grown at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium containing and were in and at and in for the cells were at for and were in a of and and a and cells were were with for was by propidium JC-1, or 4-HPR were with and 4-HPR 4-HPR was in at a of and at were to the with growth medium Fumonisin were with 1 fumonisin was in at a of 1 and at were to the with growth medium were with was in at a of and at were to the with growth medium were with different and as provided by S. M. from from from The for the Transfection were The cells were and for Transfection of cells was an and in Transfection of cells was by in Transfection of cells was a cells were a Transfection was in including of GB3/CD77 LASS1, LASS2, and LASS4, were from of CLN9-deficient cells. of LASS5, and were from of CLN9-deficient cells. were an of cells with were grown and with for of were at The of of was The of apoptotic cells was The of of was and were and was iodide; was used to the in membrane treatment with is a that forms with the apoptosis are a in the from to This the of apoptosis. of cells with were grown The cells were with 1 and with 1 of for at 37 °C and in 5% The cells were in and the a with apoptotic and cells in different of were The of apoptotic of was as well as and was of Ceramide of normal and CLN9-deficient fibroblasts were grown and in a containing acid and at °C for were in by for in were a with 1 of and with a Ceramide and Dihydroceramide and dihydroceramide levels were by of to Ceramide and dihydroceramide levels were also by diacylglycerol from and of for and and the Dihydroceramide synthase activity was by de novo dihydroceramide cells with with were with a and in 1 of and and were by a were used SM synthesis was as described. The of protein, of and of in a of of The was at 37 °C by the of 1 of 1 of and of were to and and SM were as the were a and were by was from normal and CLN9-deficient fibroblasts the were out with synthesis was and to the in a The mRNA levels of were used as CLN8 and in CLN9-deficient fibroblasts were with CLN3, CLN6, and CLN8 genes and were for growth and to apoptosis. with out of all NCL genes, partially corrected growth in CLN9-deficient cells 1, A and CLN9-deficient cells with CLN8 a in growth with CLN9-deficient cells with still that of normal fibroblasts with Transfection with CLN8 decreased to apoptosis in CLN9-deficient with CLN8 or were with of with CLN8 significantly with Dihydroceramide Dihydroceramide and Ceramide Low in CLN9-deficient cells have significantly decreased dihydroceramide synthase as by the decreased levels of dihydroceramide and ceramide in CLN9-deficient cells. The most were in and and and with cells A and and levels were and with normal Dihydroceramide synthase activity was by cells with and dihydroceramide Lag1 Sc Ceramide and and in CLN9-deficient with Lag1 Sc increased ceramide levels in CLN9-deficient cells levels in cells with This was by an increase in the ceramide not Lag1 Sc decreased growth Lag1 Sc also decreased to apoptosis by treatment with CLN9-deficient cell lines a and apoptotic with with a and apoptotic with Lag1 Sc the other increased apoptosis with ceramide levels were in normal fibroblasts Lag1 Sc to ceramide levels. yeast Lag1 Sc partially corrected ceramide levels, growth, and apoptosis in CLN9-deficient cells. Lag1 and in CLN9-deficient is the human homolog to Lag1 LASS1 increased ceramide levels 4-fold in CLN9-deficient cells, as by an increase in de novo ceramide synthesis as well not LASS1 increased of and by in normal fibroblasts and in CLN9-deficient fibroblasts This is with LASS1 ceramide LASS1 partially corrected growth in CLN9-deficient cells and decreased cell as by not LASS1 decreased CLN9-deficient cell to apoptosis as observed by and not LASS1 were normal in CLN9-deficient cells. human Lag1 homologs were for their to the CLN9 phenotype. partially corrected growth and diminished apoptosis in CLN9-deficient cells not LASS1, LASS2, LASS4, LASS5, and LASS6 were normal in CLN9-deficient cells. expression levels were normal, and LASS1, and -6 expression levels were increased and the that the CLN9 gene could be one of the known human Lag1 4-HPR Dihydroceramide Activity and Dihydroceramide and LASS1 by cells were with or 4-HPR, a dihydroceramide synthase activator H. C.P. Res. 2001; Google Scholar). with 4-HPR increased dihydroceramide levels in both normal and CLN9-deficient fibroblasts an increase of all dihydroceramide CLN9-deficient cells an