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A novel monoclonal antibody recognizes lysosome-like structures and reflects regional and age-related differences in the rat dentate gyrus
Neuroscience Letters 330 (2002) 275–279 www.elsevier.com/locate/neulet
A novel monoclonal antibody recognizes lysosome-like structures and reflects regional and age-related differences in the rat dentate gyrus Seishi Maeda a,*, Keigo Kawabata a, Akinori Takanaga a, Koichi Tanaka a, Hisao Ito b, Tetsu Hayakawa a, Makoto Seki a a Department of Anatomy, Hyogo College of Medicine, Mukogawa 1-1, Nishinomiya, Hyogo 663-8501, Japan The Institute of Experimental Animal Sciences, Hyogo College of Medicine, Mukogawa 1-1, Nishinomiya, Hyogo 663-8501, Japan
b
Received 15 May 2002; received in revised form 8 July 2002; accepted 15 July 2002
Abstract The granule cells (GCs) of dentate gyrus exhibit regionally specific morphology, and continue to be born and to develop well into adult life. We used a novel monoclonal antibody, MAb2G7, elicited by immunization of a mouse with a microsome fraction of the hippocampus, to evaluate regional and age-related differences in GCs immunohistochemically. Weak cytoplasmic reactions were observed in many neurons, but intense MAb2G7-positive dots were observed only in GCs. Using electron microscopy, we observed that these dots were localized in the internal droplets of secondary lysosome-like structures in GCs. The MAb2G7-positive granules were quantitatively analyzed in young adult and middle-aged rats. Larger numbers of reactive granules were observed in the infrapyramidal blade (IPB) than in the suprapyramidal blade (SPB) and the numbers of positive granules were proportionally reduced in the two areas in middle-aged rats. The changes in the MAb2G7 immunoreactivity may reflect different activation or neurogeneration of GCs in the IPB versus the SPB, and in middle-aged versus young adult rats. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Age; Dentate gyrus; Granule cell; Monoclonal antibody; Secondary lysosome
The granule cells (GCs) are the principal neurons of the dentate gyrus and are arranged in a densely packed layer with two sections called the suprapyramidal blade (SPB) and the infrapyramidal blade (IPB) [1]. A characteristic feature of these neurons is the laminated termination of afferents on their dendrites. The distal dendrites are contacted by associational and commissural fiber systems [16]. The vast majority (85%) of these cells are born postnatally, and the neurogenesis and migration of newborn GCs appears to extend well into adult life [2,13,20]. The mechanisms controlling the development and maintenance of neurons, including the GCs, are largely unknown but may involve the breakdown of intracellular materials as part of the biochemical machinery. Lysosomes are membrane-delimited organelles that can digest essentially all macromolecules. Secondary lysosomes contain heterogeneous contents, and in a further transformation, secondary lysosomes condense spontaneously into lipofuscin in neuronal cells [8]. Biological changes in lysosomes * Corresponding author. Tel.: 181-798-45-6484; fax: 181-79845-6485. E-mail address: [email protected] (S. Maeda).
correlate with aging and age-associated pathologies [15]. Therefore, alterations of lysosomal numbers and activity could be an indicator of the cellular vitality and maturity, especially in neurons. In this study, we have used a mouse monoclonal antibody, MAb2G7, to investigate the distribution of lysosomal structures in the hippocampi of young adult and middle-aged rats. The monoclonal antibody MAb2G7 was prepared by immunizing mice with centrifugal microsome fraction of rat hippocampal homogenate as antigen. Hippocampi from six adult rats were minced and homogenized in 10 mM Tris– HCl (pH 7.4) containing 320 mM sucrose and 1 mM MgCl2 by Potter-Elvehjem homogenizer. Homogenate was filtered through Nylon bolting cloth (pore size 40 mm) and centrifuged in 9–15% gradient OptiPrep e (Nycomed Pharma, Norway) at 18 000 £ g for 20 min at 48C by Beckman Optima XL-90 with swing rotor SW28. Microsome-rich fraction was collected, mixed with adjuvant, and injected to mice. The splenocytes from an immunized mouse were fused to myeloma NSO cells and the hybridomas were
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 81 5- 7
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obtained. Cloned hybridoma produced MAb2G7 was transplanted to mouse, intraperitoneal, and ascites was collected. For immunohistochemistry with MAb2G7, young adult male rats (2 months old, n ¼ 3) and middle-aged rats (12 months old, n ¼ 3) were deeply anesthetized and perfused with phosphate buffer saline (PBS) followed by 4% paraformaldehyde and 7% sucrose in PB (pH 7.4). The brains were removed, cut into several septo-temporal pieces, and fixed with the same fixative for an additional 4 h at 48C. To observe and count the cells clearly, the brains were embedded in paraffin and sectioned at 6 mm, and then they were put onto glass slides. Immunohistochemistry was performed using the avidin-biotin complex method with a Vector Laboratories kit (Burlingame, CA, USA). Sections were treated with the hightemperature epitope-activating method: the sections were serially autoclaved in 10 mM citric acid (pH 7.0) for 15 min at 1058C and for 15 min at 1168C. After rinsing with deionized water, these sections were immersed in 0.1% NaN3 in PBS for 10 min to block intrinsic peroxidase and blocked with Block Ace e (Dainippon Pharmaceuticals Co. Ltd., Japan) containing 