- Email: [email protected]
Fibroblast growth factor (FGF)-9 immunoreactivity in senile plaques
Brain Research 814 Ž1998. 222–225
Short communication
Fibroblast growth factor ŽFGF .-9 immunoreactivity in senile plaques Satoshi Nakamura a
a,b,)
, Kunimasa Arima a , Seiichi Haga a , Takako Aizawa a , Yumiko Motoi a , Mieko Otsuka b , Akira Ueki b , Kazuhiko Ikeda a
Department of Ultrastructure and Histochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya, Tokyo 156-8585, Japan b Department of Neurology, Jichi Medical School Omiya Medical Center, Omiya, Japan Accepted 29 September 1998
Abstract We examined fibroblast growth factor ŽFGF.-9 immunoreactivity in human hippocampal sections of Alzheimer’s disease ŽAD.. FGF-9 immunoreactivity was observed in dystrophic neurites of senile plaques in AD and control cases, in addition to the hippocampal and cortical neurons. The amyloid core and neurofibrillary tangles lacked immunoreactivity. FGF-9 immunoreactive astrocytes were conspicuous in AD brains. FGF-9 may be involved in the neuropathology of AD. q 1998 Elsevier Science B.V. All rights reserved. Keywords: FGF-9 immunoreactivity; Fibroblast growth factor; Alzheimer’s disease; Senile plaque; Dystrophic neurite
Fibroblast growth factor ŽFGF.-9 was initially purified from the culture medium of human glioma cell line NMCG1 w12x. FGF-9 is a heparin-binding and secretory growth factor despite its lack of a signal sequence in N-terminus w10,12x. It has been reported that FGF-9 mRNA is expressed in neurons of widespread regions of rat brain w20x. Recently, we reported that FGF-9 immunoreactivity ŽFGF9-IR. was present mainly in neurons of widespread regions of human and rat brain w21x. Since basic FGFrFGF-2 w3,19x and acidic FGFrFGF-1 w22,26x immunoreactivity has been found in association with Alzheimer’s disease ŽAD. neuropathology, we performed immunohistochemistry of human autopsy brain by using a specific anti-FGF-9 antibody. Hippocampal sections including occipitotemporal neocortex from 10 AD cases and 10 age-matched controls with non-neurological diseases were cut from formalinfixed, paraffin-embedded blocks, and subjected to immunohistochemistry. A rabbit polyclonal antibody against a synthetic peptide corresponding to amino acid residues 87–102 of human FGF-9 w10x was used. This anti-FGF-9 antibody has been extensively characterized for its specificity w11x. Immunohistochemistry was performed by stan-
) Corresponding author. Department of Ultrastructure and Histochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya, Tokyo 156-8585, Japan. Fax: q81-3-3329-8035; E-mail: [email protected]
dard streptoavidin–biotin peroxidase method as described previously w21x with pretreatment with formic acid. Diaminobezidine was used as a chromogen. Counter staining with hematoxylin was performed. Adjacent sections, pretreated with autoclave, were immunostained with anti-tau antibody tau-2 ŽSigma, St. Louis, MO. to identify the dystrophic neurites of senile plaques ŽSPs. and neurofibrillary tangles ŽNFTs.. Control experiments were performed using the anti-FGF-9 antibody preincubated with the synthetic peptide or the preimmune rabbit serum as primary antibody. In both AD and control sections, moderate to strong FGF9-IR was observed in the pyramidal and granular cells of hippocampus ŽFig. 1A.. FGF9-IR was localized in the cytoplasm, and staining pattern was granular ŽFig. 1B.. Neocortical neurons also showed weak to moderate cytoplasmic FGF9-IR. No obvious difference of neuronal staining intensity was observed between AD and control sections. Weak neuropil staining was also observed. In AD cases, spotted FGF9-IR was conspicuous in hippocampal gyrus, subiculum and parahippocampal gyrus ŽFig. 2A.. Spotted FGF9-IR was also observed in the occipitotemporal neocortex. At higher magnification, dystrophic neurites composing diffuse plaques were found strongly immunostained by anti-FGF-9 antibody ŽFig. 2B.. Dystrophic neurites of typical plaques with amyloid core were also FGF-9 immunoreactive, while amyloid core was not or only weakly stained ŽFig. 2C.. The comparison of the adjacent sections stained with either tau-2 ŽFig. 2D. or
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 0 4 2 - 7
S. Nakamura et al.r Brain Research 814 (1998) 222–225
Fig. 1. FGF-9 immunoreactivity ŽFGF9-IR. in the hippocampus of AD patients. ŽA. Strong FGF9-IR was observed within the cytoplasm of pyramidal and granule cells of hippocampus. =20. ŽB. At higher magnification, staining pattern of pyramidal cells was granular. =200.
