ARTICLE |
Correspondence to: Tamao Endo, Dept. of Glycobiology, Tokyo Metropolitan Inst. of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan. E-mail: endo@tmig.or.jp
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Summary |
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We examined the distribution of sialoglycoconjugates in the cerebellum of 9-week-old and 30-month-old rats using light microscopy and electron microscopy in combination with two lectins, Maackia amurensis lectin (MAL) for Sia23Gal and Sambucus sieboldiana agglutinin (SSA) for Sia
26Gal. Each lectin showed characteristic staining patterns. In young adult rats, MAL stained a strongly granular layer, a weakly molecular layer, and the medullary lamina, while SSA more strongly stained the medullary lamina than the molecular and granular layers. After aging, different staining patterns were obtained. Intense SSA reactivity was observed in the granular layer and intense MAL reactivity was observed in the medullary lamina of the aged groups. The reactivity of Purkinje cells with MAL was downregulated in the aged rats. These results indicated that Sia
23Gal and Sia
26Gal were expressed in distinct regions of the rat cerebellum and that their expression patterns changed in the aged brain. (J Histochem Cytochem 50:11791186, 2002)
Key Words: sialoglycoconjugate, cerebellum, aging, granular layer, Purkinje cell
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Introduction |
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Sialoglycoconjugates are sugar chains in animals in which the non-reducing end contains sialic acid, an acidic 9-carbon sugar. These molecules not only are responsible for the negative charge and hydrophilicity of the cell surface but also act as specific ligands that play important roles in intercellular and/or intermolecular recognition phenomena in a linkage-specific manner (23,
26,- and
28-ketosidic linkages. Whereas much is known about the location and function of the
28 sialyl linkage (
23 and
26 sialyl linkages in the nervous system remain unclear.
Cerebellar neurons are arranged in a highly regular manner that results from repetition of the same basic circuit module (
Because the biosynthesis of glycans is not controlled by the interaction of a template and depends on the concerted action of glycosyltransferases, the structures of glycans are much more variable than those of proteins and nucleic acids. Therefore, the structures of glycans can be easily altered by the physiological conditions of the cells (23 and
26-sialoglycoproteins were downregulated in several regions of the aged rat hippocampus (
In this study we examined the distribution of sialoglycoconjugates in the cerebellum of 9-week-old rats and 30-month-old rats using light microscopy and electron microscopy in combination with two lectins, MAL and SSA.
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Materials and Methods |
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Animals
Five each of 9-week-old and 30-month-old female Fischer rats were used in this study. All experimental procedures using laboratory animals were approved by the Animal Care and Use Committee of Tokyo Metropolitan Institute of Gerontology. All efforts were made to minimize the number of animals used and their suffering. Because the same results were found in all five rats in each group, only the data obtained for one sample in each group is described in detail here.
Lectin Staining for Light Microscopy
Lectin staining was carried out as previously described (
Lectin Staining for Electron Microscopy
Under pentobarbital anesthesia, the rats were perfused transcardially with 10 mM PBS, pH 7.4, and then with 4% formaldehydePBS at RT over 20 min. The brains were immediately dissected out and placed in 4% formaldehydePBS at 4C for 2 hr. Lectin staining for electron microscopy was carried out according to
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Results |
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Light Microscopic Study
Staining with MAL and SSA, which specifically bind to Sia23Gal and Sia
26Gal groups, respectively, showed that the Sia
23Gal and Sia
26Gal groups were expressed in distinct regions of the rat cerebellum. MAL staining was intense in the granular layer and weak in the molecular layer and the medullary lamina (Fig 1A), whereas SSA staining was intense in the medullary lamina and weak in the granular and molecular layers (Fig 1D).