10% normal goat serum for 1 h at room temperature (RT). The sections were incubated with diluted MAb2G7 ascites (1:500) for 3 days at 48C. After washing with PBS containing 0.05% Tween 20 (PBS/T), sections were incubated in biotinylated goat anti-mouse IgM (ICN Pharmaceuticals Inc., USA) for 2 h, then in avidin-biotin complex solution for 1 h at RT. The peroxidase reaction was carried out with 0.05% diaminobenzidine and 0.03% H2O2 in PBS/T for exactly 10 min at RT. The sections were dehydrated, mounted, and observed under light microscopy. In a control experiment, the first antibody was omitted, and no positive reactions were observed. The number of immunologically positive dots and the number of GCs in the dentate gyrus were counted and the rate per cell was calculated. Statistical significance was assessed using a one-way analysis of variance with region or age as factors, followed by a Student’s t-test. Data are expressed as mean ^ SE if not otherwise stated and P-values ,0.05 were considered significant. For an immunoelectron microscopic study, 0.05% glutaraldehyde was added to the fixative mentioned above and sectioned by microslicer, and detergent-free PBS was used instead of PBS/T. After immunohistochemical staining, the sections were additionally fixed with 2% osmium tetra-oxide in PB, dehydrated and flat-embedded in Spurr’s resin (TAAB, Germany) between acular films (Nissin EM, Japan). The sections were observed under light microscopy, trimmed, and ultra-thin sectioned by Ultra-Cut microtome (Leica, Germany). The immunoreactivity was observed with JEM 1200EX transmission electron microscopy at 80 kV. To detect the relative molecular weight of and age-related changes in the MAb2G7 antigen, immunoblotting was performed. The homogenized hippocampi of 2- and 12month old male rats, at a concentrations of 10 mg/ml, were electrophoresed in a 7.5% sodium dodecyl sulfate-
polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. The membrane was washed with PBS/T and blocked with Block Ace e for 1 h at RT. Diluted MAb2G7 (1:5000) was applied to the membrane and incubated for 2 h at RT. After washing, the membrane was incubated with peroxidase-conjugated goat antibody. Chemiluminescent detection was performed with ECL Plus e (Amersham Pharmacia Biotech, UK) according to the manufacturer’s protocol. A plot profile of the detected bands was processed using NIH-Image software (Scion Image, USA). Immunohistochemical localization of MAb2G7 in the dentate gyrus is shown in Fig. 1. MAb2G7 binding was observed in the GC layer of the SPB and IPB in 2-monthold rats (Figs. 1A,B), and appeared both as intense dots and as diffuse background staining in the GC cytoplasm. While numerous positive dots were observed in both blades, there were somewhat more in the IPB (Fig. 2). We counted approximately one to three per cell in the sections, but cells with no dots were also seen. Since less-positive cells were intermingled with cells containing several dots, the estimation of the number of dots per cell may not be exact, but may be sufficient to reflect significant differences in the populations between the blades. In the CA1 and CA3 other of the hippocampal formation, positive dots were observed but they were scarce and less intense than in the dentate gyrus of rats of the same age (Figs. 1E,F). Thus, the localization of numerous MAb2G7-positive dots may be a distinctive characteristic of dentate GCs. In fact, very few or no definitive MAb2G7-positive reactions were found in other brain regions such as cortex and thalamus, except for weak cytoplasmic staining of neurons. A remarkable difference in the numbers of MAb2G7-positive dots was found between young adult rats and middleaged rats (Figs. 1A–D and 2). MAb2G7-positive dots were not only decreased in quantity in 12-month-old rats compared to 2-month-old rats, but were also of weaker intensity. These quantitative declines were observed in both the SPB and IPB. Similar to the results in the young adult rats, in the older rats more MAb2G7-positive dots were seen in the IPB than in the SPB. The differences between the blades may therefore be maintained despite the decreasing amount of the antigen with age. These results may reflect differences in the strength of antigen processing or in activation between SPB and IPB neurons, and between younger and older rats. Immunoelectron microscopic observations indicated that MAb2G7 was localized exclusively on secondary lysosome-like structures (Fig. 1G). These structures showed membrane-delimited spheroid or ellipsoid bodies containing various sizes of droplets and folding lamella, and the MAb2G7 reaction product was distributed on the internal lipid droplets (Fig. 1G, insert). The reaction intensity reflected the droplet sizes and quantities. None of the positive reactions was observed in the first antibody omitted negative control (Fig. 1H). Since these droplets are likely to be derived from other organelles taken up by the second-
S. Maeda et al. / Neuroscience Letters 330 (2002) 275–279
ary lysosomes, the antigen recognized by MAb2G7 may be involved in the proteolysis of these organelles. Immunoblotting showed that the relative molecular weight of the antigen recognized with MAb2G7 was approximately 68 kDa (Fig. 3). The same band was detected in both 2- and 12-month-old rats, but the immunoreactivity was slightly less intense in the older rats. However, the difference in the intensity between the immunoblot bands in the rats of the two ages was not as great as the difference seen with immunohistochemistry.