anti-FGF-9 antibody ŽFig. 2E. revealed that the SPs with FGF-9 immunoreactive dystrophic neurites were almost equal in number to the SPs with tau positive dystrophic neurites. No FGF9-IR was observed in the NFTs Ždata not shown.. In control sections, several FGF-9 immunoreactive SPs were also observed, however, the number of FGF-9 immunoreactive SPs was much smaller than in AD sections. In AD sections, FGF-9 immunoreactive astrocytes were scattered in the gray matter. There was a tendency that FGF-9 immunoreactive astrocytes were more abundant surrounding SPs ŽFig. 3A.. Numerous FGF-9 immunoreactive astrocytes were seen in the subcortical white matter ŽFig. 3B. of AD sections. In contrast, small numbers of FGF-9 immunoreactive astrocytes in the gray matter and no apparent FGF-9 immunoreactive astrocytes in the white matter were observed in control sections. Absorption of the anti-FGF-9 antibody with the synthetic peptide completely abolished the staining, and preimmune serum did not show any staining in neurons, SPs and astrocytes Ždata not shown.. We previously reported that FGF9-IR was observed in neurons of cerebral cortex, hippocampus, brainstem and cerebellum in the human brain w21x. In the present study, we demonstrated FGF9-IR in neurons of hippocampus and occipitotemporal neocortex of AD brain. The intensity of immunostaining varied between cases. This may be due to the differences of postmortem interval and fixation time. However, there was a tendency that the intensity of neu-
223
ronal FGF9-IR was almost same between in AD and control sections. It seems that obvious increase or decrease of FGF-9 in neurons may not be occurred in hippocampus and occipitotemporal neocortex of AD brain. As concerned with neuropathological changes of AD, marked FGF9-IR was observed in dystrophic neurites of SPs both in AD and control brain. FGF9-IR was not found in amyloid core of SPs and NFTs. FGF-9 immunoreactive astrocytes were observed in both the gray and white matter of AD brain, while a few FGF-9 immunoreactive astrocytes were observed only in the gray matter of control brain. In the gray matter, FGF-9 immunoreactive astrocytes were more frequently observed surrounding SPs. Numerous FGF-9 immunoreactive astrocytes were observed in the subcortical white matter. Immunoreactivity of prototypic FGFs has been found in association with neuropathology of AD w3,19,22,26x. Basic FGF immunoreactivity has been observed within the neuritic plaques and NFTs present within neuronal perikarya w3,19x. In addition, substantial increase in the overall specific staining of astrocytes and neurons has been reported in AD cases w19x. It has been considered that basic FGF may be upregulated to support neuronal survival and prevent neuronal degeneration w19x. The possibility has also
Fig. 2. FGF9-IR within senile plaques ŽSPs.. ŽA. Many SPs displayed FGF9-IR in subiculum of AD. =100. ŽB. Dystrophic neurites of diffuse plaque displayed strong FGF9-IR. =100. ŽC. Amyloid core of typical plaques did not display FGF9-IR, while dystrophic neurites displayed apparent FGF9-IR. =100. ŽD, E. Comparison of the adjacent sections immunostained with either anti-tau antibody tau-2 ŽD. or anti-FGF-9 antibody ŽE.. SPs with FGF-9 positive dystrophic neurites were almost equal in number of SPs with tau positive dystrophic neurites. =50.
