Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects

Abstract

Gangliosides are a family of sialic acid-containing glycosphingolipids highly enriched in the mammalian nervous system. Although they are the major sialoglycoconjugates in the brain, their neurobiological functions remain poorly defined. By disrupting the gene for a key enzyme in complex ganglioside biosynthesis (GM2/GD2 synthase; EC 2.4.1.92) we generated mice that express only simple gangliosides (GM3/GD3) and examined their central and peripheral nervous systems. The complex ganglioside knockout mice display decreased central myelination, axonal degeneration in both the central and peripheral nervous systems, and demyelination in peripheral nerves. The pathological features of their nervous system closely resemble those reported in mice with a disrupted gene for myelin-associated glycoprotein (MAG), a myelin receptor that binds to complex brain gangliosides in vitro. Furthermore, GM2/GD2 synthase knockout mice have reduced MAG expression in the central nervous system. These results indicate that complex gangliosides function in central myelination and maintaining the integrity of axons and myelin. They also support the theory that complex gangliosides are endogenous ligands for MAG. The data extend and clarify prior observations on a similar mouse model, which reported only subtle conduction defects in their nervous system [Takamiya, K., Yamamoto, A., Furukawa, K., Yamashiro, S., Shin, M., Okada, M., Fukumoto, S., Haraguchi, M., Takeda, N., Fujimura, K., et al. (1996) Proc. Natl. Acad. Sci. USA 93, 10662-10667].

Figure 1

Targeted disruption of the GalNAcT locus and brain glycosphingolipid analysis of offspring. (A) GalNAcT targeting vector (Top), GalNAcT locus (Middle), and predicted homologously recombined locus (Bottom). (B) Biosynthetic pathways for major brain gangliosides. The biosynthetic relationships between major brain gangliosides and their precursors is shown schematically, along with the ganglioside nomenclature of Svennerholm (1). The block in ganglioside biosynthesis because of disruption at the GalNAcT locus is indicated by a double line. GalNAcT^–/– mice lack all major brain gangliosides, expressing instead a corresponding increase in GM3 and GD3 (17).

Figure 2

Pathological features in sciatic nerves of GalNAcT^−/− mice. (A) Toluidine blue-stained 1-μm epon section showing several myelinated fibers undergoing axonal degeneration (arrowheads) and a myelinated fiber surrounded by supernumerary Schwann cell processes (arrow). (Bar = 5 μm.) (B) Low-power EM image showing myelin figures and collapsed myelin at different stages of degeneration (arrowheads) and a thinly myelinated fiber surrounded by supernumerary Schwann cell processes (arrow) (Bar = 2.5 μm.) (C) EM image showing a macrophage containing myelin debris (arrow) and a minor onion bulb around a myelinated fiber (arrowheads point to Schwann cell processes). An endoneurial fibroblast also is apparent (∗) (Bar = 1 μm.) (D) EM image showing a thinly myelinated fiber and an axonal sprout (∗) invested by a Schwann cell (Bar = 200 nm). Mice were 12–16 weeks of age at the time nerves were harvested.

Figure 3

Pathological features in optic nerves of GalNAcT^−/− mice. EM image demonstrating numerous unmyelinated axons (*) of varying caliber (A) (Bar = 1 μm), a pocket of relatively large-caliber unmyelinated axons (B) (∗) (Bar = 1 μm), axonal degeneration and resultant myelin figures (C) (a number of unmyelinated axons also can be seen; Bar = 200 nm), and a doubly myelinated axon (arrow) with cytoplasm between the two compact myelin sheaths (D) (Bar = 200 nm). Mice were 12–16 weeks of age at the time nerves were harvested.

Figure 4

Morphometric analyses of sciatic and optic nerves. (A) Axonal diameters of myelinated fibers in sciatic nerves from GalNAcT^+/− (control, gray line) and GalNAcT^−/− (black line) mice. (B) Neurofilament nearest-neighbor distances in sciatic nerve axons from GalNAcT^−/− (black line) and control (gray line) mice. (C) Histogram of the diameters of unmyelinated axons in optic nerves from GalNAcT^−/− (black line) and control (gray line) mice. (D) Myelination threshold (percentage of total fibers remaining unmyelinated for each 0.1-μm increment in axonal diameter) in optic nerves from GalNAcT^−/− (black line) and control (gray line) mice. Mice were 12–16 weeks of age at the time nerves were harvested.

Figure 5

Expression of MAG in GalNAcT^−/− and control mice. Equivalent amounts of myelin from wild-type (+/+), GalNAcT^+/−, and GalNAcT^−/− mice (1.3 μg protein for immunoblotting or 5 μg protein for Coomassie staining) were subjected to SDS/PAGE (28). The positions of molecular mass standards are indicated in kDa. (A) Immunodetection of MAG. After resolution on a 10% polyacrylamide gel, proteins were transferred to a poly(vinylidene difluoride) membrane by using a semidry transfer apparatus. MAG, which migrates at 100 kDa, was detected by incubation of the blot with GenS3 mAb followed by an alkaline phosphatase-conjugated secondary antibody. Antibody binding was detected by development with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. (B) Staining of major myelin proteins. After resolution on an 8–16% polyacrylamide gradient gel, proteins were detected by using Coomassie brilliant blue stain. The major myelin proteins myelin basic protein (MBP) and proteolipid protein (PLP) are indicated. Mice were 15–23 weeks of age when brain tissue was collected for analysis.