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In the present study, we showed that the MAb2G7 antibody specifically recognized secondary lysosome-like structures in GCs of the rat dentate gyrus. The distribution of these structures differed quantitatively between the SPB and IPB, and declined with age. However, the location of MAb2G7 reactivity within the cell, and its relative distribution between the SPB and IPB, did not change with age. Therefore, the reduction of the histochemical reaction in relatively aged rats may be caused by a decline in production of the antigen in GCs. The adult dentate gyrus (consist-
Fig. 1. The MAb2G7 immunostaining of young adult and middle-aged rat hippocampus. MAb2G7-positive dots can be observed in the granule cell layers of the SPB (A); and the IPB (B) in 2-month-old rats. More immunopositive dots were detected in IPB granule cells than in SPB granule cells. The reactions decreased in number and intensity in the SPB (C); and IPB (D) of 12-month-old rats. In the pyramidal cell layers of CA1 (E); and CA3 (F) in 2-month-old rats, some positive reactions were detected, but they were lower in quantity and intensity than in the dentate gyrus. Bar ¼ 20 mm in A–F. (G) Immunoelectron microscopy of a MAb2G7-positive granule cell in 2-monthold rat IPB. Electron-dense 3,3 0 -diaminobenzidine precipitates were observed in the lysosome-like bodies of the cell (arrowheads). No reactions were observed in other types of organelles. A high magnification of the typical appearance of a positive organelle is shown in the inset. The positive organelle contains droplets and folding lamellae of various sizes, and the reactions are localized to the internal droplets. These structural peculiarities have a resemblance to secondary lysosomes or lipofuscin. (H) MAb2G7 omitted negative control image. A lysosomal granule (arrowhead) is somewhat dense, but no positive reaction is observed (insert). Bar ¼ 2 mm in G and H, and 200 nm in inserts. Abbreviations are as follows: ml, molecular layer; gl, granule cell layer; pl, polymorphic cell layer; so, stratum oriens; pcl, pyramidal cell layer; and sl, stratum lucidum.
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Fig. 2. A quantitative graph of the number of MAb2G7 positive dots per cell in the dentate gyrus of 2- and 12-month-old rats. We counted the number of positive dots and cells in the granule cell layers of the 6-mm thick sections in a field of vision under light microscopy. A total of 30–35 fields were counted for each animal (n ¼ 3). The stippled bar indicates SPB granule cells of 2-monthold rats, the bar with vertical lines indicates IPB granule cells of 2-month-old rats, the bar with horizontal lines indicates SPB granule cells of 12-month-old rats, and the bar with diagonal lines indicates IPB granule cells of 12-month-old rats. *P , 0:05 compared with SPB. **P , 0:05 compared with 2month-old rats.
ing SPB and IPB) is constantly renewed through a process of neurogenesis and migration of GCs in temporal-to-septal, and superficial-to-deep gradients [2,6,20]. Dendritic trees of GCs located in the SPB tend, on average, to be longer than those in the IPB [5,7], suggesting that the GCs in SPB and those in IPB may have different metabolic activities or functions during their development and maintenance. This is also supported by our finding that MAb2G7-positive lysosomal granules are distributed unevenly between the SPB
Fig. 3. Immunoblotting of rat hippocampus extracts reacted with the MAb2G7 antibody. Contents of the lanes are as follows: lane 1, young adult (2-month-old) rats; and lane 2, middle-aged (12month-old) rats. Immunoreactivity of the 68 kDa bands (arrowhead) was slightly decreased in quantity in middle-aged rats. The plot profile was analyzed using NIH Image software. The molecular weight was calculated from standard protein makers run in parallel (letters at right).