224
S. Nakamura et al.r Brain Research 814 (1998) 222–225
Fig. 3. FGF-9 immunoreactive astrocytes observed in sections of AD patients. ŽA. FGF-9 immunoreactive astrocytes Žarrowheads. were seen surrounding SPs in the gray matter. =200. ŽB. Numerous FGF-9 immunoreactive astrocytes were seen in the subcortical white matter. =200.
been suggested that basic FGF may accelerate neuropathological changes of AD by increasing production of the amyloid brA4 protein by its ability to increase the secretion of the b-amyloid precursor protein w14,16x or by decrease bioavailability due to sequestration by heparan sulfate proteoglycans ŽHSPGs. integrated within the amyloidotic lesions of AD w19x. Involvement of basic FGF in the formation of neuritic plaques has also been indicated w3x. In the case of acidic FGF, significant number of astrocytes were positively stained for acidic FGF in both gray and white matter of AD brain w22,26x. These intensely stained astrocytes were frequently observed surrounding SPs. It has been suggested that acidic FGF may be upregulated in areas of Alzheimer pathology and concerned with the proliferation of astrocytes surrounding SPs and the protection of degenerating cells and processes in the SPs w22x. The meaning of FGF9-IR in dystrophic neurites is at present unclear. Because FGF-9 is a heparin-binding protein w12x and HSPGs present in SPs w17,18x, FGF-9 seems to bind HSPGs in dystrophic neurites non-specifically. Indeed, externally administered basic FGF has been shown to bind HSPGs within SPs and NFTs w8x. However, there was no apparent FGF9-IR in both amyloid core of SPs and NFTs, which containing HSPGs w17,18x. FGF9-IR within dystrophic neurites may be considered to reflect specific neuropathological change. It is suggested that FGF-9 may be accumulated in dystrophic neurites in the process of formation of SPs and contribute to repair of damaged
neurites by autocrine or paracrine mechanism. We recently observed the neurotrophic effect of FGF-9 in our preliminary experiments Žunpublished data., this finding supported this hypothesis. FGF-9 has been shown to promote astrocytic proliferation in vitro w12x. Recently, FGF-9 has shown to bind with high affinity to FGF receptor ŽFGFR.-3 and FGFR-2 w6,15x. Both FGFR-3 and FGFR-2 mRNA are expressed in glial cells w2,13x. It is also suggested that FGF-9 in dystrophic neurites may promote the proliferation and activation of astrocytes surrounding SPs, and may influence the neuropathological process of AD. Because the production of FGF-9 has been reported almost exclusively in neurons but not in astrocytes w11,20x, FGF9-IR in astrocytes may be due to the increase of FGF-9 uptake from a local source, such as neurons. However, FGF-9 was initially purified from human glioma cell line w12x. Recently, we observed the expression of FGF-9 mRNA in astrocytes in rat spinal cord by in situ hybridization Žin preparation.. It is suggested that, under normal condition, some astrocytes may synthesize a small amount of FGF-9, so only a few astrocytes display FGF9-IR. It has been well known that the expression of basic FGF in astrocytes increases reacting to several injuries including mechanical lesion w3,4x, ischemia w9x, seizure w7x and so on. Increase astrocytic expression of basic FGF has been considered to provide trophic support to neurons and participate in injury-induced plasticity w4x. Like basic FGF, FGF-9 may be upregulated within astrocytes under pathological condition such as AD brain to provide trophic support to degenerating neurons directly andror indirectly by promotion of astrocytic proliferation and activation. Indeed, we recently observed the marked appearance of FGF-9 immunoreactive astrocytes in the corticospinal tract and anterior horn of spinal cord with amyotrophic lateral sclerosis Žin preparation.. On the other hand, it is also considered that upregulated FGF-9 may be involved in gliosis by its strong activity to promote astrocytic proliferation w12x especially in the white matter. In AD brain, cholinergic neurons of basal forebrain providing widespread innervation of the cerebral cortex are impaired w24,25x. Basic FGF has been shown to have a neurotrophic effect on cholinergic neurons of rat basal forebrain in vitro w5,23x and in vivo w1x. Further examinations, including about neurotrophic effect on cholinergic neurons of basal forebrain, will be required to determine the role of FGF-9 concerned with neuropathological changes of AD.
