Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth Richard Jennemann*†, Roger Sandhoff*, Shijun Wang*, Eva Kiss*, Norbert Gretz‡, Cecilia Zuliani§, Ana Martin-Villalba§, Richard Ja¨ ger¶, Hubert Schorle¶, Marc Kenzelmann*, Mahnaz Bonrouhi*, Herbert Wiegandt*, and Hermann-Josef Gro¨ ne*† Abteilungen *Zellula¨re und Molekulare Pathologie and §Immungenetik, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; ‡Zentrum fu¨r Medizinische Forschung, Ruprecht-Karls-Universita¨t Heidelberg, Theodor-Kutzer-Ufer, 68167 Mannheim, Germany; and ¶Abteilung Entwicklungspathologie, Institut fu¨r Pathologie, Universita¨tsklinikum Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany Edited by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, and approved July 11, 2005 (received for review February 3, 2005) Sialic acid-containing glycosphingolipids, i.e., gangliosides, consti- resulting neural cell-specific disruption of the formation of gluco- tute a major component of neuronal cells and are thought to be cerebroside-derived GSLs did not impair late embryonic develop- essential for brain function. UDP-glucose:ceramide glucosyltrans- ment. However, all Ugcg-deficient mice died between postnatal day ferase (Ugcg) catalyzes the initial step of glycosphingolipid (GSL) 11 (P11) and P24. Our results indicate that GSLs are required for biosynthesis. To gain insight into the role of GSLs in brain devel- brain maturation after birth. opment and function, a cell-specific disruption of Ugcg was per- formed as indicated by the absence of virtually all glucosylceramide- Materials and Methods based GSLs. Shortly after birth, mice showed dysfunction of Construction of the Targeting Vector. An Ϸ8-kb Sst I͞Sal I DNA cerebellum and peripheral nerves, associated with structural de- fragment of the Ugcg gene (National Center for Biotechnology fects. Axon branching of Purkinje cells was significantly reduced. In Information accession no. NP 035803), containing exon 5 to exon primary cultures of neurons, dendritic complexity was clearly Ј diminished, and pruning occurred early. Myelin sheaths of periph- 9 and a part of the 3 ORF, was cloned into pBluescript eral nerves were broadened and focally severely disorganized. GSL (Stratagene). A HSVtk–PGKneo selection cassette, which was Ј deficiency also led to a down-regulation of gene expression sets flanked by FLP recombination target (FRT) sites and one 5 involved in brain development and homeostasis. Mice died Ϸ3 single loxP site, was inserted into a Nhe I site between exons 5 Ј weeks after birth. These results imply that GSLs are essential for and 6 (Fig. 1C). A diagnostic Nde I site was introduced at the 5 brain maturation. end of the HSVtk–PGKneo cassette. A second loxP site and a preceding diagnostic Spe I site were inserted into Eco 47 III glycosphingolipid ͉ ceramide ͉ neuron differentiation ͉ myelin between exons 8 and 9 (Fig. 1C). E14 ES cells were transfected in the presence of the linearized lycosphingolipids (GSLs)ʈ with their lipophilic ceramide Ugcg-targeting vector and cultivated as described in ref. 12. For Glinked to a great variety of complex carbohydrate residues genotyping, see Fig. 1 D and E and Supporting Methods, which is are typical amphipathic membrane constituents of eukaryotic published as supporting information on the PNAS web site. cells. A major portion of the GSLs is located on the outer leaflet of the cellular plasma membrane where they may act in the Generation of Germ-Line Chimeras, as Well as Null and Floxed Mice. assembly of signaling molecules (2, 3), modulation of cell Positive ES cells were injected into blastocysts from C57BL͞6 mice. adhesion (4), and differentiation (5). Intracellularly, GSLs may Mutant mice were characterized by Southern and PCR analysis of be important for protein trafficking (6). genomic DNA of tail biopsies as described in Supporting Methods. BIOLOGY All mammalian GSLs are synthesized either on the cytoplas- Ugcg-null and -floxed mice were generated by breeding Ugcgflox-tkneo DEVELOPMENTAL mic surface of the Golgi complex by enzyme-catalyzed addition mice with cre- (13) and FLP-deleter mice (14) (Fig. 1 F–H). of UDP-activated glucose to ceramide, or on the luminal side of Neural cell-specific Ugcg-deficient animals were generated by the endoplasmic reticulum with UDP-galactose (7) (Fig. 1 A and breeding mice expressing Cre recombinase under control of B). The resulting gluco- or galactocerebrosides may be further the nestin gene promoter (11) with heterozygous Ugcgnull/ϩmice. substituted by enzyme-catalyzed addition of monosaccharides at The resulting Ugcgnull/ϩ//NesCre mice were further mated with the luminal side of the Golgi complex. Acidic GSLs, i.e., Ugcgflox/flox mice, resulting in Ugcgnull/flox//NesCre mice. sulfatides and gangliosides, are formed by addition of sulfate or neuraminic acid residues, respectively. Whereas only compara- tively few GSLs are derived from galactocerebroside, hundreds Isolation of GSLs and Electrospray Ionization͞Tandem Mass Spectrom- of