and IPB, although it remain to be clarified what percentage of secondary lysosomes are MAb2G7-positive in dentate gyrus GCs Neuronal lysosomes are sometimes involved in agedependent neuropathologies, due to lysosomal metabolic malfunction, such as accumulation of lipofuscin [10,13,17], deposition of amyloid precursor protein in Alzheimer’s disease [3,11,12], and gangliosidoses [9,19]. Age-related changes in the activity of the lysosomal enzyme cathepsin have been reported [4,18]. Nakanishi et al. Examined age-related changes in the lysosomal cathepsins, cathepsin D, B, and L. An age-related decline of activity was observed only for cathepsin L, whereas the activity of cathepsin D and B increased in aged rats [18]. Since immunohistochemical reactivity of cathepsin L showed only a slight reduction in aged rats, its localization and activity may not always be coincident with MAb2G7 reactivity. Bi et al. [4] showed that the expression of lysosomal cathepsin D and B changes in an age-related and regionally selective manner in the rat brain. This suggests that proteolysis in lysosomes may be regulated in age- and region-specific manners, and by extension, that the antigen recognized by MAb2G7 is involved in proteolysis in lysosomes. However, it is not known whether the antigen recognized by MAb2G7 is a native lysosomal enzyme or a substrate being processed in them. However, since not many molecules are known that are characteristic of dentate gyrus GCs, studying the molecular makeup and metabolic cascade of this antigen will be useful for learning about the regional and age-related metabolism of dentate GCs. There are three distinct temporal gradients in the early morphogenesis of the granular cell layer: SPB to IPB, caudal to rostral, and in all regions of the granular layer the more superficial neurons to the deeper ones [20]. GC progenitors have been shown to migrate from the dentate hilus region for at least 11 months postnatally [14], suggesting that a neurogenetic gradient of the granular neurons could be found for several months after birth. However, neurogenesis is drastically reduced in 27-month-old rats compared to 6-month-old controls [14]. The pattern of MAb2G7 antibody staining in the present study is consistent with this drastic postnatal reduction of the GC neurogenesis. Therefore, our novel antibody, MAb2G7 that can be used to visualize the lysosome-like granules in the GCs, might be an indicator of GC neurogenesis. [1] Amaral, D.G. and Witter, M.P., Hippocampal formation, In G. Paxinos (Ed.), The Rat Nervous System, 21, Academic Press, Inc, New York, 1995, pp. 443–493. [2] Bayer, S.A., Development of the hippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autoradiography, J. Comp. Neurol., 190 (1980) 87–114. [3] Benowitz, L.I., Rodriguez, W., Paskevich, P., Mufson, E.J., Schenk, D. and Neve, R.L., The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects, Exp. Neurol., 106 (1989) 237–250. [4] Bi, X., Yong, A.P., Zhou, J., Gall, C.M. and Lynch, G., Regionally selective changes in brain lysosomes occur in
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the transition from young adulthood to middle age in rats, Neuroscience, 97 (2000) 395–404. Claiborne, B.J., Amaral, D.G. and Cowan, W.M., A quantitative three-dimensional analysis of granule cell dendrites in the rat dentate gyrus, J. Comp. Neurol., 302 (1990) 206–219. Crespo, D., Stanfield, B.B. and Cowan, W.M., Evidence that late-generated granule cells do not simply replace earlier formed neurons in the rat dentate gyrus, Exp. Brain Res., 62 (1986) 541–548. Desmond, N.L. and Levy, W.B., Granule cell dendritic spine density in the rat hippocampus varies with spine shape and location, Neurosci. Lett., 54 (1985) 219–224. Elleder, M., A histochemical and ultrastructural study of stored material in neuronal ceroid lipofuscinosis, Virchows Arch. B Cell Pathol., 28 (1978) 167–178. Goebel, H.H., Morphology of the gangliosidoses, Neuropediatrics, 15 (1984) 97–106. Ivy, G.O., Protease inhibitors as a model for NCL disease, with special emphasis on the infantile and adult forms, Am. J. Med. Genet., 42 (1992) 555–560. Ivy, G.O., Schottler, F., Wenzel, J., Baudry, M. and Lynch, G., Inhibitors of lysosomal enzymes: accumulation of lipofuscin-like dense bodies in the brain, Science, 226 (1984) 985–987. Kalanj-Bognar, S., Rundek, T., Furac, I., Demarin, V. and Cosovic, C., Leukocyte lysosomal enzymes in Alzheimer’s disease and Down’s syndrome, J. Gerontol. A Biol. Sci. Med. Sci., 57 (2002) B16–B21.
