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Expression of β-amyloid precursor protein in the developing human spinal cord
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BRAIN RESEARGH ELSEVIER
Brain Research 642 (1994) 132-136
Research Report
Expression of/3-amyloid precursor protein in the developing human spinal cord Hiroyuki Arai
a,b,*, Susumu Higuchi b, Sachio Matsushita b, Takefumi Yuzuriha b, John Q. Trojanowski c, Virginia M.-Y. Lee c
a Department of Neurology, Motojima General Hospital, Ota, Gunma 373, Japan, b Department of Psychiatry, National Institute On Alcoholism, Kurihama National Hospital, Yokosuka, Kanagawa 239, Japan, c Department of Pathology and Laboratory Medicine, Division of Anatomic Pathology, Hospital of University of Pennsylvania, PA 19104, USA (Accepted 16 December 1993)
Abstract
Human fetal spinal cords and other non-neural tissues from cases with gestational age from 6 to 21 weeks were examined with a panel of antibodies to different domains of/3-amyloid precursor proteins (fl-APPs). In the early developmental stages, the /3-APPs were expressed in three distinct layers, i.e., primitive neuroepithelial cell layer, mantle layer and marginal layer, fl-APP immunoreactivity was most prominent in cell bodies of putative neuroblasts located in the outer ventral part of the mantle layer. /3-APP expression diminished as the spinal cord matured and a weak residual immunoreactivity was detected exclusively in a subset of the anterior horn cells by 21 weeks gestational age. Throughout the gestational ages examined, no convincing fl/A4 immunostaining was seen in any of the spinal cord regions. Outside the spinal cord, /3-APP immunostaining was consistently present in (1) cell bodies and proximal nerves of immature neurons of dorsal root ganglia and in (2) myotubules, although these cells were devoid of fl/A4 immunoreactivity. Western blot analysis of fetal spinal cord revealed immunoreactive bands with apparant molecular weight between 100 and 140 kDa in the membrane-associated fraction, whi!e soluble proteins with a molecular mass centered on 115 kDa were detected in the cytosolic fraction. Our results indicate that: (1) one or more isoforms of full length /3-APPs are expressed at very early gestational ages in the developing human spinal cord; (2) the normal metabolism of/3-APPs does not result in accumulations of/3/A4 fragments.
Key words:/3-Amyloidprecursor protein; Human fetal spinal cord; Development; Immunostaining; Western blot analysis
1. Introduction
The brains of Alzheimer's disease (AD) patients are characterized by senile plaques (SPs), neurofibrillary tangles, neuropil threads and vascular amyloidosis in the central nervous system [24,29]. SPs are extracellular amyloid deposits consisting of 39-42 amino acid (aa) long peptides t e r m e d / 3 / A 4 [7]./3/A4 is derived from one or more larger precursor proteins, i.e., /3-amyloid precursor proteins (/3-APPs) [11]./3/A4 deposition has been noted in brains of aged monkeys, aged dogs and polar bears [10,17]. Notably, the /3-APPs and amyloid precursor-like proteins (APLPs) [20,26] have been
* Corresponding author at address a. Fax: (81) (22) 276-4682. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 6 4 3 - H
identified in a variety of tissues and cells including brains of rats, mice and even drosophila, suggesting that the /3-APPs a n d / o r APLPs are highly conserved proteins encoded by a multigene family and they probably are involved in important normal biological functions [12,15,22]. Furthermore, it is now known that /3/A4 is the product of normal metabolic events in human neurons and in the brains of AD patients as well as in the brains of normal elderly individuals [18,19,28]. These ideas naturally lead us to define the developmental a n d / o r evolutional expression of the /3-APPs. Recently, the evolutional origin of a Kunitztype protease inhibitor domain inserted in the /3-APP was analyzed [9]. Although mRNAs encoding the /3APPs have been demonstrated in human fetal tissues [11], /3-APP expression has not been examined at the protein level. The present study was undertaken to
1t. Arai et aL / Brain Research 642 (1994) 132-136
develop insights into the expression of the fl-APPs during human spinal cord development.
