- Email: [email protected]
Enhanced localization of amyloid β precursor protein in the rat hippocampus following long-term adrenalectomy
Brain Research 806 Ž1998. 108–112
Short communication
Enhanced localization of amyloid b precursor protein in the rat hippocampus following long-term adrenalectomy A. Islam a , R.N. Kalaria a
b,c,)
, B. Winblad a , A. Adem
a
Department of Clinical Neuroscience and Family Medicine, Karolinska Institute, KFC NoÕum, S-14186 Huddinge, Sweden b Department of Neurology, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106, USA c Department of Pathology, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106, USA Accepted 7 July 1998
Abstract Using various antibodies to the amyloid ß precursor protein ŽAPP. associated with Alzheimer’s disease, we investigated changes in the distribution of APP in the hippocampus and neocortex of adrenalectomized ŽADX. rats. In contrast to sham-operated controls, ADX rats euthanised after a survival period of 5 months showed striking APP reactivity in the CA1–CA4 fields and in the surviving cells in the dentate gyrus. Our results suggest the enhanced APP reactivity in hippocampal neurons may pertain to previous observations on the accumulation of APP fragments in the neocortex during ischemic or traumatic injury. Thus, long-term hormone deprivation would be another factor, which may influence the expression of APP in brain. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Adrenalectomy; Alzheimer’s disease; Amyloid protein precursor; Glucocorticoid; Hippocampus; Vascular cell
The expression and metabolism of the amyloid b protein precursor ŽAPP. has gained significant importance in evaluating the pathological mechanisms of Alzheimer’s disease. Recent studies have demonstrated the rapid induction of APP particularly in reactive astrocytes and hippocampal neurons subsequent to neuronal damage caused by intrathecal or intraparenchymal injections of excitotoxins and by physical injury w13–15,20,22x. APP mRNA was also induced after heat shock in cultured lymphoblastoid cells w1x. There have also been studies on the expression of APP in persistent focal ischemia. Abe et al. w2x demonstrated the selective induction of the Kunitz inhibitor domain-containing APP mRNA at 4 days after the ischemic insult. We previously demonstrated the accumulation of APP fragments in reactive astroglia, dystrophic axons and neurons localized in the perifocal regions of infarcted area in rats subjected to cerebral ischemia w9,10x. Enhanced APP immunoreactivity was similarly observed in injured neurons and axons in traumatic head injury ŽM. Buzek, W.D. Lust and R.N. Kalaria, unpublished observations.. Similar findings were also reported in a rat middle cerebral
) Corresponding author. CBV Path Group, MRC Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, UK. Fax: q44-191-272-5291; E-mail: [email protected]
artery occlusion model w25x and in gerbils subjected to global ischemia w16,19x. However, apart from a recent study suggesting that hippocampal APP mRNA expression is modulated by the ovarian sex steroids in old female rats w5x, there is little knowledge on the expression or metabolism of APP during hormonal dysregulation. In an effort to evaluate this, we assessed APP and other markers of AD lesions in rat brains after long-term adrenalectomy ŽADX. which results in chronic corticosteroid deprivation w3,8,23x. Using various antibodies, in this study, we describe the immunocytochemical localization of APP and other antigens in hippocampus of rats subjected to ADX and sham-operated controls. Adult male Sprague–Dawley rats Žmean weights" 220 " 10 g. were bilaterally adrenalectomized essentially as described w3x. We performed adrenalectomy in a total of 30 rats consisting of the experimental group and 10 others were used as sham-operated controls. The rats were let to survive 5 months after surgery during which were given free access to food and water containing 0.9% NaCl. To prevent death after the adrenalectomies, rats were also given graded doses of corticosterone Ž20 to 5 mgrml. in the drinking water for the first three weeks after surgery. At the selected survival time, rats were killed by decapitation and the brains immediately frozen in dry-ice cooled isopentane. Successful adrenalectomies were confirmed by
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 0 7 1 1 - 2
A. Islam et al.r Brain Research 806 (1998) 108–112