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[13] Kaplan, M.S. and Hinds, J.W., Neurogenesis in the adult rat; Elocron microscopic analysis of light radioautographs, Science, 197 (1977) 1092–1094. [14] Kuhn, H.G., Dickinson-Anson, H. and Gage, F.H., Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation, J. Neurosci., 16 (1996) 2027–2033. [15] Landfield, P.W., Braun, L.D., Pitler, T.A., Lindsey, J.D. and Lynch, G., Hippocampal aging in rats: a morphometric study of multiple variables in semithin sections, Neurobiol. Aging, 2 (1981) 265–275. [16] Lubbers, K. and Frotscher, M., Fine structure and synaptic connections of identified neurons in the rat fascia dentata, Anat. Embryol. (Berl.), 177 (1987) 1–14. [17] Moore, W.A. and Ivy, G.O., Implications of increased intercellular variability of lipofuscin content with age in dentate gyrus granule cells in the mouse, Gerontology, 41 (1995) 187–199. [18] Nakanishi, H., Tominaga, K., Amano, T., Hirotsu, I., Inoue, T. and Yamamoto, K., Age-related changes in activities and localizations of cathepsins D, E, B, and L in the rat brain tissues, Exp. Neurol., 126 (1994) 119–128. [19] Sandhoff, K. and Conzelmann, E., The biochemical basis of gangliosidoses, Neuropediatrics, 15 (1984) 85–92. [20] Schlessinger, A.R., Cowan, W.M. and Gottlieb, D.I., An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat, J. Comp. Neurol., 159 (1975) 149–176.
A novel monoclonal antibody recognizes lysosome-like structures and reflects regional and age-related differences in the rat dentate gyrus Seishi Maeda a,*, Keigo Kawabata a, Akinori Takanaga a, Koichi Tanaka a, Hisao Ito b, Tetsu Hayakawa a, Makoto Seki a a Department of Anatomy, Hyogo College of Medicine, Mukogawa 1-1, Nishinomiya, Hyogo 663-8501, Japan The Institute of Experimental Animal Sciences, Hyogo College of Medicine, Mukogawa 1-1, Nishinomiya, Hyogo 663-8501, Japan
b
Received 15 May 2002; received in revised form 8 July 2002; accepted 15 July 2002
Abstract The granule cells (GCs) of dentate gyrus exhibit regionally specific morphology, and continue to be born and to develop well into adult life. We used a novel monoclonal antibody, MAb2G7, elicited by immunization of a mouse with a microsome fraction of the hippocampus, to evaluate regional and age-related differences in GCs immunohistochemically. Weak cytoplasmic reactions were observed in many neurons, but intense MAb2G7-positive dots were observed only in GCs. Using electron microscopy, we observed that these dots were localized in the internal droplets of secondary lysosome-like structures in GCs. The MAb2G7-positive granules were quantitatively analyzed in young adult and middle-aged rats. Larger numbers of reactive granules were observed in the infrapyramidal blade (IPB) than in the suprapyramidal blade (SPB) and the numbers of positive granules were proportionally reduced in the two areas in middle-aged rats. The changes in the MAb2G7 immunoreactivity may reflect different activation or neurogeneration of GCs in the IPB versus the SPB, and in middle-aged versus young adult rats. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Age; Dentate gyrus; Granule cell; Monoclonal antibody; Secondary lysosome
The granule cells (GCs) are the principal neurons of the dentate gyrus and are arranged in a densely packed layer with two sections called the suprapyramidal blade (SPB) and the infrapyramidal blade (IPB) [1]. A characteristic feature of these neurons is the laminated termination of afferents on their dendrites. The distal dendrites are contacted by associational and commissural fiber systems [16]. The vast majority (85%) of these cells are born postnatally, and the neurogenesis and migration of newborn GCs appears to extend well into adult life [2,13,20]. The mechanisms controlling the development and maintenance of neurons, including the GCs, are largely unknown but may involve the breakdown of intracellular materials as part of the biochemical machinery. Lysosomes are membrane-delimited organelles that can digest essentially all macromolecules. Secondary lysosomes contain heterogeneous contents, and in a further transformation, secondary lysosomes condense spontaneously into lipofuscin in neuronal cells [8]. Biological changes in lysosomes * Corresponding author. Tel.: 181-798-45-6484; fax: 181-79845-6485. E-mail address: [email protected] (S. Maeda).
