b-series Ganglioside deficiency exhibits no definite changes in the neurogenesis and the sensitivity to Fas-mediated apoptosis but impairs regeneration of the lesioned hypoglossal nerve.

The polymorphic carbohydrate structures of gangliosides play regulatory roles. In particular, b-series gangliosides, all of which contain alpha-2,8 sialic acids, have been considered to be critical in various biological events such as adhesion, toxin binding, neurite extension, cell growth, and apoptosis. To clarify the physiological functions of b-series gangliosides in vivo, we have established a gene knockout mouse of GD3 synthase. Although all b-series structures were deleted in the mutant mice, they showed an almost complete nervous tissue morphology with no apparent abnormal behavior. Moreover, no differences in Fas-mediated apoptotic reaction of lymphocytes between wild type and the mutant mice were detected. However, the mutant mice exhibited clearly reduced regeneration of axotomized hypoglossal nerves compared with the wild type, suggesting that b-series gangliosides are more important in the repair rather than in the differentiation of the nervous system and apoptotic process induced via Fas.

To clarify the physiological functions of these b-series gangliosides in vivo, we have established a gene knockout mice line for ␣-2,8-sialyltransferase (9), which is responsible for the generation of all b-series gangliosides as well as c-series gangliosides. As expected, all b-series structures were deleted in the mutant mice, and a-series species were slightly increased instead. However, these b-series ganglioside-lacking mice showed an almost intact morphology of the brain and other nervous tissues, and no clear abnormal behaviors were detectable during the early period of life. Despite that GD3 has been considered to mediate Fas-induced apoptosis in lymphoid cells (8,10), no differences in the apoptotic reaction of lymphocytes between wild-type and mutant mice were detected. However, the mutant mice exhibited clearly reduced regeneration of axotomized hypoglossal nerves compared with the wild type, suggesting that b-series gangliosides are important in the repair of damaged nerves rather than in the differentiation of the nervous system.

EXPERIMENTAL PROCEDURES
Generation of GD3 Synthase Gene Knockout Mice-The chromosomal GD3 synthase gene was isolated from the gt11 phage library using GD3 synthase cDNA (pD3T-31) and mapped as described previously (11). To distinguish the true GD3 synthase gene from pseudo-genes, in situ hybridization was performed, and the identity was confirmed based on the correspondence of the gene assignment between humans and mice. The neo r gene was inserted between the BalI and AccI sites in exon 1 of the gene, and a 9.5-kb gene fragment was used as a targeting vector as shown in Fig. 1A. The diphtheria toxin A gene was attached to eliminate nonhomologous recombinants as described previously (12). Homologous recombination was confirmed by Southern blotting using a probe as shown in Fig. 1, generating 4.5-and 2.8-kb fragments by BamHI and 4.6-and 3.1-kb fragments by HindIII digestion in the wild-type and the recombinant allele, respectively (Fig. 1A).
Preparation of Thymocytes and Spleen Cells-The thymus and spleen were dispersed using syringe needles, and the cell number was counted using a hemocytometer. Cells were used for flow cytometry and proliferation and apoptosis assays. Spleen cells were used after elimination of adhesive cells with plastic dishes.
Flow Cytometry-Ganglioside expression was analyzed by flow cytometry with monoclonal antibodies (mAbs) for individual structures as described previously (14). T cell subsets were analyzed by fluorescein isothiocyanate-conjugated anti-CD4 mAb and phycoerythrin-conjugated anti-CD8 mAb as described previously (14). Cell proliferation was * This study was supported by Grants-in-aid for the Center of Excellence Research (10CE2006) and Scientific Research of Priority Areas (13216047, 13218060, and 13470021) from the Ministry of Education, Science, Sports and Culture of Japan and by a grant-in-aid from the Mizutani Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Apoptosis Induction Assay-Apoptosis induction was performed by adding anti-Fas mAb Jo2 (1-1,000 ng/ml) to the culture medium of thymocytes or spleen cells treated with cycloheximide (30 g/ml) as described by Ogasawara et al. (15). The apoptotic cell population was evaluated by flow cytometry after propidium iodide staining.
Hypoglossal Nerve Regeneration Assay-Regeneration of axotomized hypoglossal nerves was examined as described by Itoh et al. (16). Briefly, mice were anesthetized by sodium pentobarbital, and the right hypoglossal nerve was cleaved. Ten weeks after treatment, 20 ml of 30% HRP (Toyobo, Osaka) in saline was injected into the tongue and perfused intracardially with heparin-Na/saline and then fixed with 10% formalin in 0.1 M phosphate buffer. The lower brain stem containing the hypoglossal nerve nuclei was dissected, and 50-m serial cross-sections were prepared and then stained by adding 3,3Ј-diaminobenzidine and H 2 O 2 .

RESULTS AND DISCUSSION
The established GD3 synthase gene knockout mice (Fig. 1C) exhibited changes in the ganglioside components as expected from biochemical enzyme analyses as shown in Fig. 1B (17). Namely, all b-series gangliosides including GD3, GD2, GD1b, GT1b, and GQ1b were deleted, and a-series gangliosides such as GM1, GD1a, and GM2 were accumulated (Fig. 1Ea). In the enzyme assay with membrane fractions of brain tissues, GD3 synthase activity completely disappeared in the mutant mice, whereas GM3 synthase and GD1a synthase activity were re-tained ( Fig. 1D, a and b). Heterozygotes showed approximately half the level of GD3 synthase activity and the corresponding profiles of gangliosides. It was also confirmed that neither GD3 synthase activity nor b-series gangliosides were detectable in the mutant embryonal brains throughout development (E10 -E18) (Fig. 1, Dc and Eb), indicating that there was no different isoform of GD3 synthase expressed in early development. Brains from newborn homozygotes were also negative for the enzyme activity and its products (data not shown). No changes in the neutral glycolipid components were detected (data not shown).
b-series ganglioside-lacking mice were born and grew up without gross abnormal behavior. They showed normal hindlimb reflex, postural changes, spontaneous motor activity, swimming ability, flinch hearing, and no sign of ataxia. Current analyses for the activity of memory and learning such as the Morris water-maze test, passive avoidance test, Y-maze test, etc. also showed no abnormal findings in the mutants (data not shown). In macroscopic and histological analysis of brain tissues, the mutant mice had almost normal size, shape, and weight of brains being indistinguishable from those of wild type. The following sites were carefully examined and exhibited almost normal morphology: cortices, striatum, callosum, hippocampus, ventricles, choroid plexus, cerebellum, and brain stem. The retina also showed no abnormalities in the laminar