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We next examined the effect of age on the staining patterns and found two differences. MAL staining in the medullary lamina was slightly stronger in 30-month-old rats than in 9-week-old rats (Fig 1A and Fig 1B), whereas SSA staining in the granular layer was much stronger in the aged rats than in the young adult rats (Fig 1D and Fig 1E). The staining intensity in the remaining parts did not change drastically with aging. MAL and SSA staining disappeared after sialidase treatment (Fig 1C and Fig 1F, respectively), indicating that the binding of each lectin to the sialic acid residue was highly specific. To examine in detail the staining of the rat cerebellum with SSA and MAL, we analyzed the whole sections with electron microscopy.
Electron Microscopic Study
In the aged group, SSA strongly stained the space between the cell bodies and neurites, where an extracellular matrix appeared to be present (Fig 2D, arrows). When the same section was examined at higher magnification, the extracellular matrix of the granular layer was confirmed to be stained with SSA (Fig 2E, asterisk). The extracellular matrix filled up the space between the membranes of cell processes. These membranes are indicated by arrowheads in Fig 2E. These results are consistent with those obtained at the light microscopic level. However, the glycoproteins whose expression patterns change with age remain to be identified. On the other hand, MAL stained the extracellular matrix of 9-week-old rats and 30-month-old rats (Fig 2A and Fig 2B, respectively, arrows), but no difference in staining intensity between them was observed.
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The cerebellar glomeruli are indicated by Gl in Fig 2. The plasma membranes of the cerebellar glomeruli and the granule cells in the granular layer from 9-week-old and 30-month-old rats were stained by MAL and SSA, although an age-associated change was not observed (Fig 2A2D, arrowheads). Nonspecific binding of HRPstreptavidin conjugates and endogenous peroxidase activity were scarcely detected under our experimental conditions because there was no difference when the sections were subjected to the staining procedure without lectin (Fig 2F) or HRPstreptavidin conjugates (data not shown). Because the electron density of myelin was high, the intensity of staining of myelin (indicated by M in Fig 2B and Fig 2F) was similar when the samples were subjected to the staining procedure with or without lectin.
The plasma membranes of the Purkinje cells were stained by both lectins at the light microscopic level, as shown in Fig 1. MAL appeared to react more weakly in aged rats than in young adult rats, although details were not clear at the light microscopic level. Therefore, we examined the staining at higher resolution with electron microscopy. The plasma membranes of the Purkinje cells from the 9-week-old (Fig 3C) and 30-month-old rats (Fig 3D) were stained by SSA (arrowheads). The Golgi apparatus of the Purkinje cells from both groups was weakly stained (Fig 3C and Fig 3D). However, no difference in staining between the 9-week-old and 30-month-old rats was observed. On the other hand, MAL staining of Purkinje cell membranes of the 30-month-old rats was less intense than that of the 9-week-old rats (Fig 3A and Fig 3B, arrowheads). Observations at the light microscopic level could not reveal whether the dendrites of Purkinje cells reacted with the lectins (Fig 1). At the electron microscopic level, the dendrites of Purkinje cells from both young and aged rats were stained by both SSA and MAL (data not shown).
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MAL staining of the Golgi apparatus of the Purkinje cells of the 9-week-old rats was stronger than that of 30-month-old rats (Fig 3A and Fig 3B). Binding of the lectins was inhibited by the addition of a hapten sugar, 0.2 M N-acetylneuraminic acid (data not shown). It is also noteworthy that nonspecific binding of HRPstreptavidin conjugates and endogenous peroxidase activity were scarcely detected (Fig 3E and Fig 3F). These results indicated that the Sia23Gal and Sia
26Gal groups were expressed on the plasma membranes of the Purkinje cells and suggested that the expression of the Sia
26Gal linkage did not change significantly during aging but that of the Sia
23Gal was downregulated.