increase in all dihydroceramide that was significantly the increase in normal cells. and levels increased the most 4-HPR treatment and were and in CLN9-deficient cells The dihydroceramide the levels in CLN9-deficient cells Dihydroceramide synthase activity was also increased by 4-HPR no increase in of de novo ceramide synthesis 4-HPR treatment in CLN9-deficient cells as by with 4-HPR decreased levels of all ceramide as by This be due to a of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. with 4-HPR corrected growth of CLN9-deficient cells in a and treatment with 4-HPR at the of growth in CLN9-deficient cells with 4-HPR not have a growth, but and 4-HPR decreased growth in a was no of 4-HPR growth of normal cells used at the not 4-HPR decreased LASS1 expression to in mRNA from CLN9-deficient cells. normal cells 4-HPR decreased LASS1 expression to expression from normal 4-HPR or CLN9-deficient from with 4-HPR CLN9-deficient cells from apoptosis. with 4-HPR significantly apoptosis by with This was 4-HPR decreased apoptosis These results were by and 4-HPR apoptosis C.P. J. 1999; PubMed Scopus (255) Google Scholar). CLN9-deficient cells were from apoptosis by 4-HPR. CLN9-deficient cells with PDMP, glucosylceramide results in an increase in ceramide A. J. M. H. A. J.A. J. Biol. Scholar). diminished apoptosis in CLN9-deficient cells. with and 4-HPR, increased apoptosis the of CLN9-deficient cells. This was by This may be due to the of an increase in ceramide due to and an increase in dihydroceramide due to 4-HPR These at different of the Fumonisin B1, a Dihydroceramide the CLN9 that the could be due to a defect in of dihydroceramide was It dihydroceramide synthase and the CLN9 phenotype. with decreased low ceramide levels in CLN9-deficient cells 4-HPR neutralized the of ceramide levels. Ceramide levels were by of 4-HPR treatment treatment accelerated growth of CLN9-deficient cells, the CLN9 phenotype. with 4-HPR this increase in growth CLN9-deficient cells with also a apoptotic cells. the of 4-HPR. The effects apoptosis were in the of as by and by Low in CLN9-deficient to a Ceramide at the is via ceramide transporter protein or ceramide transporter protein to the of the Golgi for to SM by sphingomyelin synthase 1 (16Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Crossref PubMed Scopus (827) Google Scholar). a ceramide trafficking defect be sphingomyelin synthase activity be synthase activity was of normal, the of a ceramide trafficking defect in CLN9-deficient cells. Also, CLN9-deficient cells, to an Golgi normal trafficking and of ceramide with to SM in the Golgi The CLN9 phenotype is by the low dihydroceramide and ceramide levels. dihydroceramide by 4-HPR or ceramide by corrected the phenotype. CLN9-deficient cells manifest increased apoptosis but accelerated growth with low ceramide levels (15Schulz A. Dhar S. Rylova S. Dbaibo G. Alroy J. Hagel C. Artacho I. Kohlschütter A. Lin S. Boustany R.M. Ann. Neurol. 2004; 56: 341-350Crossref Scopus (53) Google Scholar). The in de novo lactosylceramide synthesis and and derived from the of one of the seven or GB3/CD77 synthase could be for CLN9. seven proteins are membrane proteins that reside in the and the latter is also the of SM synthesis from ceramide and lactosylceramide synthesis from glucosylceramide M. R. T. H. 1999; PubMed Scopus Google Scholar, T. Cell. Mol. Sci. 2002; PubMed Scopus Google Scholar). A defect in been described in a patient with a H. S. E. J. J. S. T. Am. J. Hum. Genet. Google Scholar). We ruled out in these genes in CLN9 patients by sequence Low sphingomyelin a possible defect in sphingomyelin synthesis. SM synthase proteins SMS1 and reside in the Golgi and the respectively K. J. EMBO J. 2004; 23: PubMed Scopus Google Scholar, S. M. T. H. T. J. Biol. 2004; Full Text Full Text PDF Scopus Google Scholar). synthase were of normal, and of both of SMS1 and were in CLN9-deficient cells. Also, SMS1 and gene expression in CLN9-deficient cells was with normal. of SMS1 or in CLN9-deficient cells no growth, out as candidate genes for CLN9. Transfection with CLN8 corrected growth and decreased apoptosis in CLN9-deficient cells. dihydroceramide synthase is still of the yeast gene Lag1 Sc and LASS1, the human Lag1 homolog for Lag1 the defect in CLN9 cells with of ceramide levels, growth, and apoptosis. The of ceramide cell growth is well known. The of Lag1 and cell growth in and cell cells derived from and S. K. S. J. W. Jiang J.C. Jazwinski S.M. B. