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
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Abstract |
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Key words: apoptosis / development / genetics / N-glycans / neurobiology
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Introduction |
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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, 1985). 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, 1989
; Kumar et al., 1990
; Sarkar et al., 1991
). 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, 1987
; D'Agostaro et al., 1995
; Schachter, 1991
).
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, 1994; Metzler et al., 1994
). 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., 1994
; Wang et al., 2001
). 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, 1996
).
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., 1998; Clark et al., 1998
; Krusius and Finne, 1977
; Zamze et al., 1998
). 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.
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Results |
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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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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 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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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., 1977; Zhu et al., 2001
).
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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 2030% 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 subunit (Kv1.1), the voltage-gated sodium channel
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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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, 1985). 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., 1994
; Tan et al., 1996
). Mice lacking a functional Mgat2 allele recapitulate the majority of CDG type IIa disease signs, including deficits in postnatal locomotor development (Wang et al., 2001
). 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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Discussion |
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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, 1994; Metzler et al., 1994
). 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., 2001
; Wang et al., 2001
).
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 cellcell 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., 1998; Clark et al., 1998
; Krusius and Finne, 1977
; Zamze et al., 1998
). 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., 2001; Zhu et al., 2001
). The frequency of recombined Mgat1
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 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 ConAlectin 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 structurefunction relationships that will be useful in efforts to determine further the molecular mechanisms by which N-glycan branching contributes to various physiologic processes.
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Materials and methods |
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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 415% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) 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 peroxidaseconjugated 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 peroxidasconjugated 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 SDSPAGE sample buffer was added to elute the proteins. After boiling 5 min, supernatants were analyzed by SDSPAGE and western blotting.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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