correlate with aging and age-associated pathologies [15]. Therefore, alterations of lysosomal numbers and activity could be an indicator of the cellular vitality and maturity, especially in neurons. In this study, we have used a mouse monoclonal antibody, MAb2G7, to investigate the distribution of lysosomal structures in the hippocampi of young adult and middle-aged rats. The monoclonal antibody MAb2G7 was prepared by immunizing mice with centrifugal microsome fraction of rat hippocampal homogenate as antigen. Hippocampi from six adult rats were minced and homogenized in 10 mM Tris– HCl (pH 7.4) containing 320 mM sucrose and 1 mM MgCl2 by Potter-Elvehjem homogenizer. Homogenate was filtered through Nylon bolting cloth (pore size 40 mm) and centrifuged in 9–15% gradient OptiPrep e (Nycomed Pharma, Norway) at 18 000 £ g for 20 min at 48C by Beckman Optima XL-90 with swing rotor SW28. Microsome-rich fraction was collected, mixed with adjuvant, and injected to mice. The splenocytes from an immunized mouse were fused to myeloma NSO cells and the hybridomas were
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 00 81 5- 7
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obtained. Cloned hybridoma produced MAb2G7 was transplanted to mouse, intraperitoneal, and ascites was collected. For immunohistochemistry with MAb2G7, young adult male rats (2 months old, n ¼ 3) and middle-aged rats (12 months old, n ¼ 3) were deeply anesthetized and perfused with phosphate buffer saline (PBS) followed by 4% paraformaldehyde and 7% sucrose in PB (pH 7.4). The brains were removed, cut into several septo-temporal pieces, and fixed with the same fixative for an additional 4 h at 48C. To observe and count the cells clearly, the brains were embedded in paraffin and sectioned at 6 mm, and then they were put onto glass slides. Immunohistochemistry was performed using the avidin-biotin complex method with a Vector Laboratories kit (Burlingame, CA, USA). Sections were treated with the hightemperature epitope-activating method: the sections were serially autoclaved in 10 mM citric acid (pH 7.0) for 15 min at 1058C and for 15 min at 1168C. After rinsing with deionized water, these sections were immersed in 0.1% NaN3 in PBS for 10 min to block intrinsic peroxidase and blocked with Block Ace e (Dainippon Pharmaceuticals Co. Ltd., Japan) containing 10% normal goat serum for 1 h at room temperature (RT). The sections were incubated with diluted MAb2G7 ascites (1:500) for 3 days at 48C. After washing with PBS containing 0.05% Tween 20 (PBS/T), sections were incubated in biotinylated goat anti-mouse IgM (ICN Pharmaceuticals Inc., USA) for 2 h, then in avidin-biotin complex solution for 1 h at RT. The peroxidase reaction was carried out with 0.05% diaminobenzidine and 0.03% H2O2 in PBS/T for exactly 10 min at RT. The sections were dehydrated, mounted, and observed under light microscopy. In a control experiment, the first antibody was omitted, and no positive reactions were observed. The number of immunologically positive dots and the number of GCs in the dentate gyrus were counted and the rate per cell was calculated. Statistical significance was assessed using a one-way analysis of variance with region or age as factors, followed by a Student’s t-test. Data are expressed as mean ^ SE if not otherwise stated and P-values ,0.05 were considered significant. For an immunoelectron microscopic study, 0.05% glutaraldehyde was added to the fixative mentioned above and sectioned by microslicer, and detergent-free PBS was used instead of PBS/T. After immunohistochemical staining, the sections were additionally fixed with 2% osmium tetra-oxide in PB, dehydrated and flat-embedded in Spurr’s resin (TAAB, Germany) between acular films (Nissin EM, Japan). The sections were observed under light microscopy, trimmed, and ultra-thin sectioned by Ultra-Cut microtome (Leica, Germany). The immunoreactivity was observed with JEM 1200EX transmission electron microscopy at 80 kV. To detect the relative molecular weight of and age-related changes in the MAb2G7 antigen, immunoblotting was performed. The homogenized hippocampi of 2- and 12month old male rats, at a concentrations of 10 mg/ml, were electrophoresed in a 7.5% sodium dodecyl sulfate-
polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. The membrane was washed with PBS/T and blocked with Block Ace e for 1 h at RT. Diluted MAb2G7 (1:5000) was applied to the membrane and incubated for 2 h at RT. After washing, the membrane was incubated with peroxidase-conjugated goat antibody. Chemiluminescent detection was performed with ECL Plus e (Amersham Pharmacia Biotech, UK) according to the manufacturer’s protocol. A plot profile of the detected bands was processed using NIH-Image software (Scion Image, USA). Immunohistochemical localization of MAb2G7 in the dentate gyrus is shown in Fig. 1. MAb2G7 binding was observed in the GC layer of the SPB and IPB in 2-monthold rats (Figs. 1A,B), and appeared both as intense dots and as diffuse background staining in the GC