N-glycan branching requirement in neuronal and postnatal viability

Zhengyi Ye and Jamey D. Marth1

Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, Glycobiology Research and Training Center, 9500 Gilman Drive-0625, University of California San Diego, La Jolla, CA 92093

Received on January 9, 2004; revised on February 26, 2004; accepted on February 26, 2004


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The structural variations among extracellular N-glycans reflect the activity of glycosyltransferases and glycosidases that operate in the Golgi apparatus. More than other types of vertebrate glycans, N-glycans are highly branched oligosaccharides with multiple antennae linked to an underlying mannose core structure. The branching patterns of N-glycans consist of three types, termed high-mannose, hybrid, and complex. Though most extracellular mammalian N-glycans are of the complex type, some cells variably express hybrid and high-mannose forms. Nevertheless, a requirement for hybrid and complex N-glycan branching exists in embryonic development and postnatal function among mice and humans inheriting defective Mgat1 or Mgat2 alleles. The resulting defects in formation N-glycan branching patterns cause multiple abnormalities, including neurologic defects, and have inferred the presence of distinct functions for hybrid and complex N-glycan branches among different cell lineages. We have further explored N-glycan structure-function relationships in vivo by using Cre-loxP conditional mutagenesis to abolish hybrid and complex N-glycan branching specifically among neuronal cells. Our findings show that hybrid N-glycan branching is an essential posttranslational modification among neurons. Loss of Mgat1 resulted in a unique pattern of neuronal glycoprotein deficiency concurrent with caspase 3 activation and apoptosis. Such animals exhibited severe locomotor deficits, tremors, paralysis, and early postnatal death. Unexpectedly, neuronal Mgat2 deletion resulting in the loss of complex but not hybrid N-glycan branching was well tolerated without phenotypic markers of neuronal or locomotor dysfunction. Structural features associated with hybrid N-glycan branching comprise a requisite posttranslational modification to neuronal glycoproteins that permits normal cellular function and viability.

Key words: apoptosis / development / genetics / N-glycans / neurobiology


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Asparagine (N)-linked oligosaccharides (N-glycans) are among the most abundant posttranslational modifications to cellular proteins. N-glycans make up a diverse repertoire of polymeric branched structures produced in the Golgi apparatus and destined for secretion or presentation on the cell surface. There has been considerable forethought given to the likely roles of glycan variation in controlling important biologic processes (Kobata, 1992Go; Rademacher et al., 1988Go; Varki, 1993Go). These deductions have provided strong incentive for the development of approaches to investigate the physiologic functions of glycans among intact organisms. In the past decade, genetic deconstruction of glycan synthetic and diversification in vivo has been suggested as one approach to determining glycan function (Marth, 1994Go). Moreover, humans bearing various and informative genetic biosynthetic defects in N-glycan synthesis have been identified (Jaeken et al., 1991Go, 2001Go). Novel functions of mammalian glycans and additional pathways in N-glycan formation have been thereby discovered with evidence that different glycan linkages play cell type–specific roles in physiology and disease (reviewed in Lowe and Marth, 2003Go).

After N-glycan processing and protein folding in the endoplasmic reticulum, transport to the Golgi occurs, where N-glycan structures are branched and diversified prior to localization among extracellular compartments. Three subtypes of secreted and cell surface N-glycans exist and are termed high-mannose, hybrid, and complex. Their expression and relative abundance is controlled by the sequential actions of specific glycotransferases and glycosidases (Kornfeld and Kornfeld, 1985Go). The conversion of high-mannose to hybrid and complex structures is initiated by the action of the GlcNAcT-I glycosyltransferase (E.C. 2.4.1.101) encoded by the Mgat1 gene (Kumar and Stanley, 1989Go; Kumar et al., 1990Go; Sarkar et al., 1991Go). The conversion of hybrid to complex structures is controlled subsequently by the Mgat2-encoded GlcNAcT-II glycosyltransferase (E.C. 2.4.1.143; Bendiak and Schachter, 1987Go; D'Agostaro et al., 1995Go; Schachter, 1991Go).

The study of N-glycan deficiency in mice and humans have indicated multiple and essential roles for hybrid and complex branch structures. Lethality among all embryos lacking Mgat1 function, and hence both hybrid and complex N-glycans, was accompanied by several morphologic and developmental abnormalities (Ioffe and Stanley, 1994Go; Metzler et al., 1994Go). Although high-mannose N-glycans are insufficient for normal ontogeny, hybrid-type branching structures represent the minimal N-glycan repertoire needed to complete embryogenesis. Nevertheless, restricting N-glycan branching to the formation of only hybrid structures by Mgat2 inactivation results in very low frequencies of survival to adulthood and disease occurrence with a postnatal phenotype in the mouse that is similar to human CDG-IIa (Jaeken et al., 1994Go; Wang et al., 2001Go). The predominance of early lethality due to the inheritance of defective Mgat1 or Mgat2 alleles has restricted the ability to discriminate among the cellular functions of mammalian N-glycan branches. The ability to ablate glycosyltransferase expression systemically and compared with conditional mutagenesis approaches could provide information pertaining to distinct roles of different N-glycan structures among specific cell lineages (Marth, 1996Go).