Acknowledgements This work was supported in part by Grants-in Aid for Scientific Research from the Health Science Research Grants from the Ministry of Health and Welfare, Japan ŽKI..
S. Nakamura et al.r Brain Research 814 (1998) 222–225
References w1x K.J. Anderson, D. Dam, S. Lee, C.W. Cotman, Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo, Nature 332 Ž1988. 360–361. w2x T. Asai, A. Wanaka, H. Kato, Y. Masana, M. Seo, M. Tohyama, Differential expression of two members of FGF receptor gene family, FGFR-1 and FGFR-2 mRNA, in the adult rat central nervous system, Mol. Brain Res. 17 Ž1993. 174–178. w3x F. Gomez-Pinilla, B.J. Cummings, C.W. Cotman, Induction of basic ´ fibroblast growth factor in Alzheimer’s disease pathology, Neuroreport 1 Ž1990. 211–214. w4x F. Gomez-Pinilla, J.W. Lee, C.W. Cotman, Basic FGF in adult rat ´ brain: cellular distribution and response to entorhinal lesion and fimbria-fornix transection, J. Neurosci. 12 Ž1992. 345–355. w5x C. Grothe, D. Otto, K. Unsicker, Basic fibroblast growth factor promotes in vitro survival and cholinergic development of rat septal neurons: comparison with the effects of nerve growth factor, Neuroscience 31 Ž1989. 649–661. w6x D. Hecht, N. Zimmerman, M. Bedford, A. Avivi, A. Yayon, Identification of fibroblast growth factor 9 ŽFGF9. as a high affinity, heparin dependent ligand for FGF receptors 3 and 2 but not for FGF receptors 1 and 4, Growth Factors 12 Ž1995. 223–233. w7x C. Humpel, A. Lippoldt, G. Chadi, D. Ganten, L. Olson, K. Fuxe, Fast and widespread increase of basic fibroblast growth factor messenger RNA and protein in the forebrain after kainate-induced seizures, Neuroscience 57 Ž1993. 913–922. w8x T. Kato, H. Sasaki, T. Katagiri, H. Sasaki, K. Koiwai, H. Youki, S. Totsuka, T. Ishii, The binding of basic fibroblast growth factor to Alzheimer’s neurofibrillary tangles and senile plaques, Neurosci. Lett. 122 Ž1991. 33–36. w9x A. Lippoldt, B. Andbjer, L. Rosen, E. Richter, D. Ganten, Y. Cao, R.F. Pettersson, K. Fuxe, Photochemically induced focal cerebral ischemia in rat: time dependent and global increase in expression of basic fibroblast growth factor mRNA, Brain Res. 625 Ž1993. 45–56. w10x M. Miyamoto, K. Naruo, C. Seko, S. Matsumoto, T. Kondo, T. Kurokawa, Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property, Mol. Cell Biol. 13 Ž1993. 4251–4259. w11x S. Nakamura, T. Todo, S. Haga, T. Aizawa, Y. Motoi, A. Ueki, T. Kurokawa, K. Ikeda, Motor neurons in human and rat spinal cord synthesize fibroblast growth factor-9, Neurosci. Lett. 221 Ž1997. 181–184. w12x K. Naruo, C. Seko, K. Kuroshima, E. Matsutani, R. Sasada, T. Kondo, T. Kurokawa, Novel secretory heparin-binding factors from human glioma cells Žglia-activating factors. involved in glial cell growth. Purification and biological properties, J. Biol. Chem. 268 Ž1993. 2857–2864.