2. Materials and methods 2.1. Tissue collection, processing and immunohistochemistry Fifteen human fetuses without evidence of disease or developmental abnormalities, ranging in gestational age (GA) from 6 to 21 weeks were collected from therapeutic abortions following criteria recommended by Japanese Society of Obstetrics and Gynecology [8]. The protocols for the present study were approved by local ethical committee at Kurihama National Hospital. The samples included six cases with GA of 6-9 weeks, five cases with GA of 10-12 weeks and four cases with GAs ranging from 13-21 weeks. The GA was determined by menstrual history. The postmortem interval was approximately 2 to 4 h and tissues were fixed in 70% ethanol with 150 mM NaCl for 24-48 h as described previously [1,2,23]. The fetuses with GAs younger than 9 weeks were processed in toto. The spinal columns were dissected with intact spinal cords in other cases except for a case with GA of 21 weeks in which the spinal cord was obtained free of spinal column. These tissues were embedded in paraffin and 6 /~m thick tissue sections were cut for staining by immunoperoxidase procedures as described previously [3,4,23,25]. Some sections were pretreated with 0.1% trypsin for 30 min at 37°C prior to
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applying primary antibodies [21]. Additional spinal cord samples of several adult patients with no neurological diseases were obtained and processed as described earlier [2]. 2. 2. Antibodies probes
A mouse monoclonal antibody (mAb), termed LN21, was raised to full-length 695 aa isoform of fl-APPs (fl-APP695) expressed in E. coli infected with baculovirus containing the coding sequence for /3-APP695 and was extensively characterized and described elsewhere [2,14]. The LN21 appears to recognize epitopes within 100 aa of N-terminal region of/3-APP69s (ref. 2, see also Lee et al., paper in preparation) and this mAb stains dystrophic neurites within senile plaques, neurons and their processes, extracellular tangles, glia cells and Lewy bodies [3,4]. An additional, well characterized mAb to the N-terminal epitope of /3-APPs (clone 22Cll) was purchased from Boehringer Mannheim [27]. A mouse mAb AMY33 and a rabbit antiserum UP107, raised to 1-28 and 1-42 aa of /3/A4 protein, respectively, were described previously [1,21]. A rabbit antiserum to 666-695 aa of /3-APP69s (W61C) was a generous gift from Dr. Yamaguchi and it was extensively characterized [12]. However, no antibodies are able to reliably distinguish each of the fl-APPs and the APLPs, except anti-/3/A4 antibodies which recognize fl-APPs, but not APLPs. Several antibodies to neuronal cytoskeletal proteins were employed to monitor neuroaxonal development as described [23]. They included RMO24, RMO254, HO14 and Alz50 which have been extensively described elsewhere [13,23].
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Fig. 1. lmmunostaining for /3-APP at different stages of maturation. Sections were immunolabeled with LN21 and counterstained with hematoxylin. A: spinal cord of GA of 8 weeks, Note that the fl-APP immunostaining was most intense in the outer ventral part of the mantle layer (indicated by arrow)• x 30; B: higher magnification of the lateral ventral aspect of the mantle layer (mt) in (A). mg: marginal layer. × 290; C: GA of 11 weeks. Fine punctate profiles were observed in diffusely stained cell bodies of anterior horn cells, x 720; D: GA of 6 weeks. Putative neuroblasts within dorsal root ganglia were intensely stained• x720; E,F: GA of 11 weeks. A subset of myoblasts was /3-APP positive. E: longitudinal section, X 290, F: transverse section, x 290.
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2.3. Westernblot analysis A spinal cord obtained from a case with a GA of 19 week was frozen at -80°C until use. An autopsied brain of 72-year old male with no neurological disease also was obtained as a control. Samples were prepared in 0.1 M MES buffer as described previously [2]. Briefly, the tissues were homogenizedin 10 vol of 0.1 M MES buffer containing 0.75 M NaCI, 0.5 mM MgCI2, 1 mM EGTA, 1 mM dithiothreitol and other protease inhibitors at pH 7.0 followed by centrifugation at 100,000 g for 60 min. The pellet was washed in 10 vol of 30% of sucrose/0.1 M MES buffer and centrifuged at 100,000 g for 60 rain. Both the supernatant and the pellet were dissolved in sample buffer and run in SDS-7.5% polyacrylamidegels, transferred to nitrocellulose replicas and probed with antibodies, as described elsewhere [1,2,25].
3. Results
3.1. Immunohistochemistry (1) [3-amyloid precursor protein and [ 3 / A 4 immunohistochemistry in the developing human spinal cord In the immunohistochemical study, comparable results were obtained with LN21 and 22Cll. In cases with G A of 6 - 9 weeks, the fl-APP immunoreactivity was present in three distinct layers, i.e., the primitive neuroepithelial layer, the mantle layer and the marginal layer. Putative neuroblasts located in the outer ventral part of the mantle layer were labeled more prominently than those located in inner dorsal region (Fig. 1A). Immature axons in the marginal layer (which develops into the white matter in later embryonic stages) also were immunoreactive (Fig. 1B). During the maturation of human spinal cord, the /3-APP immunoreactivity was further localized to the outer ventral neuroblasts, whereas immunostaining in the other spinal cord regions diminished. Cell bodies and possibly proximal processes of the neuroblasts were labeled in a diffuse manner which was focally punctate (Fig. 1C). Presumptive gila cells were unlabeled. At a G A of 21 weeks, a residual immunoreactivity was detected in neuronal cell bodies and proximal processes of a subset of anterior horn cells. However, the immunoreactivity f o r / 3 - A P P was markedly diminished in other cells of both gray and white matter. In adult spinal anterior horn cells, neuronal cell bodies as well as proximal processes also were immunopositive [2]. W61C labeled profiles similar to those produced by LN21 and 22C11. Finally, no convincing / 3 / A 4 immunostaining with AMY33 and UP107 was present throughout the GAs examined here.