Glycans in the mammalian brain have been previously analyzed, and results indicate the presence of unique structures as well as a greater abundance of high mannose N-glycans as compared with other tissues (Chen et al., 1998Go; Clark et al., 1998Go; Krusius and Finne, 1977Go; Zamze et al., 1998Go). It seemed possible that neurons may be less reliant than other cell types on the formation of hybrid and complex N-glycans. We pursued this possibility by eliminating Mgat1 and Mgat2 genes specifically in neuronal cell types using Cre-loxP mutagenesis. Our data reveal that hybrid N-glycans are essential for neuronal and postnatal viability in mice, whereas complex N-glycans appear dispensable in this cell lineage.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Mgat1 gene targeting for conditional mutagenesis
The Mgat1 gene exists as a single protein-encoding exon and was previously disrupted in the mouse germ line by conventional gene-targeting approaches resulting in lethality during embryonic day 9 of development (Ioffe and Stanley, 1994Go; Metzler et al., 1994Go; Pownall et al., 1992Go). In this study, genomic DNA encoding the mouse Mgat1 gene was used to construct a targeting vector in which conditional mutagenesis could be achieved (Figure 1A). Targeted ES cell clones bearing the Mgat1F[tkneo] allele were confirmed by genomic Southern blotting (Figure 1B). Transient Cre expression followed by selection by ganciclovir resistance was then used to isolate ES cell subclones bearing either the loxP-flanked Mgat1 allele (Mgat1F) or the deleted null allele (Mgat1{Delta}). These allelic structures were further characterized by genomic Southern blotting using the loxP probe (Figure 1C). Chimeric mice generated from ES clone 7.1.2T2 were bred in producing mice heterozygous and homozygous for the Mgat1F alllele. All analyses of mutant animals were accomplished with littermate controls and the Syn1-Cre transgene by itself did not alter mouse development or viability.



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Fig. 1. Mgat1 mutagenesis. (A) A mouse genomic DNA clone containing the Mgat1 gene was isolated and used in constructing the Mgat1 targeting vector with use of pflox as indicated. Homologous recombination in ES cells formed the Mgat1F[tkneo] allele. Following Cre transfection and ganciclovir selection, the Mgat1{Delta} or Mgat1F alleles are produced. (B) Southern blot analysis of ES clones 7.1 and 1.5 containing Mgat1wt and Mgat1F[tkneo] alleles using a genomic probe. (C) Southern blot analysis with a loxP probe revealing Mgat1{Delta} (T1) and Mgat1F (T2) alleles in subclones used to produce chimeric mice. Restriction enzyme sites: K, Kpn1; E, EcoR1; Bg, BglII; Pf, PfIM1; X, Xho1; Xb, Xba1; B, BamH1.

 
Neurological defects of Mgat1F/F/Syn1-Cre mice
Neuronal-specific deletion of loxP-flanked DNA is achieved by breeding loxP-flanked alleles with transgenic mice bearing the Syn1-Cre transgene, as previously described (DeFalco et al., 2001Go; Zhu et al., 2001Go). The rat synapsin-1 promoter is capable of eliciting transgene expression specifically in neurons beginning at ~ embryonic day 13 and typically proceeding to encompass the majority of neurons during synaptogenesis by day 20 of postnatal development (Hoesche et al., 1993Go). The Syn1-Cre transgenic mouse was crossed with mates that were heterozygous for the Mgat1F allele. Offspring heterozygous for the Mgat1F allele and hemizygous for the Syn1-Cre transgene were unremarkable and were bred to produce offspring that were homozygous for Mgat1F allele and hemizygous for the Syn1-Cre transgene. These were studied further along with control littermates that lacked the Syn1-Cre transgene.

Newborn mice with the Mgat1F/F/Syn1-Cre genotype were obtained at normal frequency and were not distinguishable from their littermates bearing either the Mgat1F/F or Syn1-Cre genotypes. However, Mgat1F/F/Syn1-Cre mice failed to thrive, and from postnatal day 12 until death, body mass was only 50% of normal (Figure 2A, B). Virtually all Mgat1F/F/Syn1-Cre mice analyzed died within 8 weeks after birth; none lived longer than 18 weeks (Figure 2C). During their brief lifespan, Mgat1F/F/Syn1-Cre mice exhibited locomotor dysfunction and tremors and developed a widespread paralysis prior to death.