structurally different GSLs, including higher sulfatides etry (ESI-MS͞MS). GSLs were isolated (15) and prepared for ESI and gangliosides, are known to contain the glucosylceramide MS͞MS quantifications as described in ref. 16. The (GlcCer) core, emphasizing the potential physiological impor- Ugcgnull/flox//NesCre mice and controls investigated were E15.5 tance of this precursor molecule (Fig. 1A). (n ϭ 2), P0 (n ϭ 2), P5 (n ϭ 3), P10 (n ϭ 5), and P15 (n ϭ 5). The mouse GlcCer synthase (Ugcg; UDP-glucose:ceramide glu- cosyltransferase, EC 2.4.1.80) gene was initially cloned by Hiraba- yashi et al. (8). It was shown that disruption of the Ugcg gene in mice This paper was submitted directly (Track II) to the PNAS office. resulted in embryonic lethality during gastrulation at embryonic Abbreviations: GSL, glycosphingolipid; Ugcg, UDP-glucose:ceramide glucosyltransferase; GlcCer, glucosylceramide; En, embryonic day n;Pn, postnatal day n; FRT, FLP recombination day 6.5 (E6.5) to E7.5 (9). To obtain more insight into the target; GalNAcT, N-acetylgalactosaminyltransferase. organ-specific function of the Ugcg gene, a cell-specific functional †To whom correspondence may be addressed. E-mail: [email protected] or deletion was performed. Central nervous tissue contains compa- [email protected]. rably high concentrations of GSLs, specifically gangliosides (10). In ʈGlycosphingolipids are abbreviated according to the recommendations of the Interna- view of the assumed importance of GSLs in the brain, mice were tional Union of Pure and Applied Chemistry–International Union of Biochemistry Joint generated that carry the nestin gene promoter-driven cre transgene Commission on Biochemical Nomenclature of Glycolipids (1). (11) in combination with an Ugcg-null and ‘‘floxed’’ allele. The © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500893102 PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12459–12464 Downloaded by guest on September 26, 2021 In Vitro Culture of Neurons. Cells from Ugcgnull/flox//NesCre embryos and Ugcgnull/flox//ϩ/ϩ controls were isolated from hippocampus at E15.5 and cultivated for 6 days (for further details, see Supporting Methods). The neurite and axonal lengths of single cells were determined as in ref. 18. Axonal and neuronal branching points were counted. Physiological Tests. A righting test was performed according to Ronca and Alberts (19). A test for the ability to maintain position on a rotating rod (Rotorod, SmartRod, AccuScan Instruments, Columbus, OH) was performed at a permanent speed of 2 cm͞s and the latency to fall was recorded three times per animal. Results Ugcgnull/flox//NesCre Mice Lack GlcCer-Based GSLs in the Brain. To verify the disruption of the Ugcg gene, brain tissue and spinal cord were analyzed to confirm the absence of these components from E15.5. All GlcCer-based gangliosides were virtually absent from brains of Ugcg-deficient mice (Fig. 2 A and C) and were already deleted at E15.5 (Fig. 7A, which is published as supporting information of the PNAS web site). Traces of GSLs (3–4% of the content in control mice) were detected, possibly because of failed deletion of the floxed Ugcg allele in cells with low Cre recombinase activity. Gangliosides were shown to be distributed throughout different brain regions at P10 (Fig. 7B). In the neutral GSL brain fraction of littermates, no significant differences could be observed (Fig. 2B). The total content of hexosylceramide (HexCer) in brain of Ugcgnull/flox//NesCre mice was similar compared with control littermates (Fig. 2D). HexCer was shown to be GalCer only, by TLC (Fig. 8, which is published as supporting information on the PNAS web site). Neutral, as well as acidic GSL fractions isolated from liver and kidney of the Ugcgnull/flox//NesCre and control littermates displayed qualitatively comparable lipid compositions (Fig. 2 A and B). Ceramide concentration in brains of Ugcgnull/flox//NesCre mice decreased from 1.7-fold at P0 to similar or lower levels at P15 as Fig. 1. Major GSL pathways and cloning strategy for Ugcg gene deletion and compared with controls (Fig. 2E). In contrast, the sphingomyelin genotyping. (A) Glucosylceramide (GlcCer)-based GSLs, including ganglio- sides, expected to be absent after disruption of the Ugcg gene in the brain. (B) content increased consistently in Ugcg-deficient mice after birth Galactosylceramide-based GSLs will not be deficient. (C) Cloning strategy for (Fig. 2F). The molar increase in sphingomyelin was approximately the disruption of the Ugcg gene. (D–G) Genotyping of mutant ES cells and mice equivalent to the loss of gangliosides (compare Fig. 2 C and F). No by Southern analysis and PCR. (D)5Ј targeted stem cells as shown by PCR were significant differences in the phosphatidylcholine levels could be indicated by the appearance of a 1.7-kb fragment. (E) The integration of the determined between Ugcgnull/flox//NesCre mice and controls (data not 3Ј single loxP site was also shown by PCR. (F and G) The correctness of Ugcgflox/ϩ shown).