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Fig. 2. Detection of /3-APPs in normal adult brain (1) and fetal spinal cord at a GA of 19 weeks (2) in Western blot. 100,000 g pellet (P) and supernatant (S) were separately analyzed. Approximately45 p.g of protein was loaded in each lane. Molecular mass markers are on the left (KD). The blots were incubated with 22Cll (1:500) as a primary antibody. Comparable results were obtained with LN21. processed in toto. Outside the spinal cord, /3-APP immunoreactivity was consistently present: in (1) cell bodies and proximal nerves of neurons in dorsal root ganglia (Fig. 1D) and in (2) myotubules (Fig. 1E,F). In the dorsal root ganglia, nearly all ganglion ceils yielded a diffuse cytoplasmic staining. The fl-APP immunostaining was more prominent in early embryonic stages than that produced at later stages, but it was still evident in the 21 week dorsal root ganglia as well as in adult ganglia [2]. Distal nerve bundles were weakly labeled. Notably, intense immunoreactive product was seen in muscle fibers. The immunoreactivity for/3-APP was present only in a subset of myotubules and even within a single muscle fiber fl-APP was focally expressed. Finally, positive fl-APP immunostairiing was never observed in intestine, lung, bone, skin and cardiac muscle tissues, fl-APP immunostaining in liver was equivocal. No convincing [3/A4 immunoreactive product was detected in any of these fetal tissues with AMY33 and UP107.
3.2. Western blot analysis of fetal spinal cord As shown in Fig. 2 (right), immunoblotting analysis verified the presence of several intact immunoreactive /3-APP bands which migrated between 100 and 140 kDa in the membrane-associated fraction of fetal spinal cord. In contrast, soluble proteins with a molecular weight centered approximately on 115 kDa were detected in the cytosolic fractions and these proteins may correspond to secreted amino-terminal /3-APP fragments. These results were comparable with those found in normal adult brain (Fig. 2, left).
(2) [3-Amyloid precursor protein immunohistochemistry outside the spinal cord
4. Discussion
Peripheral tissues also were examined with the antibodies to different domains of/3-APPs mainly in cases with GAs younger than 9 week where the fetuses were
This is the first study to describe the developmental expression and distribution of /3-APPs in the human
H. Arai et al. /Brain Research 642 (1994) 132-136
fetal spinal cord at the different stages of maturation. Human neurogenesis and gliogenesis is a highly orchestrated processes in which neuron-specific and gliaspecific gene products are differentially induced [23]. Although postmortem proteolysis is a potential methodological problem inherent in the kind of study described here, our efforts were focused on short postmortem intervals (i.e., 2-4 h). Further, to better preserve the antigenicity of /3-APPs we used ethanol - 150 mM NaC1 as the fixative, since this has been shown to optimally retain/3-APP immunoreactivity [1,2]. It was shown here that one or more isoforms of full length/3-APP extending from amino to carboxy terminus was expressed in both white and gray matter in the early stages of spinal cord development, but the expression dinimished with maturation and it was localized exclusively to the anterior horn cells as the spinal cord matured. This work is an extension of previous studies by Tohyama et al. [23] and Lee et al. [13] in which the onset of expression of several of the most well-characterized developmentally regulated neuronal and glial proteins were extensively observed at different GAs. These proteins include neurofilament (NF) triplet proteins, microtubule-associated protein (MAP)-2, MAP-5 and normal ~', modified AD ~- protein (A68), p75 nerve growth factor (p75NGF) receptor, synaptic proteins and glial proteins. Of these proteins, the expression pattern of /3-APP is similar to that of the p75NGF receptor, MAP-5, z and A68, whereas it is quite different from that of NF, MAP-2, synaptic and glial proteins. It has been suggested that /3-APP might be involved in the process of neuritic outgrowth either as a trophic factor or adhesion molecule [16]. In fact,/3-APP mediates neuronal cell to cell and cell surface adhesion [5]. Notably, /3-APP contains features characteristic of a cell surface receptor [11]. Taking these findings together, the present study suggests that /3-APPs might be developmentally regulated molecules and fundamentally required for neurite outgrowth especially at early maturational stages. Another important finding is that AMY33 and UP107-positive SPs or other types of deposits were never found in the spinal cord or other tissues at any of the GAs studied here. This is also the case in adult non-neural peripheral tissues [2]. In addition, /3-APP a n d / o r its fragments were shown to be secreted as soluble proteins by Western blot analysis. It is possible that the/3-APP might be cleaved within /3/A4 region by constitutive secretary pathway [6], thus leaving no accumulation of amyloidogenic proteins in the developing human spinal cord. On the other hand, it is likely that /3/A4 is being produced and secreted as a byproduct of normal metabolic pathways in the fetal spinal cord as is known to occur in the adult central nervous system [18,19], while the function of normally
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produced /3/A4 and /3-APPs remain elusive. The developmentally regulated, restricted expression of /3APP695 by neurons as they attain maturity suggests a role for /3-APP695 in the acquisition of the mature neuronal phenotype [28].