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Fig. 2. Reduced viability, growth retardation, and locomotor defects associated with Mgat1F/F mice bearing the Syn1-Cre transgene. (A) Mgat1F/F/Syn1-Cre mice (4 weeks of age shown) are runted in comparison with littermates. (B) Mgat1F/F/Syn1-Cre mice are distinguishable by reduced body weight at various postnatal ages compared with Mgat1F/F littermates. A cohort of 25 mice including littermates of indicated genotypes are presented. Mean weights and SDs are plotted. (C) Plot of Mgat1F/F/Syn1-Cre mouse viability after birth following a cohort of more than 200 Mgat1F/F/Syn1-Cre mice. Triangles represent days when viability was measured. (D) In the wire (grip) test, Mgat1F/F/Syn1-Cre mice have reduced muscular strength measured by average time able to support weight (mean and SD plotted, n = 12 mice of each genotype). (E) In the rotarod test, Mgat1F/F/Syn1-Cre mice exhibited a significant decrease in locomotor and coordination ability as they fall off the rotating rod before the speed reaching to 20 rpm, whereas all littermate controls remained on the rod at the maximal speed of 40 rpm (*). The mean and standard deviation are plotted for Mgat1F/F/Syn1-Cre mice (n = 6 of each genotype).

 
Using a test of muscular strength that involves gripping onto a wire mesh screen while being suspended, 4-week-old Mgat1F/F/Syn1-Cre mice exhibited to a significant reduction in the time they were able to hold their body weight (Figure 2D). Locomotor test of coordination involving the rotarod also revealed a significant deficit, involving the ability of Mgat1F/F/Syn1-Cre mice to remain on the rotating rod when compared to control littermates (Figure 2E). Although reduced muscle fiber size (atrophy) was observed by histologic analyses, no evidence of muscle degeneration was found (data not shown).

Mgat1 deletion with altered glycosylation in the brain and spinal cord
Brain and spinal cord morphology were not significantly altered in Mgat1F/F/Syn1-Cre mice (Figure 3A). Nevertheless, Cre recombination with production of the Mgat1{Delta} allele was found to occur specifically in these two tissues as expected (Figure 3B). The efficiency of Cre recombination was quantitatively investigated by densitometry and found to typically encompass ~20% of Mgat1 alleles. This value is close to the approximate contribution of neuronal cell types among these two neural tissues suggesting that the majority of neuronal Mgat1F alleles had undergone Cre recombination, resulting in Mgat1 deletion and thereby loss of GlcNAcT-I activity.



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Fig. 3. Neuronal Mgat1 gene deletion alters glycosylation in the brain and spinal cord. (A) Mgat1F/F/Syn1-Cre mice exhibit normal brain and spinal cord anatomical structures. (B) Cre-mediated Mgat1 deletion occurs among neuronal cells in the brain and spinal cord of Mgat1F/F/Syn1-Cre mice. (C) Expected increase in high-mannose type N-glycans in Mgat1F/F/Syn1-Cre was obtained by enhanced binding of ConA lectin. M1, M2: Mgat1F/F/Syn1-Cre mice. C1, C2: Mgat1F/F littermate controls. (D) Decreased E-PHA-FITC binding in brain hippocampal regions of Mgat1F/F/Syn1-Cre mice. CA1-3: fields of Ammon's horn, DG: dentate gyrus; LM: lacunosum molecular layer of the hippocampus, SR: stratum radiatum of the hippocampus, SL: stratum lucidum of the hippocampus, PY: pyramidal cell layer of the hippocampus, OR: oriens layer, hippocampus, ML: molecular layer of the dentate gyrus, GR: granule cell layer of the dentate gyrus, HL: hilus of dentate gyrus.

 
To detect alterations in N-glycosylation, the plant lectin Concanavalin A (ConA) has been used as a probe for mannose residues that are exposed by the absence of hybrid and complex type N-glycan branching (Campbell et al., 1995Go; Metzler et al., 1994Go). Brain and spinal cord proteins were isolated, electrophoresed, and analyzed for ConA binding. In all samples ConA binding was increased among glycoproteins derived from Mgat1F/F/Syn1-Cre mice, with the largest observed elevation involving an unidentified glycoprotein migrating at ~40–50 kDa (Figure 3C). The lectins erythro-phytohemagluttinin (E-PHA) and leuko-phytohemagluttinin (L-PHA) were also used to detect N-glycosylation changes, as binding of these to mouse glycoproteins and tissues requires hybrid and complex N-glycan branching in vivo (Chui et al., 1997Go; Wang et al., 2001Go). A significant decrease in E-PHA binding was observed among Mgat1F/F/Syn1-Cre mice in histological analyses of neuronal-enriched areas among hippocampal regions of the brain (Figure 3D) and the ventral horn of the spinal cord (data not shown). Similar results were generated using L-PHA (data not shown).