visual inspection of the adrenals and corticosterone assay of the plasma w3x. Frozen brain blocks containing the hippocampal formation were cut into coronal sections of 15 mM thickness and were mounted on gelatin subbed slides. The tissue sections were fixed in ice cold 10% formalin for 5 min and then either single or double immunostained using the avidin– biotin complex ŽABC. method essentially as described previously w9,12x. To detect APP, we used the following polyclonal and monoclonal antibodies: mAb 22C11 to amino-terminal ŽAPP51 – 95 ; Boehringher, NJ., aC6 to carboxyl ŽC.-terminal ŽAPP591 – 695 ; Athena, CA., 369W to C-terminal ŽAPP645 – 694 ; courtesy of S. Gandy, NY., 4G8 to Aß17 – 24 ; Drs. Kim and Wisniewski, NYSBRI, NY.. In some experiments, amyloid b associated proteins or cells were immunodetected using antibodies to the following proteins: apolipoprotein E ŽCourtesy of H. Mori, TIP., C3d ŽDako, CA., presenlin 1 ŽH. Mori, TIP., glial acidic fibrillary protein ŽGFAP; Dako, CA. and OX-42 ŽC3bi receptor, Seralab, NY.. The dilutions of all the antibodies were between 1:500 to 1:2000, except 22C11 and OX-42 which were 1:10. The specificity of all the antibodies used in the described experiments has been well-established previously w11,12x. Upon immunoblotting the antibodies recognized the expected bandŽs. of proteins in solubilized preparations of neocortical or cerebral microvascular homogenates. The immunoreactivity was absorbed by the respective peptides. Antibodies to both amino- and C-terminal domains of APP were used to evaluate changes in the distribution of APP reactivity, as well as other markers in the brains of rats subjected to chronic glucorticoid deprivation. Compared to the sham-operated controls, we observed enhanced APP immunoreactivity in the brains of ADX rats killed 5 months after the surgery ŽFig. 1.. The hippocampus and the parietal and piriform cortical areas were particularly striking apparent with antibodies to the Cterminal of APP. In the hippocampus, APP immunoreactivity to the C-terminal antibodies Ž aC6 and 369W., was prominent in neurons in the CA fields ŽFig. 1A.. The CA1, CA3 and CA4 fields were readily immunostained. In contrast, there was relatively sparse APP reactivity in the granule cells of the dentate gyrus ŽFig. 1A–C.. However, a number of the surviving cells showed accumulation of the C-terminal fragments of APP ŽFig. 1C.. The general absence of APP reactivity in the dentate gyrus presumably reflected the resultant ADX induced cell loss. The antibody to the amino-terminal of APP, 22C11 was only weakly reactive and did not show the same distribution as the C-terminal antibodies Žnot shown.. Occasionally, we observed intracellular granular accumulations of APP in the CA3 field that were not apparent elsewhere. However, we also noted numerous APP positive extracellular filaments in the CA3 field ŽFig. 1E.. These immunocytochemical observations on APP reactivity were not apparent with
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any of the antibodies tested in any of the sham-operated control rats ŽFig. 1D.. Similarly, control experiments showed that tissue sections incubated with preimmune rabbit serum and antibodies absorbed with APP Ž aC6. gave only diffuse background staining. Antibodies to the C-terminal of APP also revealed accumulations of APP in pyramidal neurons of the parietal and piriform cortices Žcf. Fig. 1C, inset.. Some APP reactivity was also apparent in the neurons within the thalamus and in three rats in the medial hypothalamus Žnot shown.. The amino-terminal APP antibody revealed similar distribution of staining but it was considerably weaker. Remarkably, none of the APP antibodies showed APP immunoreactivity in astrocytes or microglial cells ŽFig. 2A.. There was no staining in the white matter of the ADX rats. However, we did observe strong staining in the smooth muscle layers of the large meningeal and intraparenchymal vessels ŽFig. 2B.. Antibody 4G8 to the amyloid ß peptide was negative and revealed no differences between the ADX and control rats Žnot shown.. In further experiments, we immunostained sections for certain cellular markers. In the majority of the ADX rats, sections stained with antibodies to GFAP did not reveal a profound astrocytic response that was different from the sham controls. However, in two ADX animals there were more astrocytic fibres in the molecular layer of the dentate