5. Acknowledgements We are grateful to Dr. Konishi, K. and Niimi, Y. for helping us to collect human aborted fetuses. Appreciation also is expressed to Dr. Motojima, T. for his presidential effort to foster our research. Dr. Davies, P. and Dr. Yamaguchi, H. kindly provided the Alz50 and W61C, respectively. We wish to add special thanks to patients and their families who made our research possible. This research was supported in part by grant provided by the Ministry of Health and Welfare, Japan.
Note added in proof. After submission of this paper, M. Ohta et al. have described the developmental expression of/3-APP like proteins in the rat brain: Dev. Brain Res., 75 (1993) 151-161.
6. References [1] Arai, H., Lee, V.M.-Y., Otvos Jr., L. Greenberg, B.D., Lowery, D.E., Sharma, S.K., Schmidt, M.L. and Trojanowski, J.Q., Defined neurofilament, ~- and /3-amyloid precursor protein epitopes distinguish Alzheimer from non-Alzheimer senile plaques, Proc. Natl. Acad. Sci. USA, 87 (1990) 2249-2253. [2] Arai, H., Lee, V.M.-Y., Messinger, M.L., Greenberg, B.D., Lowery, D.E. and Trojanowski, J.Q., Expression patterns of /3-amyloid precursor protein (/3-APP) in neural and nonneural human tissues from Alzheimer's disease and control subjects, Ann. Neurol., 30 (1991) 686-693. [3] Arai, H., Schmidt, M.L., Lee, V.M.-Y., Hurtig, H.I., Greenberg, B.D., Adler, C.H. and Trojanowski, J.Q., Epitope analysis of senile plaque components in the hippocampus of patients with Parkinson's disease, Neurology, 42 (1992) 1315-1322. [4] Arai, H., Lee, V.M.-Y., Hill, W.D., Greenberg, B.D. and Trojanowski, J.Q., Lewy bodies contain beta-amyloid precursor proteins of Alzheimer's disease, Brain Res., 585 (1992) 386-390. [5] Breen, K.C., Bruce, M. and Anderton, B.H., Beta amyloid precursor protein mediates neuronal cell-cell and cell-surface adhesion, J. Neurosci. Res., 28 (1991) 90-100. [6] Esch, F.S., Keim, P.S., Beattie, E.C., Blacker, R.W., Culwell, A.R., Oltersdolf, T., McClure, D. and Ward, P.J., Cleavage of amyloid /3 peptide during constitutive processing of its precursor, Science, 248 (1990) 1122-1124. [7] Glenner, G.G. and Wong, C.W., Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular fbril protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. [8] Iizuka, R., Criteria for use of aborted fetuses (English translation), Acta. Obst. Gynaecol. Jpn., 45 (1993) 25. [9] Ikeo, K., Takahashi, K. and Gojobori, T., Evolutional origin of a kunitz-type trypsin inhibitor domain inserted in the amyloid /3 precursor protein of Alzheimer's disease, J. Mol. Evol., 34 (1992) 536-543. [10] Ishihara, T., Gondo, T., Takahashi, M., Uchino, F., Ikeda, S., Allsop, D. and Imai, K., Immunohistochemical and immuno-
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electron microscopical characterization of cerebrovascular and senile plaque amyloid in aged dogs' brains, Brain Res., 548 (1991) 196-205. [11] Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeshik, K.H., Multhaup, G., Beyreuther, K. and Muller-Hill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell surface receptor, Nature, 325 (1987) 733-736. [12] Kawarabayashi, T., Shoji, M., Harigaya, Y., Yamaguchi, H. and Hirai, S., Amyloid/3/A4 protein precursor is widely distributed in both the central and peripheral nervous systems of the mouse, Brain Res., 552 (1991) 1-7. [13] Lee, J.H., Goedert, M., Hill, W.D., Lee, V.M.-Y. and Trojanowski, J.Q., Tau proteins are abnormally expressed in olfactory epithelium of Alzheimer patients and developmentally regulated in human fetal spinal cord, Exp. Neurol., 121 (1993) 93-105. [14] Lowery, D.E., Pasternak, J., Gonzalez-DeWhitt, P.A., ZurcherNeely, H., Tomich, C.-S.C., Altman, R.A., Fairbanks, M.B., Heinrikson, R.L., Younkin, S.G. and Greenberg, B.D., Alzheimer amyloid precursor protein produced by recombinant baculovirus expression: proteolytic processing and protease inhibitory properties, J. Biol. Chem., 226 (1991) 19842-19850. [15] Luo, L., Tully, T. and White, K., Human amyloid precursor protein ameliorates behavioral deficit of flies deleted for Appl gene, Neuron, 9 (1992) 595-605. [16] Masliah, E., Mallory, M., Ge, N. and Saitoh, T., Amyloid precursor protein is localized in growing neurites of neonatal rat brain, Brain Res., 593 (1992) 323-328. [17] Selkoe, D.J., Bell, D.S., Podlisny, M.B., Price, D.J. and Cork, L.C., Conservation of brain amyloid proteins in aged mammals and humans with Alzheimer's disease, Science, 235 (1987) 873877. [18] Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J. and Swindlehurst, C., MeCormack, R., Wolfert, R., Selkoe, D., Lieberburg, I., Schenk, D., Isolation and quantification of soluble Alzheimer's/3-peptide from biological fluids, Nature, 359 (1992) 325-327. [19] Shoji, M., Golde, T.E., Ghiso, J., Cheung, T.T., Estus, S., Shaffer, L.M., Cai, X.-D., McKay, D.M., Tintner, R., Frangione, B. and Younkin, S.G., Production of the Alzheimer amyloid /3 protein by normal proteolytic processing, Science, 258 (1992) 126-129.