These findings indicate that neuronal Mgat1 deletion, as expected, results in an increase in mannose exposure with a deficit of hybrid and complex type N-glycans among glycoproteins of the brain and spinal cord. Although the overall glycosylation defect appears small in these brain and spinal cord analyses, the majority of hybrid and complex N- glycans are derived from the more abundant nonneuronal cell types and processes that compose these tissues, and therefore are not susceptible to Cre recombination due to absence of Syn1-Cre transgene expression.

Gliosis and neuronal apoptosis
Further histological inspection of various brain regions revealed a significant increase in pyknotic bodies concurrent with an apparent reduction in neuronal cell abundance specifically among Mgat1F/F/Syn1-Cre mice (Figure 4A). These findings were typical of mice in which the neurologic phenotype had progressed and become severe. When we examined the brain of Mgat1F/F/Syn1-Cre mice for expression of Glial Fibrillary Acidic Protein, all brain regions exhibited extensive astrogliosis, which is typically observed in the context of widespread neuronal damage and death (Figure 4B; Ridet et al., 1977Go; Zhu et al., 2001Go).



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Fig. 4. Pyknotic bodies and gliosis in the Mgat1F/F/Syn1-Cre brain. (A) In thalamus, hippocampus and cortex, neuronal cells seem reduced in abundance and pyknotic bodies are observed (cresyl violet staining). Boxed areas are magnified further to the right of each tissue panel. (B) Extensive reactive astrogliosis is evident throughout regions of the brain in Mgat1F/F/Syn1-Cre brain as visualized by glial fibrillary acidic protein antibody binding (brown). Hemotoxylin was used to counterstain nuclei.

 
Pyknotic bodies are condensed nuclei that can result from cell death by apoptosis. We performed a terminal deoxynucleotidyl transferase dUTP nicked end labeling (TUNEL) assay to detect the presence of DNA fragmentation occurring during apoptosis. In all regions where pyknosis and astrogliosis were observed, Mgat1F/F/Syn1-Cre mice exhibited a significant increase TUNEL staining (Figures 5A and B). Costaining with a neurofilament antibody and TUNEL indicated that neuronal cells were specifically labeled (Figure 5C).



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Fig. 5. Neuronal apoptosis in Mgat1F/F/Syn1-Cre mice with increased Caspase 3 activation. (A) Increased cell death by apoptosis is indicated by TUNEL in Mgat1F/F/Syn1-Cre mice. TUNEL-positive staining is correlated with areas of neuronal cell loss. (B) The pyknotic bodies revealed by cresyl violet staining match the TUNEL-positive nuclei. (C) TUNEL (green) and neurofilament (SMI32 antibody, red) coexpression indicates apoptotic cells are neurons. (D) Activation of caspase-3 (green) is observed using a cleaved caspase-3 (Asp175) antibody in brain sections of Mgat1F/F/Syn1-Cre mice. Nuclei are labeled blue (DAPI).

 
The level of caspase activation in various brain regions of Mgat1F/F/Syn1-Cre mice was investigated to determine if the observed neuronal death was due to apoptosis. Using an antibody to caspase-3 (Asp175) that only binds after caspase 3 has been cleaved and activated, immunohistological analyses revealed a significant induction in cleaved caspase 3 in the brain among all Mgat1F/F/Syn1-Cre mice analyzed (n = 15), even at ages prior to onset of the most severe aspects of the neurologic phenotype (Figure 5D and data not shown).

Altered expression and glycosylation of neuronal glycoproteins
The apoptotic death of neuronal cells due to Mgat1 deletion indicates deficits in the expression or function of one or more essential neuronal N-glycoproteins. We examined the expression of several important neuron-specific glycoproteins. Of those studied, the amyloid precursor protein contains two N-glycosylation sites. Nerve growth factor receptor (TrkA) contains 15 N-glycosylation sites. Among synaptic vesicle glycoproteins studied, SV2 contains three N-glycosylation sites, whereas synaptophysin (Syp) and synaptotagmin (Syt) contain one N-glycosylation site each. Voltage-sensitive ion channels are highly N- and O-glycosylated at many different sites with between 20–30% of channel molecular mass resulting from glycan linkages.

Unexpectedly, significant reductions in expression levels and altered electrophoretic mobility were observed among some but not all neuronal glycoproteins analyzed (Figures 6A and B). Although no significant change in abundance was found among amyloid precursor protein, the nerve growth factor receptor, synaptophysin, and synaptotagmin, we observed significantly reduced levels of expression involving synaptic vesicle protein, the voltage-gated potassium channel {alpha} subunit (Kv1.1), the voltage-gated sodium channel {alpha} subunit (SCNA1), and the voltage-gated sodium channel ß2 subunit (Naß2). In addition, electrophoretic mobility shifts were observed among TrkA, Syp, SV2, and Kv1.1 consistent with loss of mass.