gyrus. The lack of active astrogliosis in the hippocampus or elsewhere in the cortex was also studied by double immunostaining ŽFig. 2A.. These observations confirmed APP reactivity was largely restricted to neurons and that there was a lack of localization of APP in astrocytes dissimilar to that found after acute ischemic or traumatic brain injury w10x. In further experiments, we noted intense OX-42 reactivity, as a mircoglial marker, in the dentate gyrus compared to other regions of the hippocampus, piriform or parietal cortex ŽFig. 2C.. This was predominantly present in the molecular layer of the dentate and absent in the sham controls ŽFig. 2D.. Interestingly, antibodies to apolipoprotein E showed diffuse immunostaining in the pyramidal cells of the CA4 in hippocampus and neocortex that was not apparent in the controls ŽFig. 1F.. Neither astrocytes nor other cells were stained by apolipoprotein E. Antiserum to complement C3d only revealed vascular staining and was not remarkable Žresults not shown.. PS1 ŽS182. antibodies only weakly stained some neurons and periventricular astrocytes in both ADX and the control rats. There were no apparent differences in the distribution of the PS1 reactivity in the two groups of animals. Consistent with previous observations showing that APP reactivity is enhanced after a variety of acute insults w6,9,20,24,25x, we observed enhanced reactivity of APP in existing pyramidal cells of the CA fields in the hippocampus, as well as in the parietal and piriform cortices of long-term ADX rats. APP reactivity in the dentate gyrus was, however, sparse and mainly associated with surviving
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Fig. 1. Immunocytochemical localization of APP in hippocampus of rats after long-term adrenalectomy ŽADX.. ŽA–C. Immunostaining of pyramidal cells with aC6 antibody in ADX rats. ŽA. Low power view showing CA fields and lack of staining in dentate gyrus Žarrow.. ŽB. High power view of the depleted dentate gyrus and CA4 neurons. ŽC. Higher power view of CA4 and some dentate neurons. Inset shows C-terminal APP reactive surviving cells in the granular layer Žinset bar s 15 mm.. ŽD. Similar field as in ŽC. from a control rat showing lack of APP immunoreactivity. ŽE. The CA3 region exhibiting degenerative ‘extracellular filamentous’ structures with APP antibodies Ž369W. in an ADX rat. ŽF. Diffuse apolipoprotein E immunoreactivity in CA4 cells and surviving cells in dentate gyrus from a long-term ADX rat. Magnification bar: A s 330 mm, B and E s 150 mm, C, D, and F s 55 mm.
granule cells. This presumably reflects the characteristic preferential destruction of the dentate granule cells after long-term deprivation of glucocorticoids w3,23x. The mechanism of this ADX induced cell loss is not currently understood but apotosis has been previously implicated w24x. Our findings on APP in other hippocampal fields relate to previously described changes in certain pyramidal cells of the CA fields w3x. The intense localization of APP in surviving cells of the hippocampal CA1, CA3, and CA4 fields and weakly in CA2 indicates the accumulation of
APP or its fragments in cells including vascular myocytes that are vulnerable as a result of chronic deprivation of the glucocorticoids w3,8x. Our observations also suggest that the accumulations of APP within neurons presumably contain more C-terminal fragments since mAb 22C11, which recognises the amino-terminal of APP only weakly stained the product in the cells. This finding is consistent with previous observations indicating enhanced accumulation of C-terminal fragments in vulnerable or degenerating neurons w9,22x. We do not know whether the observed changes in protein in long-term ADX are accompanied by alter-
A. Islam et al.r Brain Research 806 (1998) 108–112
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Fig. 2. Glial and vascular immunoreactivity in the hippocampus of ADX rats. ŽA. High power view of double-stained section for APP and GFAP. Intense APP reactivity observed in CA4 cells and some cells in the dentate gyrus but there was no profound astrocytic reaction. ŽB. Vascular APP reactivity in an ADX rat. Smooth muscle cells within the media were clearly stained. ŽC. Section immunostained with OX-42, a microglial marker. Intense reactivity was limited to the dentate gyrus. ŽD. Section from a control rat stained with OX-42. Magnification bar: A and B s 55 mm, C and D s 380 mm.