[20] Sprecher, C.A., Grant, F.J., Grimm, G., O'Hara, P.J., Norris, F., Norris, K. and Foster, D.C., Molecular cloning of the cDNA for a human amyloid precursor protein homolog: Evidence eor a multugene family, Biochemistry, 32 (1993) 4481-4486. [21] Stern, R.A., Otvos Jr., L., Trojanowski, J.Q. and Lee, V.M.-Y., Monoclonal antibodies to a synthetic peptide homologous with the first 28 amino acids of Alzheimer's disease/3-protein recognize amyloid and diverse glial and neuronal cell types in the central nervous system, Am. J. Pathol., 134 (1989) 973-978. [22] Takio, K., Hasegawa, M., Titani, K. and Ihara, Y., Identification of/3 protein precursor in newborn rat brain, Biochem. Biophys. Res. Commun., 160 (1989) 1296-1301. [23] Tohyama, T., Lee, V.M.-Y., Rorke, L.B. and Trojanowski, J.Q., Molecular milestones that signal axonal maturation and the commitment of human spinal cord precursor cells to the neuronal or glial phenotype in development, J. Comp. Neurol., 310 (1991) 285-299. [24] Trojanowski, J.Q., Schmidt, M.L., Otvos, L. Jr, Arai, H., Hill, W.D. and Lee, V.M.-Y., Vulnerability of the neuronal cytoskeleton in aging and Alzheimer's disease: widespread involvement of all three major filament systems, Annu. Rev. Gerontol. Geriatr., 10 (1990) 167-182. [25] Trojanowski, J.Q., Schuck, T., Schmidt, M.L. and Lee, V.M.-Y., Distribution of phosphate-independent MAP2 epitopes revealed with monoclonal antibodies in microwave-denatured human nervous system tissues, Z Neurosci. Methods, 29 (1989) 171-180. [26] Wasco, W., Gurubhagavatula, S., Paradis, M.D., Romano, D.M., Sisodia, S.S., Hyman, B.T., Neve, R.L. and Tanzi, R.E, Isolation and characterization of APLP2 encoding a homologue of the Alzheimer's associated amyloid /3 protein precursor, Nature genetics, 5 (1993) 95-100. [27] Weidemann, A., Konig, G., Bunke, D., Fisher, P., Salbaum, J.M., Masters, C.L. and Beyreuther, K., Identification, biogenesis and localization of precursor of Alzheimer's disease A4 amyloid protein, Cell, 57 (1989) 115-126. [28] Wertkin, A.M., Turner, R.S., Pleasure, S.J., Golde, T.E., Younkin, S.G., Trojanowski, J.Q. and Lee, V.M.-Y., Human neurons derived from a teratocarcinoma cell line express solely APP695 and produce intracellular/3/A4 peptides, Proc. Natl. Acad. Sci. USA, 90 (1993) 9513-9517. [29] Yankner, B.A. and Mesulam, M.M., /3-amyloid and the pathogenesis of Alzheimer's disease, N. Engl. J. Med., 325 (1991) 1849-1857.