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w13x K. Peters, D. Ornitz, S. Werner, L. Williams, Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis, Dev. Biol. 155 Ž1993. 423–430. w14x D. Quon, R. Catalano, B. Cordell, Fibroblast growth factor induces b-amyloid precursor mRNA in glial but not neuronal cultured cells, Biochem. Biophys. Res. Commun. 167 Ž1990. 96–102. w15x S. Santos-Ocampo, J.S. Colvin, A. Chellaiah, D.M. Ornitz, Expression and biological activity of mouse fibroblast growth factor-9, J. Biol. Chem. 271 Ž1996. 1726–1731. w16x D. Schubert, L.W. Jin, T. Saitoh, G. Cole, The regulation of amyloid b protein precursor secretion and its modulatory role in cell adhesion, Neuron 3 Ž1989. 689–694. w17x A.D. Snow, H. Mar, D. Nochlin, K. Kimata, M. Kato, S. Suzuki, J. Hassell, T.N. Wight, The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimer’s disease, Am. J. Pathol. 133 Ž1988. 456–463. w18x A.D. Snow, H. Mar, D. Nochlin, R.T. Sekiguchi, K. Kimata, Y. Koike, T.N. Wight, Early accumulation of heparan sulfate in neurons and in the b-amyloid protein-containing lesions of Alzheimer’s disease and Down’s syndrome, Am. J. Pathol. 137 Ž1990. 1253– 1270. w19x E.G. Stopa, A.M. Gonzalez, R. Chorsky, R.J. Corona, J. Alvarez, E.D. Bird, A. Baird, Basic fibroblast growth factor in Alzheimer’s disease, Biochem. Biophys. Res. Commun. 171 Ž1990. 690–696. w20x S. Tagashira, K. Ozaki, M. Ohta, N. Itoh, Localization of fibroblast growth factor-9 mRNA in the rat brain, Mol. Brain Res. 30 Ž1995. 233–241. w21x T. Todo, T. Kondo, S. Nakamura, T. Kirino, T. Kurokawa, K. Ikeda, Neuronal localization of fibroblast growth factor-9 immunoreactivity in human and rat brain, Brain Res. 783 Ž1998. 179–187. w22x I. Tooyama, H. Akiyama, P.L. McGeer, Y. Hara, O. Yasuhara, H. Kimura, Acidic fibroblast growth factor-like immunoreactivity in brain of Alzheimer patients, Neurosci. Lett. 121 Ž1991. 155–158. w23x P.A. Walicke, Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions, J. Neurosci. 8 Ž1988. 2618–2627. w24x P.J. Whitehouse, D.L. Price, A.W. Clark, J.T. Coyle, M.R. DeLong, Alzheimer disease: evidence for selective loss of cholinergic neurons in the nucleus basalis, Ann. Neurol. 10 Ž1981. 122–126. w25x P.J. Whitehouse, D.L. Price, R.G. Struble, A.W. Clark, J.T. Coyle, M.R. DeLong, Alzheimer’s disease and senile dementia: loss of neurons in the basal forebrain, Science 215 Ž1982. 1237–1239. w26x O. Yasuhara, I. Tooyama, H. Akiyama, I. Akiguchi, J. Kimura, P.L. McGeer, Y. Hara, H. Kimura, Reactive astrocytes express acidic fibroblast growth factor in Alzheimer’s disease brain, Dementia 2 Ž1991. 64–70.