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Fig. 6. Alterations in the abundance and glycosylation of specific neuronal glycoproteins in Mgat1F/F/Syn1-Cre mice. (A) The expression of key neuronal glycoproteins was studied using western blotting on total membrane protein extracts from brain and spinal cord. Decreased neuronal glycoprotein levels was noted for voltage-gated sodium channel {alpha} subunit (SCNA1) and ß2 subunit (Naß2), the voltage-gated potassium channel {alpha} subunit (Kv1.1), the synaptic vesicle protein SV2, and nerve growth factor receptor (TrkA), but not for synaptic vesicle protein synaptophysin (Syp) and synatotagmin (Syt); nor was a decrease observed in amyloid precursor protein (APP). (B) App, Syp, and Syt contained a significant increase in high-mannose N-glycans among Mgat1F/F/Syn1-Cre mice, revealed by ConA precipitation of equivalent amounts of total protein extract. M1, M2: Mgat1F/F/Syn1-Cre mice. C1, C2: Mgat1F/F littermate control mice.

 
ConA lectin binding was used in glycoprotein blotting experiments to confirm that neuronal glycoproteins were deficient in N-glycan branching. A significant increase in ConA binding was observed for App, Syp, and Syt (Figure 6C). These results indicate that hybrid and/or complex N-glycans are normally present among these glycoproteins and that Mgat1 deletion has eliminated these forms of N-glycan branching.

Neuron-specific deletion of the Mgat2 gene
Subsequent to Mgat1 function in N-glycan biosynthesis, the Mgat2-encoded GlcNAcT-II glycosyltransferase is essential for the production of complex N-glycans (Kornfeld and Kornfeld, 1985Go). Inheritance of homozygous null mutations of the Mgat2 locus result in a severe human childhood disease termed congenital deficiency of glycosylation (CDG) type IIa (Jaeken et al., 1994Go; Tan et al., 1996Go). Mice lacking a functional Mgat2 allele recapitulate the majority of CDG type IIa disease signs, including deficits in postnatal locomotor development (Wang et al., 2001Go). To assess the contribution of complex type N-glycans to neuronal function and viability, we bred mice bearing the loxP-flanked Mgat2 allele with the Syn1-Cre transgenic line.

Unlike results obtained with the identical approach involving neuronal Mgat1 deletion, Mgat2F/F/Syn1-Cre mice were not distinguishable from littermate controls (Figure 7A). Nevertheless, loxP-flanked Mgat2 gene recombination by the Syn1-Cre transgene occurred at frequencies identical to those observed among Mgat1F/F/Syn1-Cre mice, indicating that the majority of neurons were devoid of a functional Mgat2 allele (Figure 7B and data not shown). Mgat2F/F/Syn1-Cre mice also have a normal lifespan without increased mortality and do not lose body mass during postnatal development (Figures 7C and D). x



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Fig. 7. Viabilty of Mgat2F/F/Syn1-Cre mice. (A) Mgat2F/F/Syn1-Cre mice are indistinguishable from Mgat2F/F littermate controls. (B) Cre-mediated recombination and excision of the Mgat2 gene by the Syn1-Cre transgene remains specific to neuronal tissues and occurs at similar frequency as in Mgat1F/F/Syn1-Cre mice. (C) Viability of Mgat2F/F/Syn1-Cre mice is normal during postnatal development among more than 100 mice analyzed. (D) Growth during postnatal development is also normal among Mgat2F/F/Syn1-Cre mice and Mgat2F/F littermates (n > 60).

 
Brain and spinal cord extracts from Mgat2F/F/Syn1-Cre mice were analyzed for L-PHA binding, apoptosis, and neuronal glycoprotein levels. Decreased L-PHA binding was observed to involve glycoproteins of 40–50 kDa and approximately 90 kDa (Figure 8A). Lectin binding decreases observed were relatively few, likely reflecting the major contribution of nonneuronal proteins among these tissues. Furthermore, no pkynotic bodies were observed, and no apoptotic phenotype was found using TUNEL or activated caspase-3 antibody among histological samples (data not shown). In contrast to results obtained by neuron-specific Mgat1 deletion, brain and spinal cord tissues of Mgat2F/F/Syn1-Cre mice maintained normal expression of neuronal glycoproteins App, TrkA, Syp, Syt, SV2, Kv1.1, SCNA1, and Naß2 (Figures 8B and C). These findings indicate that Mgat2 function is dispensable for neuronal viability and postnatal development.