cytoplasm. While numerous positive dots were observed in both blades, there were somewhat more in the IPB (Fig. 2). We counted approximately one to three per cell in the sections, but cells with no dots were also seen. Since less-positive cells were intermingled with cells containing several dots, the estimation of the number of dots per cell may not be exact, but may be sufficient to reflect significant differences in the populations between the blades. In the CA1 and CA3 other of the hippocampal formation, positive dots were observed but they were scarce and less intense than in the dentate gyrus of rats of the same age (Figs. 1E,F). Thus, the localization of numerous MAb2G7-positive dots may be a distinctive characteristic of dentate GCs. In fact, very few or no definitive MAb2G7-positive reactions were found in other brain regions such as cortex and thalamus, except for weak cytoplasmic staining of neurons. A remarkable difference in the numbers of MAb2G7-positive dots was found between young adult rats and middleaged rats (Figs. 1A–D and 2). MAb2G7-positive dots were not only decreased in quantity in 12-month-old rats compared to 2-month-old rats, but were also of weaker intensity. These quantitative declines were observed in both the SPB and IPB. Similar to the results in the young adult rats, in the older rats more MAb2G7-positive dots were seen in the IPB than in the SPB. The differences between the blades may therefore be maintained despite the decreasing amount of the antigen with age. These results may reflect differences in the strength of antigen processing or in activation between SPB and IPB neurons, and between younger and older rats. Immunoelectron microscopic observations indicated that MAb2G7 was localized exclusively on secondary lysosome-like structures (Fig. 1G). These structures showed membrane-delimited spheroid or ellipsoid bodies containing various sizes of droplets and folding lamella, and the MAb2G7 reaction product was distributed on the internal lipid droplets (Fig. 1G, insert). The reaction intensity reflected the droplet sizes and quantities. None of the positive reactions was observed in the first antibody omitted negative control (Fig. 1H). Since these droplets are likely to be derived from other organelles taken up by the second-
S. Maeda et al. / Neuroscience Letters 330 (2002) 275–279
ary lysosomes, the antigen recognized by MAb2G7 may be involved in the proteolysis of these organelles. Immunoblotting showed that the relative molecular weight of the antigen recognized with MAb2G7 was approximately 68 kDa (Fig. 3). The same band was detected in both 2- and 12-month-old rats, but the immunoreactivity was slightly less intense in the older rats. However, the difference in the intensity between the immunoblot bands in the rats of the two ages was not as great as the difference seen with immunohistochemistry.
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In the present study, we showed that the MAb2G7 antibody specifically recognized secondary lysosome-like structures in GCs of the rat dentate gyrus. The distribution of these structures differed quantitatively between the SPB and IPB, and declined with age. However, the location of MAb2G7 reactivity within the cell, and its relative distribution between the SPB and IPB, did not change with age. Therefore, the reduction of the histochemical reaction in relatively aged rats may be caused by a decline in production of the antigen in GCs. The adult dentate gyrus (consist-
Fig. 1. The MAb2G7 immunostaining of young adult and middle-aged rat hippocampus. MAb2G7-positive dots can be observed in the granule cell layers of the SPB (A); and the IPB (B) in 2-month-old rats. More immunopositive dots were detected in IPB granule cells than in SPB granule cells. The reactions decreased in number and intensity in the SPB (C); and IPB (D) of 12-month-old rats. In the pyramidal cell layers of CA1 (E); and CA3 (F) in 2-month-old rats, some positive reactions were detected, but they were lower in quantity and intensity than in the dentate gyrus. Bar ¼ 20 mm in A–F. (G) Immunoelectron microscopy of a MAb2G7-positive granule cell in 2-monthold rat IPB. Electron-dense 3,3 0 -diaminobenzidine precipitates were observed in the lysosome-like bodies of the cell (arrowheads). No reactions were observed in other types of organelles. A high magnification of the typical appearance of a positive organelle is shown in the inset. The positive organelle contains droplets and folding lamellae of various sizes, and the reactions are localized to the internal droplets. These structural peculiarities have a resemblance to secondary lysosomes or lipofuscin. (H) MAb2G7 omitted negative control image. A lysosomal granule (arrowhead) is somewhat dense, but no positive reaction is observed (insert). Bar ¼ 2 mm in G and H, and 200 nm in inserts. Abbreviations are as follows: ml, molecular layer; gl, granule cell layer; pl, polymorphic cell layer; so, stratum oriens; pcl, pyramidal cell layer; and sl, stratum lucidum.