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V
BRAIN RESEARGH ELSEVIER
Brain Research 642 (1994) 132-136
Research Report
Expression of/3-amyloid precursor protein in the developing human spinal cord Hiroyuki Arai
a,b,*, Susumu Higuchi b, Sachio Matsushita b, Takefumi Yuzuriha b, John Q. Trojanowski c, Virginia M.-Y. Lee c
a Department of Neurology, Motojima General Hospital, Ota, Gunma 373, Japan, b Department of Psychiatry, National Institute On Alcoholism, Kurihama National Hospital, Yokosuka, Kanagawa 239, Japan, c Department of Pathology and Laboratory Medicine, Division of Anatomic Pathology, Hospital of University of Pennsylvania, PA 19104, USA (Accepted 16 December 1993)
Abstract
Human fetal spinal cords and other non-neural tissues from cases with gestational age from 6 to 21 weeks were examined with a panel of antibodies to different domains of/3-amyloid precursor proteins (fl-APPs). In the early developmental stages, the /3-APPs were expressed in three distinct layers, i.e., primitive neuroepithelial cell layer, mantle layer and marginal layer, fl-APP immunoreactivity was most prominent in cell bodies of putative neuroblasts located in the outer ventral part of the mantle layer. /3-APP expression diminished as the spinal cord matured and a weak residual immunoreactivity was detected exclusively in a subset of the anterior horn cells by 21 weeks gestational age. Throughout the gestational ages examined, no convincing fl/A4 immunostaining was seen in any of the spinal cord regions. Outside the spinal cord, /3-APP immunostaining was consistently present in (1) cell bodies and proximal nerves of immature neurons of dorsal root ganglia and in (2) myotubules, although these cells were devoid of fl/A4 immunoreactivity. Western blot analysis of fetal spinal cord revealed immunoreactive bands with apparant molecular weight between 100 and 140 kDa in the membrane-associated fraction, whi!e soluble proteins with a molecular mass centered on 115 kDa were detected in the cytosolic fraction. Our results indicate that: (1) one or more isoforms of full length /3-APPs are expressed at very early gestational ages in the developing human spinal cord; (2) the normal metabolism of/3-APPs does not result in accumulations of/3/A4 fragments.
Key words:/3-Amyloidprecursor protein; Human fetal spinal cord; Development; Immunostaining; Western blot analysis
1. Introduction
The brains of Alzheimer's disease (AD) patients are characterized by senile plaques (SPs), neurofibrillary tangles, neuropil threads and vascular amyloidosis in the central nervous system [24,29]. SPs are extracellular amyloid deposits consisting of 39-42 amino acid (aa) long peptides t e r m e d / 3 / A 4 [7]./3/A4 is derived from one or more larger precursor proteins, i.e., /3-amyloid precursor proteins (/3-APPs) [11]./3/A4 deposition has been noted in brains of aged monkeys, aged dogs and polar bears [10,17]. Notably, the /3-APPs and amyloid precursor-like proteins (APLPs) [20,26] have been
* Corresponding author at address a. Fax: (81) (22) 276-4682. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 6 4 3 - H
identified in a variety of tissues and cells including brains of rats, mice and even drosophila, suggesting that the /3-APPs a n d / o r APLPs are highly conserved proteins encoded by a multigene family and they probably are involved in important normal biological functions [12,15,22]. Furthermore, it is now known that /3/A4 is the product of normal metabolic events in human neurons and in the brains of AD patients as well as in the brains of normal elderly individuals [18,19,28]. These ideas naturally lead us to define the developmental a n d / o r evolutional expression of the /3-APPs. Recently, the evolutional origin of a Kunitztype protease inhibitor domain inserted in the /3-APP was analyzed [9]. Although mRNAs encoding the /3APPs have been demonstrated in human fetal tissues [11], /3-APP expression has not been examined at the protein level. The present study was undertaken to
1t. Arai et aL / Brain Research 642 (1994) 132-136
develop insights into the expression of the fl-APPs during human spinal cord development.
2. Materials and methods 2.1. Tissue collection, processing and immunohistochemistry Fifteen human fetuses without evidence of disease or developmental abnormalities, ranging in gestational age (GA) from 6 to 21 weeks were collected from therapeutic abortions following criteria recommended by Japanese Society of Obstetrics and Gynecology [8]. The protocols for the present study were approved by local ethical committee at Kurihama National Hospital. The samples included six cases with GA of 6-9 weeks, five cases with GA of 10-12 weeks and four cases with GAs ranging from 13-21 weeks. The GA was determined by menstrual history. The postmortem interval was approximately 2 to 4 h and tissues were fixed in 70% ethanol with 150 mM NaCl for 24-48 h as described previously [1,2,23]. The fetuses with GAs younger than 9 weeks were processed in toto. The spinal columns were dissected with intact spinal cords in other cases except for a case with GA of 21 weeks in which the spinal cord was obtained free of spinal column. These tissues were embedded in paraffin and 6 /~m thick tissue sections were cut for staining by immunoperoxidase procedures as described previously [3,4,23,25]. Some sections were pretreated with 0.1% trypsin for 30 min at 37°C prior to
133
applying primary antibodies [21]. Additional spinal cord samples of several adult patients with no neurological diseases were obtained and processed as described earlier [2]. 2. 2. Antibodies probes
A mouse monoclonal antibody (mAb), termed LN21, was raised to full-length 695 aa isoform of fl-APPs (fl-APP695) expressed in E. coli infected with baculovirus containing the coding sequence for /3-APP695 and was extensively characterized and described elsewhere [2,14]. The LN21 appears to recognize epitopes within 100 aa of N-terminal region of/3-APP69s (ref. 2, see also Lee et al., paper in preparation) and this mAb stains dystrophic neurites within senile plaques, neurons and their processes, extracellular tangles, glia cells and Lewy bodies [3,4]. An additional, well characterized mAb to the N-terminal epitope of /3-APPs (clone 22Cll) was purchased from Boehringer Mannheim [27]. A mouse mAb AMY33 and a rabbit antiserum UP107, raised to 1-28 and 1-42 aa of /3/A4 protein, respectively, were described previously [1,21]. A rabbit antiserum to 666-695 aa of /3-APP69s (W61C) was a generous gift from Dr. Yamaguchi and it was extensively characterized [12]. However, no antibodies are able to reliably distinguish each of the fl-APPs and the APLPs, except anti-/3/A4 antibodies which recognize fl-APPs, but not APLPs. Several antibodies to neuronal cytoskeletal proteins were employed to monitor neuroaxonal development as described [23]. They included RMO24, RMO254, HO14 and Alz50 which have been extensively described elsewhere [13,23].