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Fig. 8. Abundance and glycosylation of specific neuronal glycoproteins in Mgat2F/F/Syn1-Cre mice. (A) Reduced L-PHA reactivity reflects the portion of total tissue extract assayed in which neuronal lineage-derived N-glycoproteins lack complex N-glycans due to loss of Mgat2 function in Mgat2F/F/Syn1-Cre mice. (B) Neuronal glycoprotein expression profiles are normal in the brain and spinal cord of Mgat2F/F/Syn1-Cre mice. M1, M2: Mgat2F/F/Syn1-Cre mice. C1, C2: Mgat2F/F littermate controls.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Although all eukaryotes use the same oligosaccharide precursor for linkage to asparagines residues on proteins in the endoplasmic reticulum, vertebrates evolved the ability to construct a diverse repertoire of N-glycans by the formation of specific branches linked to the processed mannose core. These branched oligosaccharide structures are subsequently expressed at the cell surface or secreted among extracellular compartments. The glycosyltransferase genes that are responsible for these branching steps have been identified and provide specific targets for exploring the amount and nature of physiologic information contained in the N-glycan repertoire (reviewed in Kornfeld and Kornfeld, 1985Go; Lowe and Marth, 2003Go; Schachter, 1991Go).

The mammalian Mgat1 and Mgat2 genes are highly conserved and encode pivotal glycosyltransferases in the formation of hybrid and complex N-glycans. Mgat1-encoded GlcNAcT-I must act on the high-mannose core to initiate the formation of hybrid N-glycans. Without this activity, N-glycans are limited to high-mannose types. As a result, mouse embryos cannot develop to term, and developmental disturbances occur involving vascularization, neural tube morphogenesis, and the formation of heart loop asymmetry (Ioffe and Stanley, 1994Go; Metzler et al., 1994Go). Mgat2-encoded GlcNAcT-II activity is essential for initiating the conversion of hybrid to complex type N-glycan branching; however, absence of this glycosyltransferase does not preclude embryogenesis in mouse or human species. Nevertheless, severe and very similar pathologic processes occur during postnatal development in mouse and human species lacking Mgat2 function indicating that hybrid N-glycans cannot compensate for the loss of complex N-glycans (Jaeken et al., 2001Go; Wang et al., 2001Go).

The altered morphology of neural ectodermal derivatives and the presence of neurological abnormalities by inherited systemic loss of Mgat1 and Mgat2 function, respectively, suggest hybrid and complex N-glycan branches are important among neural tissues and cell types. Neuronal cells express membrane glycoproteins, such as ion channels and synaptic molecules, that bear N-glycans and are involved in key features of development, synaptic function, and cell–cell recognition. The N-glycan repertoires of the mammalian brain and spinal cord have been studied, and neural tissues appear to have unique structures with a larger proportion of high-mannose N-glycan types (Chen et al., 1998Go; Clark et al., 1998Go; Krusius and Finne, 1977Go; Zamze et al., 1998Go). Our findings indicate that hybrid branches are nevertheless an essential component of the neuronal N-glycan repertoire.

Neuron-specific loss of Mgat1 function relied on the activity of the rat Synapsin-1 promoter and was found to be highly efficient as previously reported (DeFalco et al., 2001Go; Zhu et al., 2001Go). The frequency of recombined Mgat1{Delta} alleles, lacking Mgat1 exonic sequence, was similar to the percent of neurons present in total brain tissue; however, embryogenesis was sustained and a normal frequency of live births was obtained. Nevertheless, Mgat1F/F/Syn1-Cre mice were severely affected and the postnatal requirement for Mgat1 function was invariant. Failure to thrive was apparent with poor locomotor skills, and all but one offspring died by 8 weeks after birth. One mutant lived almost 6 months before death; however, the reason for the increased survival time in this single case is not presently known. Death was typically preceded by the increasing occurrence of tremors and general paralysis subsequently ensued. In brain tissue studies, widespread reactive gliosis was evident and may reflect the observation that astrocytes undergo hypertrophy, including up-regulation of Glial Fibrillary Acidic Protein in response to neuronal damage (Ridet et al. 1997). Among Mgat1F/F/Syn1-Cre mice, brain regions typically rich in neuronal cell abundance contained pyknotic bodies that were found to be dying and dead neuronal cells. Moreover, this loss of viable neurons appeared due to the activation of an apoptosis program that was apparent from evidence of widespread caspase-3 activation.

Neuronal apoptosis attenuates synaptic connections. The induction of neuronal apoptosis on Mgat1 deletion intimated the involvement of one or more glycoproteins that may normally control this process. Our analyses of several essential neuron-specific glycoproteins provided unexpected findings relating to aberrant expression and electrophoretic mobility upon reduced N-glycan branching. Among Mgat1F/F/Syn1-Cre brain and spinal cord tissues, a decrease of glycoprotein abundance was observed for the synaptic vesicle protein SV2, the nerve growth factor receptor TrkA, the voltage-gated potassium channel Kv1.1, and the voltage-gated sodium channel {alpha} and ß2 subunits. The profile of Kv1.1 expression was intriguing and may reflect a proteolytic event in the spinal cord and to a lesser extent in the brain, or perhaps this is simply due to reduced N-glycan mass and conformation associated with a greater than average shift in electrophoretic mobility. Additional studies will be needed to determine among these possibilities. In contrast, other neuron-specific glycoproteins, such as App and Syt, were not altered in abundance even though they were aberrantly glycosylated with a deficit in branching and increased mannose residue exposure detected by ConA–lectin binding.