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Fig. 2. A quantitative graph of the number of MAb2G7 positive dots per cell in the dentate gyrus of 2- and 12-month-old rats. We counted the number of positive dots and cells in the granule cell layers of the 6-mm thick sections in a field of vision under light microscopy. A total of 30–35 fields were counted for each animal (n ¼ 3). The stippled bar indicates SPB granule cells of 2-monthold rats, the bar with vertical lines indicates IPB granule cells of 2-month-old rats, the bar with horizontal lines indicates SPB granule cells of 12-month-old rats, and the bar with diagonal lines indicates IPB granule cells of 12-month-old rats. *P , 0:05 compared with SPB. **P , 0:05 compared with 2month-old rats.
ing SPB and IPB) is constantly renewed through a process of neurogenesis and migration of GCs in temporal-to-septal, and superficial-to-deep gradients [2,6,20]. Dendritic trees of GCs located in the SPB tend, on average, to be longer than those in the IPB [5,7], suggesting that the GCs in SPB and those in IPB may have different metabolic activities or functions during their development and maintenance. This is also supported by our finding that MAb2G7-positive lysosomal granules are distributed unevenly between the SPB
Fig. 3. Immunoblotting of rat hippocampus extracts reacted with the MAb2G7 antibody. Contents of the lanes are as follows: lane 1, young adult (2-month-old) rats; and lane 2, middle-aged (12month-old) rats. Immunoreactivity of the 68 kDa bands (arrowhead) was slightly decreased in quantity in middle-aged rats. The plot profile was analyzed using NIH Image software. The molecular weight was calculated from standard protein makers run in parallel (letters at right).
and IPB, although it remain to be clarified what percentage of secondary lysosomes are MAb2G7-positive in dentate gyrus GCs Neuronal lysosomes are sometimes involved in agedependent neuropathologies, due to lysosomal metabolic malfunction, such as accumulation of lipofuscin [10,13,17], deposition of amyloid precursor protein in Alzheimer’s disease [3,11,12], and gangliosidoses [9,19]. Age-related changes in the activity of the lysosomal enzyme cathepsin have been reported [4,18]. Nakanishi et al. Examined age-related changes in the lysosomal cathepsins, cathepsin D, B, and L. An age-related decline of activity was observed only for cathepsin L, whereas the activity of cathepsin D and B increased in aged rats [18]. Since immunohistochemical reactivity of cathepsin L showed only a slight reduction in aged rats, its localization and activity may not always be coincident with MAb2G7 reactivity. Bi et al. [4] showed that the expression of lysosomal cathepsin D and B changes in an age-related and regionally selective manner in the rat brain. This suggests that proteolysis in lysosomes may be regulated in age- and region-specific manners, and by extension, that the antigen recognized by MAb2G7 is involved in proteolysis in lysosomes. However, it is not known whether the antigen recognized by MAb2G7 is a native lysosomal enzyme or a substrate being processed in them. However, since not many molecules are known that are characteristic of dentate gyrus GCs, studying the molecular makeup and metabolic cascade of this antigen will be useful for learning about the regional and age-related metabolism of dentate GCs. There are three distinct temporal gradients in the early morphogenesis of the granular cell layer: SPB to IPB, caudal to rostral, and in all regions of the granular layer the more superficial neurons to the deeper ones [20]. GC progenitors have been shown to migrate from the dentate hilus region for at least 11 months postnatally [14], suggesting that a neurogenetic gradient of the granular neurons could be found for several months after birth. However, neurogenesis is drastically reduced in 27-month-old rats compared to 6-month-old controls [14]. The pattern of MAb2G7 antibody staining in the present study is consistent with this drastic postnatal reduction of the GC neurogenesis. Therefore, our novel antibody, MAb2G7 that can be used to visualize the lysosome-like granules in the GCs, might be an indicator of GC neurogenesis. [1] Amaral, D.G. and Witter, M.P., Hippocampal formation, In G. Paxinos (Ed.), The Rat Nervous System, 21, Academic Press, Inc, New York, 1995, pp. 443–493. [2] Bayer, S.A., Development of the hippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autoradiography, J. Comp. Neurol., 190 (1980) 87–114. [3] Benowitz, L.I., Rodriguez, W., Paskevich, P., Mufson, E.J., Schenk, D. and Neve, R.L., The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects, Exp. Neurol., 106 (1989) 237–250. [4] Bi, X., Yong, A.P., Zhou, J., Gall, C.M. and Lynch, G., Regionally selective changes in brain lysosomes occur in
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