ations in APP mRNA. However, a previous report showed that hippocampal APP mRNA was not altered at 7 days survival period after ADX w5x. It is plausible that three mechanisms may be involved in the observed change in the localization and distribution of APP reactivity analogous to that seen in neurons subjected to acute insults. First, APP is expressed and accumulates in cells, which become vulnerable w18,21x because of secondary events related to hormone deprivation. Second, the glucocorticoids may have a direct effect on APP regulation although this has not, thus far, been studied in vitro. Third, APP may be increased indirectly via inflammatory mechanisms by the action of cytokines or other inflammatory agents. Previous studies have shown that APP is regulated by certain inflammatory cytokines w7x and that cerebral APP expression is enhanced after lipopolysaccharide treatments w4x. Therefore, it is possible that some of the increased APP reactivity we observed in cortical areas may arise from the inflammatory reactions associated with ADX w8x. We did not observe APP reactivity in astrocytes or microglial cells. This is in contrast to previous studies where acute insults induced both by excitotoxic lesions and cerebral ischemia w16,19,20,23x result in striking APP
reactivity within astrocyte cell bodies and cellular processes besides neurons. The reason for lack of astrocytic localization of APP in the ADX model could be two fold. One, that ADX induces an astrocytic reaction, which may have been protracted from the changes in APP expression and two, unlike the other models, hormonal effect is perhaps limited to APP modulation in neurons. However, we are not certain that long-term ADX induces a profound astrocytic proliferation in the hippocampus although a microglial reaction was apparent. The investigation of the brains at earlier survival times would be necessary to evaluate whether astrogliosis is concomitant with APP expression in the ADX model. We also noted diffuse apolipoprotein E immunoreactivity that was localized to CA neurons in the hippocampus. While it is suggested that astrocytes and macrophages may synthesize and release apolipoprotein E within damaged areas to scavenge cholesterol and phospholipids from cellular debris, it is possible that the neuronal localization of protein reflects lipid mobilization in vulnerable or at risk cells in the hippocampus w17x. In summary, we observed enhanced APP reactivity in the CA fields and remaining granule cells of the dentate gyrus in rats subjected to ADX. Cerebrovascular myocytes
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and certain pyramidal neurons in the parietal and piriform cortices were also strongly reactive to antibodies to the C-terminal of APP. These cellular changes were not associated with a profound astrocytic response as shown by GFAP immunocytochemistry. While our observations are related to enhanced APP accumulation in other experimental conditions, long-term hormonal deprivation might be another factor that may directly or indirectly modulate the expression of APP in brain.
Acknowledgements We thank Dawn Cohen, Dr. Isam Suliman and Dr. Adlan Elhassan for technical assistance. We also thank Dr. Hiroshi Mori, Tokyo Institute of Psychiatry ŽTIP. for the antibodies. We acknowledge financial support from Loo och Hans Ostermans fond, Swedish Society for Medical Research, Stiftelsen Sigurd och Elsa Goljes Minne, KI fonder, Greta Lindenau Hansells Minnesfond, Gamla Tjanarinnor Foundation, the Alzheimer’s Association ŽChicago. and the USPHS for grants ŽRNK. AG08992 and AG10030.