Not all neuronal glycoproteins that normally bear hybrid or complex type N-glycan branches are reduced in expression by Mgat1 inactivation. The decrease in levels of a subset of neuron-specific glycoproteins suggests the presence of a selective glycan-dependent mechanism that may promote the expression of certain N-glycoproteins. Such a mechanism (if it exists) would likely originate in the Golgi where the Mgat1-encoded glycosyltransferase operates. An alternative possibility is that upon neuronal cell apoptosis, some glycoproteins are depleted first perhaps due to lesser nonglycan-dependent half-lives and thereby yield the expression profiles we observed. Future studies are needed to resolve among these possibilities.

Unlike the essential role of Mgat1 in neuronal and postnatal viability, Mgat2F/F/Syn1-Cre mice did not exhibit growth abnormalities, locomotor defects, or phenotypic markers of neuronal dysfunction. Mgat2F/F/Syn1-Cre mice were unremarkable in comparison to littermate controls. No evidence of neuronal apoptosis was found among brain tissues analyzed from Mgat2F/F/Syn1-Cre mice. In addition, the same neuronal glycoproteins found depleted on Mgat1 deletion were expressed at normal levels among brain and spinal cord tissues that had undergone neuronal Mgat2 deletion. Nevertheless, we found that Mgat2 deletion by Cre recombination occurred at similar frequencies among brain and spinal cord tissues as compared with Mgat1F/F/Syn1-Cre mice. These findings appear to contrast with reported neurological abnormalities associated with inherited and systemic Mgat2 deficiency in mice and humans. However, inherited mutations operate earlier in embryogenesis than is achieved by the use of the Syn1-Cre transgene. It is relevant to consider the timing of Mgat1 deletion as controlled by rat Synapsin-1 promoter. In the Syn1-Cre mice, Cre expression and recombination occur mainly among differentiated neurons during synaptogenesis, and not among neuronal precursors in early morphologic development of the brain. In addition, it is possible that the continued presence of complex N-glycans among nonneuronal compartments may in some manner modulate neuronal processes and phenotype development in Mgat2F/F/Syn1-Cre mice.

By eliminating sequential steps in N-glycan biosynthesis among experimentally defined cell types in vivo, we observe an essential physiologic role for Mgat1-dependent hybrid N-glycan branching in the viability of neurons and the survival of mice during early postnatal development. The resulting neuronal apoptosis and the depletion of essential neuronal glycoproteins on Mgat1 deficiency were not reproduced by similarly abolishing neuronal Mgat2-dependent complex N-glycan branching. This differential requirement for Mgat1 and hybrid type N-glycan branching may exist among other cell types as well. Studies involving lymphocyte-specific deletion of Mgat1 and Mgat2 indicate a failure of Mgat1-deficient T cells to survive in vivo, whereas Mgat2 deficiency is well tolerated (data not shown). Genetic modeling of N-glycan branching and function in vivo provides a method for discovering structure–function relationships that will be useful in efforts to determine further the molecular mechanisms by which N-glycan branching contributes to various physiologic processes.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Mgat1 gene targeting
The mouse Mgat1 gene was cloned from a mouse 129/SvJ genomic library and used to construct a targeting vector with the pflox plasmid vector (Figure 1A). The targeted vector was transferred into R1 ES cells by electroporation, and clones were isolated as described previously (Chui et al., 1997Go). ES cells bearing the loxP-flanked Mgat1 allele were used to generate chimeric mice by blastocyst injection of 7.1.2 T2 ES cells. Mice bearing the heterozygous loxP-flanked Mgat1 allele were crossed with Syn1-Cre mice to generate Mgat1F/WT/Syn1-Cre mice. Mice bearing the Mgat1F/WT/Syn1-Cre genotype were generated from paternal Mgat1F/WT/Syn1-Cre and Mgat1F/F genotypes.

Locomotor and muscular function
The wire test was used to assess muscular strength involved in grasping. Mice were allowed to grasp a wire net and then suspended 40 cm above a padded surface. The mean latency to fall over three trials was determined. To assess locomotor function, mice were placed on a rotating drum that was accelerated to 40 rpm in 4 min. Latency to fall in three trials was used, and the rpm at fall was plotted.