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Fig. 1. lmmunostaining for /3-APP at different stages of maturation. Sections were immunolabeled with LN21 and counterstained with hematoxylin. A: spinal cord of GA of 8 weeks, Note that the fl-APP immunostaining was most intense in the outer ventral part of the mantle layer (indicated by arrow)• x 30; B: higher magnification of the lateral ventral aspect of the mantle layer (mt) in (A). mg: marginal layer. × 290; C: GA of 11 weeks. Fine punctate profiles were observed in diffusely stained cell bodies of anterior horn cells, x 720; D: GA of 6 weeks. Putative neuroblasts within dorsal root ganglia were intensely stained• x720; E,F: GA of 11 weeks. A subset of myoblasts was /3-APP positive. E: longitudinal section, X 290, F: transverse section, x 290.
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2.3. Westernblot analysis A spinal cord obtained from a case with a GA of 19 week was frozen at -80°C until use. An autopsied brain of 72-year old male with no neurological disease also was obtained as a control. Samples were prepared in 0.1 M MES buffer as described previously [2]. Briefly, the tissues were homogenizedin 10 vol of 0.1 M MES buffer containing 0.75 M NaCI, 0.5 mM MgCI2, 1 mM EGTA, 1 mM dithiothreitol and other protease inhibitors at pH 7.0 followed by centrifugation at 100,000 g for 60 min. The pellet was washed in 10 vol of 30% of sucrose/0.1 M MES buffer and centrifuged at 100,000 g for 60 rain. Both the supernatant and the pellet were dissolved in sample buffer and run in SDS-7.5% polyacrylamidegels, transferred to nitrocellulose replicas and probed with antibodies, as described elsewhere [1,2,25].
3. Results
3.1. Immunohistochemistry (1) [3-amyloid precursor protein and [ 3 / A 4 immunohistochemistry in the developing human spinal cord In the immunohistochemical study, comparable results were obtained with LN21 and 22Cll. In cases with G A of 6 - 9 weeks, the fl-APP immunoreactivity was present in three distinct layers, i.e., the primitive neuroepithelial layer, the mantle layer and the marginal layer. Putative neuroblasts located in the outer ventral part of the mantle layer were labeled more prominently than those located in inner dorsal region (Fig. 1A). Immature axons in the marginal layer (which develops into the white matter in later embryonic stages) also were immunoreactive (Fig. 1B). During the maturation of human spinal cord, the /3-APP immunoreactivity was further localized to the outer ventral neuroblasts, whereas immunostaining in the other spinal cord regions diminished. Cell bodies and possibly proximal processes of the neuroblasts were labeled in a diffuse manner which was focally punctate (Fig. 1C). Presumptive gila cells were unlabeled. At a G A of 21 weeks, a residual immunoreactivity was detected in neuronal cell bodies and proximal processes of a subset of anterior horn cells. However, the immunoreactivity f o r / 3 - A P P was markedly diminished in other cells of both gray and white matter. In adult spinal anterior horn cells, neuronal cell bodies as well as proximal processes also were immunopositive [2]. W61C labeled profiles similar to those produced by LN21 and 22C11. Finally, no convincing / 3 / A 4 immunostaining with AMY33 and UP107 was present throughout the GAs examined here.
KD
// 2
1
200"-"
97" P
S
P
S
Fig. 2. Detection of /3-APPs in normal adult brain (1) and fetal spinal cord at a GA of 19 weeks (2) in Western blot. 100,000 g pellet (P) and supernatant (S) were separately analyzed. Approximately45 p.g of protein was loaded in each lane. Molecular mass markers are on the left (KD). The blots were incubated with 22Cll (1:500) as a primary antibody. Comparable results were obtained with LN21. processed in toto. Outside the spinal cord, /3-APP immunoreactivity was consistently present: in (1) cell bodies and proximal nerves of neurons in dorsal root ganglia (Fig. 1D) and in (2) myotubules (Fig. 1E,F). In the dorsal root ganglia, nearly all ganglion ceils yielded a diffuse cytoplasmic staining. The fl-APP immunostaining was more prominent in early embryonic stages than that produced at later stages, but it was still evident in the 21 week dorsal root ganglia as well as in adult ganglia [2]. Distal nerve bundles were weakly labeled. Notably, intense immunoreactive product was seen in muscle fibers. The immunoreactivity for/3-APP was present only in a subset of myotubules and even within a single muscle fiber fl-APP was focally expressed. Finally, positive fl-APP immunostairiing was never observed in intestine, lung, bone, skin and cardiac muscle tissues, fl-APP immunostaining in liver was equivocal. No convincing [3/A4 immunoreactive product was detected in any of these fetal tissues with AMY33 and UP107.