It should be noted that conspicuous lipofuscin accumulation was observed only in the cytoplasm of the 30-month-old rats (Fig 3B and Fig 3D3F, indicated by L). Lipofuscin, which has high electron density, is known to accumulate in the cytoplasm of aged cells only (
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Discussion |
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The present study demonstrates that the expression patterns of sialoglycoconjugates containing the Sia2 3Gal and Sia
26Gal groups are region-specific in the rat cerebellum and change in the aged brain (Table 1).
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The granular layer contains a vast number of densely packed small neurons, mostly small granule cells. The granular layer also contains small clear spaces called cerebellar glomeruli, in which the bulbous terminal of a mossy fiber makes synaptic contact with Golgi and granule cell axons (26 group at the extracellular matrix in the granular layer remains to be solved. The increase of the Sia
26 group in the granular layer in the aged brain may be due to an increase of the activity of
26-sialyltransferases, which are involved in the formation of the Sia
26 group (
It should be noted that the expression levels of 23 and
26-sialoglycoproteins are downregulated in the aged rat hippocampus (
26 group at the extracellular matrix in the granular layer and the Sia
23 group in the medullary lamina are upregulated. These results suggest that the expression of sialylglycoconjugates is regulated in a region-specific manner.
The Purkinje cells are characterized by their rich and extensive dendritic tree in one plane. This structure is designed to accommodate an enormous synaptic input. The Purkinje cells are responsible for the only output signal from the cerebellar cortex, and the activity of the Purkinje cells is highly regulated by several excitatory and inhibitory afferent inputs. In this study we found that the staining intensity of the Purkinje cells by MAL was downregulated in the aged rats. Because such a decrease in the sialic acid residue reduces cellcell repulsion and enhances cellcell contact, the transmission efficiency of synaptic input and output signals may be affected. Interestingly, the expression of the Sia23 group decreased and that of the Sia
26 group did not change during aging. One possible explanation for this is that there is an increment in the activity of a novel sialidase that acts on the Sia
23 group but not on the Sia
26 group. Although we cannot completely rule this explanation out, it seems unlikely because such a sialidase has not been identified in mammals. Another possibility is that the decreased expression of Sia
23 group is due to a depression in the activity of
23-sialyltransferase. Recently, the mRNAs of seven sialyltransferases in the mouse hippocampus were found to be differentially expressed. Furthermore, some sialyltransferase mRNAs were observed to be upregulated and others were found to be downregulated (
Our finding that the expression of the Sia23Gal group and Sia
26Gal group changed during aging is biologically significant for three reasons. First, because sialic acids are ubiquitous components of cell surface glycoconjugates, a change of sialic acid expression during aging may modulate molecular and cellular interactions by changing the electrostatic potential of cells. Second, a change of expression of sialic acid during aging may disturb cellcell recognition via various sialic acid-binding molecules. One type of such molecules are the siglec proteins, which are endogenous lectins that recognize specific sialic acid linkages (
23Galß13GalNAc linkage (
23-linked sialic acid residues on CD24, which, in turn, induces neurite outgrowth (
We previously compared young and aged rat cerebellums using the lectins SSA and MAL after SDS-PAGE (26 linkage after aging. We are starting to purify and identify the glycoprotein bands detected by SSA. Future studies are required to determine the full significance of the change in sialic acid during aging.
The present results clearly reveal a change of sialic acids during aging. Therefore, the change of sialoglycoconjugates during aging probably affects intercellular adhesion via a sialic acid-binding molecule, such as one of the siglec proteins, and the disturbance of adhesion then affects brain activity. Such a change may induce a deterioration of navigational brain functions, such as those involved in reaching an object, exploring a new environment, and acquiring knowledge of one's spatial orientation. These functions are believed to require undisturbed cerebellar activity (
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Acknowledgments |
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Supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, the Mizutani Foundation, and the Naito Foundation.
We thank Mr M. Fukuda and Ms T. Shibata (Laboratory for Electron Microscopy and Department of Anatomy, Kyorin University School of Medicine) for their technical assistance.
Received for publication December 26, 2001; accepted April 10, 2002.
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