Short communication
Fibroblast growth factor ŽFGF .-9 immunoreactivity in senile plaques Satoshi Nakamura a
a,b,)
, Kunimasa Arima a , Seiichi Haga a , Takako Aizawa a , Yumiko Motoi a , Mieko Otsuka b , Akira Ueki b , Kazuhiko Ikeda a
Department of Ultrastructure and Histochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya, Tokyo 156-8585, Japan b Department of Neurology, Jichi Medical School Omiya Medical Center, Omiya, Japan Accepted 29 September 1998
Abstract We examined fibroblast growth factor ŽFGF.-9 immunoreactivity in human hippocampal sections of Alzheimer’s disease ŽAD.. FGF-9 immunoreactivity was observed in dystrophic neurites of senile plaques in AD and control cases, in addition to the hippocampal and cortical neurons. The amyloid core and neurofibrillary tangles lacked immunoreactivity. FGF-9 immunoreactive astrocytes were conspicuous in AD brains. FGF-9 may be involved in the neuropathology of AD. q 1998 Elsevier Science B.V. All rights reserved. Keywords: FGF-9 immunoreactivity; Fibroblast growth factor; Alzheimer’s disease; Senile plaque; Dystrophic neurite
Fibroblast growth factor ŽFGF.-9 was initially purified from the culture medium of human glioma cell line NMCG1 w12x. FGF-9 is a heparin-binding and secretory growth factor despite its lack of a signal sequence in N-terminus w10,12x. It has been reported that FGF-9 mRNA is expressed in neurons of widespread regions of rat brain w20x. Recently, we reported that FGF-9 immunoreactivity ŽFGF9-IR. was present mainly in neurons of widespread regions of human and rat brain w21x. Since basic FGFrFGF-2 w3,19x and acidic FGFrFGF-1 w22,26x immunoreactivity has been found in association with Alzheimer’s disease ŽAD. neuropathology, we performed immunohistochemistry of human autopsy brain by using a specific anti-FGF-9 antibody. Hippocampal sections including occipitotemporal neocortex from 10 AD cases and 10 age-matched controls with non-neurological diseases were cut from formalinfixed, paraffin-embedded blocks, and subjected to immunohistochemistry. A rabbit polyclonal antibody against a synthetic peptide corresponding to amino acid residues 87–102 of human FGF-9 w10x was used. This anti-FGF-9 antibody has been extensively characterized for its specificity w11x. Immunohistochemistry was performed by stan-
) Corresponding author. Department of Ultrastructure and Histochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya, Tokyo 156-8585, Japan. Fax: q81-3-3329-8035; E-mail: [email protected]
dard streptoavidin–biotin peroxidase method as described previously w21x with pretreatment with formic acid. Diaminobezidine was used as a chromogen. Counter staining with hematoxylin was performed. Adjacent sections, pretreated with autoclave, were immunostained with anti-tau antibody tau-2 ŽSigma, St. Louis, MO. to identify the dystrophic neurites of senile plaques ŽSPs. and neurofibrillary tangles ŽNFTs.. Control experiments were performed using the anti-FGF-9 antibody preincubated with the synthetic peptide or the preimmune rabbit serum as primary antibody. In both AD and control sections, moderate to strong FGF9-IR was observed in the pyramidal and granular cells of hippocampus ŽFig. 1A.. FGF9-IR was localized in the cytoplasm, and staining pattern was granular ŽFig. 1B.. Neocortical neurons also showed weak to moderate cytoplasmic FGF9-IR. No obvious difference of neuronal staining intensity was observed between AD and control sections. Weak neuropil staining was also observed. In AD cases, spotted FGF9-IR was conspicuous in hippocampal gyrus, subiculum and parahippocampal gyrus ŽFig. 2A.. Spotted FGF9-IR was also observed in the occipitotemporal neocortex. At higher magnification, dystrophic neurites composing diffuse plaques were found strongly immunostained by anti-FGF-9 antibody ŽFig. 2B.. Dystrophic neurites of typical plaques with amyloid core were also FGF-9 immunoreactive, while amyloid core was not or only weakly stained ŽFig. 2C.. The comparison of the adjacent sections stained with either tau-2 ŽFig. 2D. or
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 0 4 2 - 7
S. Nakamura et al.r Brain Research 814 (1998) 222–225
Fig. 1. FGF-9 immunoreactivity ŽFGF9-IR. in the hippocampus of AD patients. ŽA. Strong FGF9-IR was observed within the cytoplasm of pyramidal and granule cells of hippocampus. =20. ŽB. At higher magnification, staining pattern of pyramidal cells was granular. =200.