3.2. Western blot analysis of fetal spinal cord As shown in Fig. 2 (right), immunoblotting analysis verified the presence of several intact immunoreactive /3-APP bands which migrated between 100 and 140 kDa in the membrane-associated fraction of fetal spinal cord. In contrast, soluble proteins with a molecular weight centered approximately on 115 kDa were detected in the cytosolic fractions and these proteins may correspond to secreted amino-terminal /3-APP fragments. These results were comparable with those found in normal adult brain (Fig. 2, left).
(2) [3-Amyloid precursor protein immunohistochemistry outside the spinal cord
4. Discussion
Peripheral tissues also were examined with the antibodies to different domains of/3-APPs mainly in cases with GAs younger than 9 week where the fetuses were
This is the first study to describe the developmental expression and distribution of /3-APPs in the human
H. Arai et al. /Brain Research 642 (1994) 132-136
fetal spinal cord at the different stages of maturation. Human neurogenesis and gliogenesis is a highly orchestrated processes in which neuron-specific and gliaspecific gene products are differentially induced [23]. Although postmortem proteolysis is a potential methodological problem inherent in the kind of study described here, our efforts were focused on short postmortem intervals (i.e., 2-4 h). Further, to better preserve the antigenicity of /3-APPs we used ethanol - 150 mM NaC1 as the fixative, since this has been shown to optimally retain/3-APP immunoreactivity [1,2]. It was shown here that one or more isoforms of full length/3-APP extending from amino to carboxy terminus was expressed in both white and gray matter in the early stages of spinal cord development, but the expression dinimished with maturation and it was localized exclusively to the anterior horn cells as the spinal cord matured. This work is an extension of previous studies by Tohyama et al. [23] and Lee et al. [13] in which the onset of expression of several of the most well-characterized developmentally regulated neuronal and glial proteins were extensively observed at different GAs. These proteins include neurofilament (NF) triplet proteins, microtubule-associated protein (MAP)-2, MAP-5 and normal ~', modified AD ~- protein (A68), p75 nerve growth factor (p75NGF) receptor, synaptic proteins and glial proteins. Of these proteins, the expression pattern of /3-APP is similar to that of the p75NGF receptor, MAP-5, z and A68, whereas it is quite different from that of NF, MAP-2, synaptic and glial proteins. It has been suggested that /3-APP might be involved in the process of neuritic outgrowth either as a trophic factor or adhesion molecule [16]. In fact,/3-APP mediates neuronal cell to cell and cell surface adhesion [5]. Notably, /3-APP contains features characteristic of a cell surface receptor [11]. Taking these findings together, the present study suggests that /3-APPs might be developmentally regulated molecules and fundamentally required for neurite outgrowth especially at early maturational stages. Another important finding is that AMY33 and UP107-positive SPs or other types of deposits were never found in the spinal cord or other tissues at any of the GAs studied here. This is also the case in adult non-neural peripheral tissues [2]. In addition, /3-APP a n d / o r its fragments were shown to be secreted as soluble proteins by Western blot analysis. It is possible that the/3-APP might be cleaved within /3/A4 region by constitutive secretary pathway [6], thus leaving no accumulation of amyloidogenic proteins in the developing human spinal cord. On the other hand, it is likely that /3/A4 is being produced and secreted as a byproduct of normal metabolic pathways in the fetal spinal cord as is known to occur in the adult central nervous system [18,19], while the function of normally
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produced /3/A4 and /3-APPs remain elusive. The developmentally regulated, restricted expression of /3APP695 by neurons as they attain maturity suggests a role for /3-APP695 in the acquisition of the mature neuronal phenotype [28].
5. Acknowledgements We are grateful to Dr. Konishi, K. and Niimi, Y. for helping us to collect human aborted fetuses. Appreciation also is expressed to Dr. Motojima, T. for his presidential effort to foster our research. Dr. Davies, P. and Dr. Yamaguchi, H. kindly provided the Alz50 and W61C, respectively. We wish to add special thanks to patients and their families who made our research possible. This research was supported in part by grant provided by the Ministry of Health and Welfare, Japan.
Note added in proof. After submission of this paper, M. Ohta et al. have described the developmental expression of/3-APP like proteins in the rat brain: Dev. Brain Res., 75 (1993) 151-161.
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