Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth

Abstract

Sialic acid-containing glycosphingolipids, i.e., gangliosides, constitute a major component of neuronal cells and are thought to be essential for brain function. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyzes the initial step of glycosphingolipid (GSL) biosynthesis. To gain insight into the role of GSLs in brain development and function, a cell-specific disruption of Ugcg was performed as indicated by the absence of virtually all glucosylceramide-based GSLs. Shortly after birth, mice showed dysfunction of cerebellum and peripheral nerves, associated with structural defects. Axon branching of Purkinje cells was significantly reduced. In primary cultures of neurons, dendritic complexity was clearly diminished, and pruning occurred early. Myelin sheaths of peripheral nerves were broadened and focally severely disorganized. GSL deficiency also led to a down-regulation of gene expression sets involved in brain development and homeostasis. Mice died approximately 3 weeks after birth. These results imply that GSLs are essential for brain maturation.

Fig. 1.

Major GSL pathways and cloning strategy for Ugcg gene deletion and genotyping. (A) Glucosylceramide (GlcCer)-based GSLs, including gangliosides, expected to be absent after disruption of the Ugcg gene in the brain. (B) Galactosylceramide-based GSLs will not be deficient. (C) Cloning strategy for the disruption of the Ugcg gene. (D–G) Genotyping of mutant ES cells and mice by Southern analysis and PCR. (D)5′ targeted stem cells as shown by PCR were indicated by the appearance of a 1.7-kb fragment. (E) The integration of the 3′ single loxP site was also shown by PCR. (F and G) The correctness of Ugcg^flox/+ mice was verified by Southern blot resulting in 5′ hybridization to a 2.0-kb Nde I fragment and in a 3′ Southern blot to a 7.0-kb Spe I fragment of the “floxed” allele, respectively. (H) Neural cell-specific Ugcg^{null/flox//NesCre} mice were characterized by PCR. n, Null; f, flox; N, nestin–Cre; +, wild type.

Fig. 2.

Sphingolipid and Ugcg mRNA analysis. (A and B) Acidic (A) and neutral (B) GSLs separated by TLC and stained as described in ref. . Deletion of gangliosides was found in Ugcg^{null/flox//NesCre} mice only (A arrow). GSL amounts corresponded to 0.5 mg of dry brain and 3.0 mg of dry liver/kidney tissue. St, standards from BB, bovine brain; HS, human spleen. (C–F) Quantitation of sphingolipids by densitometry (C) and electrospray ionization/tandem mass spectrometry (D–F). (G–J) mRNA levels in mutant animals: brain (G), liver (H), kidney (I), and heart (J). Results are presented as mean ± SEM. **, P < 0.001; ***, P < 0.0001. Amount of Ugcg mRNA of Ugcg^{null/flox//+/+} and Ugcg^{null/flox//NesCre} mice in H–J was ≈50% of Ugcg^flox/+//+/+ animals. Solely mRNA from brain of Ugcg^{null/flox//NesCre} mice was almost completely erased (G). n, Null; f, flox; N, nestin–Cre; +, wild type.

Fig. 3.

Body weight, survival, and flight reflex. (A) With advancing age, the bodyweight of Ugcg^{null/flox//NesCre} mice lagged increasingly behind that of their control littermates. (B) Survival rate of Ugcg^{null/flox//NesCre} mice. (C) When hung by their tails, Ugcg^{null/flox//NesCre} mice brought their hind limbs to midline, whereas control animals splayed them widely.

Fig. 4.

Brain morphology. (A–D) Sagittal view of a Ugcg^{null/flox//+/+} (control) (A and C) and Ugcg^{null/flox//NesCre} brain (B and D) at P15 with similar morphology. (A–D) Hematoxylin/eosin staining of cerebellum, brainstem, and part of the cortex (A and B); hippocampus (C and D). (E and F) Glial fibrillary acidic protein immunohistochemistry showed no significant differences between controls (E) and the Ugcg-deficient animals (F) at P15. igl, internal granular layer; ml, molecular layer of the cerebellum. (G–J) Anti-calbindin staining of the Purkinje cell layer. Dendritic tree of Purkinje cells is generally thinner and less complex in Ugcg^{null/flox//NesCre} mice (H and J) as compared with the controls (G and I). (Scale bars: 100 μm.) (K and L) Apoptotic cells verified by TUNEL assay, in cerebellum (K) and cortex (L). Solely in cerebellum of Ugcg-deficient mice (n/f//N), a slight statistical difference was observed at P10.

Fig. 5.

Electron micrographs of peripheral nerves showed degenerations in axons and myelin. (A–D) Relative percentage distribution of areas from femoral nerve axons (n = 419; A and B) and myelin (C and D). Ugcg^{null/flox//NesCre} mice (A and C) and controls (B and D) at P10 to P15. Ugcg^{null/flox//NesCre} mice showed an increased number of hypertrophic nerve axons (A) with larger myelin areas (C) as compared with their control littermates (B and D), with respective significant differences P < 0.0001; Wilcoxon test. Controls (E–G) showed thinner myelin sheaths than Ugcg^{null/flox//NesCre} mice (H–J). Some of the investigated Ugcg^{null/flox//NesCre} animals showed pronounced degenerations of nerve axons and myelin sheaths with extensive splitting of the broad myelin sheath (I and J).

Fig. 6.

Primary cultured neuronal cells have restricted potential to form dendritic extensions. Neurite length and axonal length from 30–40 representative neuronal cells isolated from hippocampus of Ugcg^{null/flox//NesCre} embryos and controls at E15.5 (n = 3 for each) were determined and calculated with photoshop (Adobe Systems, San Jose, CA) at culture-day 6. Numbers of dendritic and axonal branches were counted (means ± SEM). Shown are photographs (A and C) and corresponding drawings (B and D) of primary neuronal cells for calculation of dendrite length (B and D) from Ugcg^{null/flox//+/+} (A and B) and Ugcg^{null/flox//NesCre} (C and D) embryos. (E) The neurite length of cultured neuronal cells from Ugcg-deficient embryos was significantly diminished to ≈1/3 of controls (P < 0.001). Significantly decreased numbers of neuronal branching points in the Ugcg-deficient cells (F) correlated well with the reduced axonal branches (G) (P < 0.000002 and P < 0.00005, respectively). n, Null; f, flox; N, nestin–Cre; +, wild type.