anti-FGF-9 antibody ŽFig. 2E. revealed that the SPs with FGF-9 immunoreactive dystrophic neurites were almost equal in number to the SPs with tau positive dystrophic neurites. No FGF9-IR was observed in the NFTs Ždata not shown.. In control sections, several FGF-9 immunoreactive SPs were also observed, however, the number of FGF-9 immunoreactive SPs was much smaller than in AD sections. In AD sections, FGF-9 immunoreactive astrocytes were scattered in the gray matter. There was a tendency that FGF-9 immunoreactive astrocytes were more abundant surrounding SPs ŽFig. 3A.. Numerous FGF-9 immunoreactive astrocytes were seen in the subcortical white matter ŽFig. 3B. of AD sections. In contrast, small numbers of FGF-9 immunoreactive astrocytes in the gray matter and no apparent FGF-9 immunoreactive astrocytes in the white matter were observed in control sections. Absorption of the anti-FGF-9 antibody with the synthetic peptide completely abolished the staining, and preimmune serum did not show any staining in neurons, SPs and astrocytes Ždata not shown.. We previously reported that FGF9-IR was observed in neurons of cerebral cortex, hippocampus, brainstem and cerebellum in the human brain w21x. In the present study, we demonstrated FGF9-IR in neurons of hippocampus and occipitotemporal neocortex of AD brain. The intensity of immunostaining varied between cases. This may be due to the differences of postmortem interval and fixation time. However, there was a tendency that the intensity of neu-
223
ronal FGF9-IR was almost same between in AD and control sections. It seems that obvious increase or decrease of FGF-9 in neurons may not be occurred in hippocampus and occipitotemporal neocortex of AD brain. As concerned with neuropathological changes of AD, marked FGF9-IR was observed in dystrophic neurites of SPs both in AD and control brain. FGF9-IR was not found in amyloid core of SPs and NFTs. FGF-9 immunoreactive astrocytes were observed in both the gray and white matter of AD brain, while a few FGF-9 immunoreactive astrocytes were observed only in the gray matter of control brain. In the gray matter, FGF-9 immunoreactive astrocytes were more frequently observed surrounding SPs. Numerous FGF-9 immunoreactive astrocytes were observed in the subcortical white matter. Immunoreactivity of prototypic FGFs has been found in association with neuropathology of AD w3,19,22,26x. Basic FGF immunoreactivity has been observed within the neuritic plaques and NFTs present within neuronal perikarya w3,19x. In addition, substantial increase in the overall specific staining of astrocytes and neurons has been reported in AD cases w19x. It has been considered that basic FGF may be upregulated to support neuronal survival and prevent neuronal degeneration w19x. The possibility has also
Fig. 2. FGF9-IR within senile plaques ŽSPs.. ŽA. Many SPs displayed FGF9-IR in subiculum of AD. =100. ŽB. Dystrophic neurites of diffuse plaque displayed strong FGF9-IR. =100. ŽC. Amyloid core of typical plaques did not display FGF9-IR, while dystrophic neurites displayed apparent FGF9-IR. =100. ŽD, E. Comparison of the adjacent sections immunostained with either anti-tau antibody tau-2 ŽD. or anti-FGF-9 antibody ŽE.. SPs with FGF-9 positive dystrophic neurites were almost equal in number of SPs with tau positive dystrophic neurites. =50.