References w1x K. Abe, P.H.St. George-Hyslop, R.E. Tanzi, K. Kogure, Induction of amyloid precursor protein mRNA after heat shock in cultured human lymphoblastoid cells, Neurosci. Lett. 125 Ž1991. 169–171. w2x K. Abe, R.E. Tanzi, K. Kogure, Selective induction of Kunitz-type protease inhibitor domain-containing amyloid precursor protein mRNA after persistent focal ischemia in rat cerebral cortex, Neurosci. Lett. 125 Ž1991. 172–174. w3x A. Adem, A. Islam, N.E. Bogdanovic, K. Carlstrom, B. Winblad, Loss of neurones after long-term adrenalectomy in the adult rat hippocampal formation, NeuroReport 5 Ž1994. 2285–2288. w4x B. Brugg, Y.L. Dubreuil, G. Huber, E.E. Wollman, N. DelhayeBouchaud, J. Mariani, Inflammatory processes induce ß-amyloid precursor protein changes in mouse brain, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 3032–3035. w5x H.M. Chao, R.L. Spencer, M. Frankfurt, B.S. McEwen, The effects of aging and hormonal manipulation on amyloid precursor protein APP695 mRNA expression in the rat hippocampus, J. Neuroendocrinol. 6 Ž1994. 517–521. w6x D. Games, J.A. Clemens, I. Lieberburg, Amyloid precursor protein in traumatic head injury in rats, Soc. Neurosci. Abstr. 18 Ž1992. 533. w7x D. Goldgaber, H.W. Harris, T. Hla, T. Maciag, R.J. Donnelly, J.S. Jacobsen, M.P. Vitek, D.C. Gajdusek, Interleukin 1 regulates synthesis of amyloid ß-protein precursor mRNAs: recognition of a novel alternatively spliced form and quantitation in Alzheimer’s disease using PCR, Proc. Natl. Acad. Sci. U.S.A. 86 Ž1989. 7606–7610. w8x A. Islam, M. Mustafa, M. Mustafa, T. Olsson, B. Winblad, A. Adem, Expression of MHC class II, CD4q and ED1 molecules in association with selective hippocampal neuronal degeneration after long-term adrenalectomy, NeuroReport 8 Ž1997. 987–990.
w9x R.N. Kalaria, S.U. Bhatti, E.A. Palatinsky, D.H. Pennington, E.R. Shelton, H.W. Chan, G. Perry, W.D. Lust, Accumulation of the ß amyloid precursor protein at sites of ischemic injury in rat brain, NeuroReport 4 Ž1993. 211–214. w10x R.N. Kalaria, S.U. Bhatti, G. Perry, W.D. Lust, The amyloid precursor protein in ischemic brain injury and chronic hypoperfusion, Ann. NY Acad. Sci. 695 Ž1993. 190–193. w11x R.N. Kalaria, D.L. Cohen, A.S. Al-Mudallal, W.D. Lust, D.R.D. Premkumar, VEGFrVPF expression in ischemic brain injury and human neurodegenerative disorders, J. Cereb. Blood Flow Metab. 15 Ž1995. S599. w12x R.N. Kalaria, D.L. Cohen, B.D. Greenberg, M.J. Savage, N.E. Bogdanovic, B. Winblad, L. Lannfelt, A. Adem, Abundance of the longer Aß 42 in neocortical and cerebrovascular amyloid ß deposits in Swedish familial Alzheimer’s disease and Down’s syndrome, NeuroReport 7 Ž1996. 1377–1381. w13x T. Kawarabayashi, M. Shoji, Y. Harigaya, H. Yamaguchi, S. Hirai, Expression of APP in the early stage of brain damage, Brain Res. 563 Ž1991. 334–338. w14x Y. Nakamura, M. Takeda, H. Niigawa, S. Hariguchi, T. Nishimura, Amyloid ß-protein precursor deposition in rat hippocampus lesioned by ibotenic acid injection, Neurosci. Lett. 136 Ž1992. 95–98. w15x N. Otsuka, M. Tomonaga, K. Ikeda, Rapid appearance of ß-amyloid precursor protein immunoreactivity in damaged axons and reactive glial cells in rat brain following needle stab injury, Brain Res. 568 Ž1991. 