Histology
After paralysis, the mutant mice and their littermate controls were euthanized with carbon dioxide and transcardially perfused with 4% phosphate buffered saline (PBS)/paraformaldehyde. Tissues were removed and fixed in 4% paraformaldehyde overnight. For paraffin sections, the tissues were dehydrated and embedded in paraffin. The serial paraffin sections were cut at 5 mm, deparaffinized, and rehydrated. The sections were either stained with cresyl violet or by immunohistochemistry. A horseradish peroxidase system (Vectastain ABC kit, Vector, Burlingame, CA) was used to visualize antibody binding to glial fibrillary acidic protein (1:1000, Sigma, St. Louis, MO), and hematoxylin was used to counterstain the nuclei. After washing with water and dehydration, the slides were mounted with Permount. TUNEL assay is performed according to the manufacturer's instructions (Apoptosis detection system, Promega, Madison, WI).

For preparation and analysis of frozen sections, tissues were cryoprotected by immersion in 30% sucrose/PBS for 24 h at 4°C, then embedded in Optimal Cutting Temperature matrix. Subsequently, 10-mm sections were cut and processed on glass slides. Sections were blocked in PBS with 1% bovine serum albumin (BSA) for 1 h and then incubated with either 10 mg/ml fluorescein isothiocyanate (FITC) conjugated E-PHA lectin (Vector) or with the cleaved Caspase-3 (Asp175) antibody (1:100; Cell Signaling, Beverly, MA). For immunostaining, FITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as the secondary antibody for primary antibody detection.

Western and lectin blotting
Brain and spinal cord tissue was isolated and homogenized in 5 ml 10 mM HEPES, pH 7.4, 1 mM ethylenediamine tetra-acetic acid (EDTA), and Roche proteinase inhibitor cocktail. The homogenized tissues were centrifuged at 1000 x g for 10 min. The supernatants were subjected to ultracentrifuge using RNA tubes at 100,000 x g for 1 h. The pellets were dissolved with Lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM Triton X-100, and proteinase inhibitors) on ice for 30 min. After 10 min centrifugation at 16,000 x g, the supernatants were transferred to an Eppendorf tube, and the protein concentration was measured by Lowry assay (BioRad, Hercules, CA).

Cellular protein (20 mg) was loaded onto 4–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels, electrophoresed, and subsequently transferred to nitrocellulose membranes (BioRad). For western analysis with antibodies, the blots were first incubated in TBST (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 0.05% NP-40) with 5% nonfat dry milk powder solution and then probed with primary antibodies in TBST with 1% milk solution. The blots were incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG or anti-mouse IgG secondary antibodies (Jackson ImmunoResearch), and ECL (Amersham Biosciences, England) was used for developing the blots.

The antibodies used for blotting were mouse anti-synaptophysin monoclonal antibody (Chemicon, Temecula, CA) at 1:1000; mouse anti-Alzheimer precursor protein A4 monoclonal antibody (Chemicon) at 1:1000; mouse anti-synaptotagmin monoclonal antibody (Chemicon) at 1:1000; mouse anti-SV2 monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa) at 1:100; mouse anti-Kv1.1 monoclonal antibody (Upstate, Lake Placid, NY) at 1:1000; rabbit anti-TrkA polyclonal antibody (Cell Signaling); rabbit anti-Na+ channel type1 polyclonal antibody (Upstate); rabbit anti-Naß2 polyclonal antibody (Alomone Labs, Jerusalem). For lectin binding analyses, the blots were incubated in TBST with 5% BSA and then probed with biotinylated ConA (50 ng/ml, Vector) or 2 mg/ml L-PHA (Vector) in TBST with 1% BSA for 1 h. After incubating with horseradish peroxidas–conjugated streptavidin (Vector) for 1 h, the blots were washed overnight and developed by enhanced chemiluminescence using the manufacturer's instructions (Amersham Biosciences).

ConA binding analysis
ConA-agarose beads (50 µl, Vector) were washed three times in TBS (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). Membrane proteins (50 µg) were added and incubated with rotation at 4°C for 1 h. Subsequently, the beads were washed in TBS three times and 20 ml of 1x SDS–PAGE sample buffer was added to elute the proteins. After boiling 5 min, supernatants were analyzed by SDS–PAGE and western blotting.


    Acknowledgements
 
We thank Holly Pieck for her work in producing the loxP-flanked Mgat1 allele. This research was funded by the National Institutes of Health (DK48247). J.D.M. acknowledges support as an investigator of the Howard Hughes Medical Institute.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: jmarth{at}ucsd.edu


    Abbreviations
 
BSA, bovine serum albumin; CDG, congenital deficiency of glycosylation; ConA, Conacanavalin A; EDTA, ethylenediamine tetra-acetic acid; E-PHA, erythro-phytohemagluttinin; FITC, fluorescein isothiocyanate; L-PHA, leuko-phytohemagluttinin; PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TUNEL, terminal deoxynucleotidyl transferase dUTP nicked end labeling


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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