224
S. Nakamura et al.r Brain Research 814 (1998) 222–225
Fig. 3. FGF-9 immunoreactive astrocytes observed in sections of AD patients. ŽA. FGF-9 immunoreactive astrocytes Žarrowheads. were seen surrounding SPs in the gray matter. =200. ŽB. Numerous FGF-9 immunoreactive astrocytes were seen in the subcortical white matter. =200.
been suggested that basic FGF may accelerate neuropathological changes of AD by increasing production of the amyloid brA4 protein by its ability to increase the secretion of the b-amyloid precursor protein w14,16x or by decrease bioavailability due to sequestration by heparan sulfate proteoglycans ŽHSPGs. integrated within the amyloidotic lesions of AD w19x. Involvement of basic FGF in the formation of neuritic plaques has also been indicated w3x. In the case of acidic FGF, significant number of astrocytes were positively stained for acidic FGF in both gray and white matter of AD brain w22,26x. These intensely stained astrocytes were frequently observed surrounding SPs. It has been suggested that acidic FGF may be upregulated in areas of Alzheimer pathology and concerned with the proliferation of astrocytes surrounding SPs and the protection of degenerating cells and processes in the SPs w22x. The meaning of FGF9-IR in dystrophic neurites is at present unclear. Because FGF-9 is a heparin-binding protein w12x and HSPGs present in SPs w17,18x, FGF-9 seems to bind HSPGs in dystrophic neurites non-specifically. Indeed, externally administered basic FGF has been shown to bind HSPGs within SPs and NFTs w8x. However, there was no apparent FGF9-IR in both amyloid core of SPs and NFTs, which containing HSPGs w17,18x. FGF9-IR within dystrophic neurites may be considered to reflect specific neuropathological change. It is suggested that FGF-9 may be accumulated in dystrophic neurites in the process of formation of SPs and contribute to repair of damaged
neurites by autocrine or paracrine mechanism. We recently observed the neurotrophic effect of FGF-9 in our preliminary experiments Žunpublished data., this finding supported this hypothesis. FGF-9 has been shown to promote astrocytic proliferation in vitro w12x. Recently, FGF-9 has shown to bind with high affinity to FGF receptor ŽFGFR.-3 and FGFR-2 w6,15x. Both FGFR-3 and FGFR-2 mRNA are expressed in glial cells w2,13x. It is also suggested that FGF-9 in dystrophic neurites may promote the proliferation and activation of astrocytes surrounding SPs, and may influence the neuropathological process of AD. Because the production of FGF-9 has been reported almost exclusively in neurons but not in astrocytes w11,20x, FGF9-IR in astrocytes may be due to the increase of FGF-9 uptake from a local source, such as neurons. However, FGF-9 was initially purified from human glioma cell line w12x. Recently, we observed the expression of FGF-9 mRNA in astrocytes in rat spinal cord by in situ hybridization Žin preparation.. It is suggested that, under normal condition, some astrocytes may synthesize a small amount of FGF-9, so only a few astrocytes display FGF9-IR. It has been well known that the expression of basic FGF in astrocytes increases reacting to several injuries including mechanical lesion w3,4x, ischemia w9x, seizure w7x and so on. Increase astrocytic expression of basic FGF has been considered to provide trophic support to neurons and participate in injury-induced plasticity w4x. Like basic FGF, FGF-9 may be upregulated within astrocytes under pathological condition such as AD brain to provide trophic support to degenerating neurons directly andror indirectly by promotion of astrocytic proliferation and activation. Indeed, we recently observed the marked appearance of FGF-9 immunoreactive astrocytes in the corticospinal tract and anterior horn of spinal cord with amyotrophic lateral sclerosis Žin preparation.. On the other hand, it is also considered that upregulated FGF-9 may be involved in gliosis by its strong activity to promote astrocytic proliferation w12x especially in the white matter. In AD brain, cholinergic neurons of basal forebrain providing widespread innervation of the cerebral cortex are impaired w24,25x. Basic FGF has been shown to have a neurotrophic effect on cholinergic neurons of rat basal forebrain in vitro w5,23x and in vivo w1x. Further examinations, including about neurotrophic effect on cholinergic neurons of basal forebrain, will be required to determine the role of FGF-9 concerned with neuropathological changes of AD.
Acknowledgements This work was supported in part by Grants-in Aid for Scientific Research from the Health Science Research Grants from the Ministry of Health and Welfare, Japan ŽKI..
S. Nakamura et al.r Brain Research 814 (1998) 222–225
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