b-series Gangliosides Function in Repair of Nervous System
architecture and number of neurons, although it was one of the sites where the GD3 synthase gene was most strongly expressed (18).
The majority of b-series gangliosides deleted in the mutant mice were also deleted in GM2/GD2 synthase knockout mice (19) except for GD3. Therefore, the phenotypes of the mutant mice might be similar to those of complex ganglioside-lacking mice. The findings described above indicated that b-series gangliosides including GD3 are not indispensable for the morphogenesis of nerve tissues as previously described (20), and probably the remaining a-series gangliosides (and asialo-series) substitute for the roles of b-series structures.
A number of studies have been performed to investigate the involvement of gangliosides in the regulation of the immune system (14,21,22). Although GD1b expression was completely deleted and GM1 expression was moderately increased in mutant thymocytes ( Fig. 2A) and splenocytes (data not shown), the thymus and spleen were normally formed, and no changes were detected in size, shape, and cell number in the mutants (Fig.  2C). The ratio of CD8 ϩ thymocytes was reduced (Fig. 2B), and the proliferative response to various stimulations in the mutant were generally increased (concanavalin A, anti-CD3) or not changed (interleukin-2) (data not shown), although the mechanisms remain to be clarified. Recently it was reported that Fas-induced apoptosis of leukocytes was mediated via GD3 (10). To analyze the resistance of b-series gangliosidelacking mice to Fas-induced apoptosis, thymocytes from the wild-type and the mutant mice were used for the induction of apoptosis. However, no reduction in the sensitivity to the Fasinduced apoptosis could be detected (Fig. 3). In the presence of cycloheximide, ϳ50% of thymocytes from the wild type underwent apoptotic cell death at 12 h of incubation with Jo2 (10 ng/ml). Roughly 55% of those from the mutant also died approximately at the same time point. The sensitivity to apoptosis with various concentrations of Jo2 was also very similar between the two genetic types (Fig. 3A). The intensities of fragmented DNA and the formation of sub-G 1 in the DNA histogram during the induced apoptosis were also very similar (Fig. 3, B and C). Thus, deletion of b-series gangliosides did not alter the sensitivity of thymocytes to Fas-induced apoptosis, although DeMaria et al. (10) reported the significant role of GD3 as a mediator of apoptosis.
We have established an experimental system to analyze the regeneration activity of damaged hypoglossal nerves (16) and reported that exogenous gangliosides were very effective for the promotion of regeneration. In the case of GD3 synthase gene FIG. 2. Immune tissues in GD3 synthase gene knockout mice. A, ganglioside expression in the mutants. Thymocytes and spleen cells were analyzed for ganglioside expression with mAbs as described under "Experimental Procedures." GD1b disappeared and GM1 slightly increased in the mutant thymocytes and spleen cells (data not shown). B, the ratio of CD4 ϩ /CD8 ϩ cells in the thymus and spleen cells was examined by two-dimensional flow cytometry. C, total numbers of thymus and spleen cells were counted, and the ratio of single positive T cells (CD8 ϩ /CD4 ϩ ) were compared between ϩ/ϩ and Ϫ/Ϫ. The results from five mice each were presented as mean Ϯ S.D. * represents p Ͻ 0.005. b-series Gangliosides Function in Repair of Nervous System 1635 knockout mice, regeneration levels were definitely reduced compared with the wild type, i.e. by ϳ45% (Fig. 4, A and C). The surviving neuron number was also reduced to ϳ50% (Fig. 4B), suggesting that b-series gangliosides are essentially needed for the protection of neuronal death and repair of damaged hypoglossal nerves. In the axotomized rat, exogenously added bseries gangliosides, i.e. GT1b and GD1b, were most effective in the regeneration (23). The present findings shown here appear to be in good agreement with those of the previous rat experiment (23). Furthermore, GM2/GD2 synthase gene transgenic mice in which b-series gangliosides were reduced also showed similar levels of reduction in the surviving neuron number and the regeneration activity (data not shown). Thus, endogenously generated b-series gangliosides turned out to be critical in the repair of damaged neural tissues in vivo, while their roles in the neurogenesis or apoptosis were not clearly elucidated. FIG. 4. Reduced regeneration of the axotomized hypoglossal nerves in the mutant mice. A, HRP-stained patterns of the hypoglossal nerve nuclei in the wild-type (ϩ/ϩ), heterozygote (ϩ/Ϫ), and homozygote mice (Ϫ/Ϫ) at 10 weeks after the nerve resection. Regeneration of the axotomized nerves was examined by staining the sections with 3,3Ј-diaminobenzidine as described under "Experimental Procedures." B, numbers of surviving neurons 10 weeks after the operation were counted and plotted. C, numbers of HRP-positive neurons were counted and plotted. B and C show the mean Ϯ S.D. (n is as indicated). * represents p Ͻ 0.005.