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). LASS1 is in with low levels of expression in and Genes involved in neurodegenerative diseases have a pattern similar to that of human LASS1 or LASS1 J.C. Kirchman P.A. M. J. Jazwinski S.M. Res. 1998; PubMed Scopus Google Scholar). of of LASS1, LASS2, LASS4, LASS5, and LASS6 that LASS1 expression was Y. A. Y. Biochem. J. 2005; PubMed Scopus Google Scholar). A normal LASS1 sequence as well as levels of gene expression in CLN9-deficient cells LASS1 from a candidate for CLN9. other human Lag1 homologs could partially growth and apoptosis in CLN9-deficient cells. contains both a Lag1 motif and a in yeast cells to the that both are for ceramide synthesis K. 2002; PubMed Scopus Google Scholar). results that in cells this is not Transfection with LASS1, variant 1 or or variant corrected growth and apoptosis in CLN9-deficient cells. variants 1 and 2 contain both a Lag1 motif and a variant the but was still to CLN9. the Lag1 motif and not the is and necessary for ceramide synthesis in human Low dihydroceramide levels and low de novo dihydroceramide synthesis and in LASS1, LASS4, and LASS6 expression by reverse suggest that CLN9 may be a modulator of dihydroceramide synthase at the protein 4-HPR, an activator of dihydroceramide increased dihydroceramide levels in CLN9-deficient cells. Dihydroceramide levels were markedly increased in both CLN9-deficient and normal fibroblasts by the 4-HPR increased de novo dihydroceramide synthesis but not increase de novo synthesis or levels of This was not as 4-HPR also dihydroceramide in dihydroceramide levels in CLN9-deficient cells corrected growth and decreased apoptosis. Dihydroceramide has been to be in cell J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The data from CLN9-deficient cells suggest that dihydroceramide may be ceramide levels are resulting in of growth and apoptosis. 4-HPR is used as a A and increase in ceramide 4-HPR treatment has been described in cell and cells, cells, and as well A. R. E. L. M. A. B. Res. 2003; Google Scholar, H. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, G. E. A. L. A. T. M. R. M.B. J. 1999; PubMed Scopus Google Scholar, S. R. D. Res. 2003; 27: PubMed Scopus Google Scholar, S. C.P. Res. 2004; PubMed Scopus Google Scholar). L. C. C.P. J. PubMed Scopus Google that 4-HPR not increase ceramide in fibroblasts and cell data also suggest that 4-HPR has different effects ceramide and dihydroceramide levels of the specific cell there is from apoptosis in CLN9-deficient cells and an increase in apoptosis in normal We that dihydroceramide and ceramide levels in cells the of 4-HPR apoptosis. Dihydroceramide levels in CLN9-deficient cells are increased by treatment with 4-HPR, but ceramide levels are ceramide but not dihydroceramide levels. of the results in from apoptosis. with both 4-HPR and increased apoptosis in CLN9-deficient cells. This is most due to a increase of dihydroceramide and ceramide levels. 4-HPR can cause both apoptosis and and cell in ceramide C.P. J. 1999; PubMed Scopus (255) Google Scholar). P.E. M. B. M. J. 2004; PubMed Scopus Google that of acid but not dihydroceramide apoptosis in cells. with both 4-HPR and an inhibitor of acid not the of 4-HPR apoptosis in CLN9-deficient fibroblasts not are two 4-HPR CLN9-deficient cells from and this in the setting of an increase in dihydroceramide and a in ceramide levels. PDMP, ceramide levels but has no dihydroceramide levels, also these cells from apoptosis. This can be in a of ceramide is necessary for the of cells, apoptosis is ceramide levels are dihydroceramide of the of ceramide, and the of ceramide and dihydroceramide or in levels of sphingolipid can explain these The of low dihydroceramide levels and of the CLN9-deficient phenotype by human that CLN9 could be a human Lag1 homolog or a regulator of dihydroceramide synthase Lag1 homolog levels of expression of human genes by reverse in dihydroceramide levels, and a in LASS1 expression in to 4-HPR, that CLN9 could be an activator of dihydroceramide synthase the in CLN9 in of a of as in a of a gene or a CLN9 resulting in binding of the inhibitor to dihydroceramide synthase may suggest that CLN9 or CLN9 could be an inhibitor of dihydroceramide synthase activity at the protein This also has and of dihydroceramide levels by can be developed as a for of new CLN9-deficient the 4-HPR, a dihydroceramide synthase activator, may also have as a treatment for CLN9-deficient patients. We the for in this