335–338. w16x G. Palacios, G. Mengod, A. Tortosa, I. Ferrer, J.M. Palacios, Increased b-amyloid precursor protein expression in astrocytes in the gerbil hippocampus following ischaemia: association with proliferation of astrocytes, Eur. J. Neurosci. 7 Ž1995. 501–510. w17x J. Poirer, A. Baccichet, D. Dea, S. Gauthier, Cholesterol synthesis and lipoprotein reuptake during synaptic remodelling in hippocampus in adult rats, Neuroscience 55 Ž1993. 81–90. w18x G.W. Roberts, S.M. Gentleman, A. Lynch, L. Murray, M. Landon, D.I. Graham, ß amyloid protein deposition in brain after severe head injury: implications for the pathogenesis of Alzheimer’s disease, J. Neurol. Neurosurg. Psychiatry 57 Ž1994. 419–425. w19x T.C. Saido, M. Yokota, K. Maruyama, W. Yamao-Harigaya, E. Tani, Y. Ihara, S. Kawashima, Spatial resolution of the primary ß-amyloidogenic process induced in postischemic hippocampus, J. Biol. Chem. 269 Ž1994. 15253–15257. w20x R. Siman, J.P. Card, R.B. Nelson, L.G. Davis, Expression of ß-amyloid precursor protein in reactive astrocytes following neuronal damage, Neuron 3 Ž1989. 275–285. w21x F.E. Sherriff, L.R. Bridges, S. Sivaloganathan, Early detection of axonal injury after human head trauma using immunocytochemistry for ß-amyloid precursor protein, Acta Neuropathol. 87 Ž1994. 55–62. w22x K. Shigematsu, P.L. McGeer, D.G. Walker, T. Ishii, E.G. McGeer, Reactive microgliarmacrophages phagocytose amyloid precursor protein produced by neurons following neural damage, J. Neurosci. Res. 31 Ž1992. 443–453. w23x R.S. Sloviter, G. Valiquette, G.M. Abrams, E.C. Ronk, A.I. Sollas, L.A. Paul, S.L. Neubort, Selective loss of hippocampal granule cells in the mature rat brain after adrenalectomy, Science 243 Ž1989. 535–538. w24x R.S. Sloviter, E. Dean, S.L. Neubort, Electron microscopic analysis of adrenalectomy-induced hippocampal granule cell degeneration in the rat, J. Comp. Neurol. 330 Ž1993. 337–351. w25x D.T. Stephenson, K. Rash, J.A. Clemens, Amyloid precursor protein accumulates in regions of neurodegeneration following focal cerebral ischemia in the rat, Brain Res. 593 Ž1992. 128–135.
Short communication
Enhanced localization of amyloid b precursor protein in the rat hippocampus following long-term adrenalectomy A. Islam a , R.N. Kalaria a
b,c,)
, B. Winblad a , A. Adem
a
Department of Clinical Neuroscience and Family Medicine, Karolinska Institute, KFC NoÕum, S-14186 Huddinge, Sweden b Department of Neurology, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106, USA c Department of Pathology, Case Western ReserÕe UniÕersity, CleÕeland, OH 44106, USA Accepted 7 July 1998
Abstract Using various antibodies to the amyloid ß precursor protein ŽAPP. associated with Alzheimer’s disease, we investigated changes in the distribution of APP in the hippocampus and neocortex of adrenalectomized ŽADX. rats. In contrast to sham-operated controls, ADX rats euthanised after a survival period of 5 months showed striking APP reactivity in the CA1–CA4 fields and in the surviving cells in the dentate gyrus. Our results suggest the enhanced APP reactivity in hippocampal neurons may pertain to previous observations on the accumulation of APP fragments in the neocortex during ischemic or traumatic injury. Thus, long-term hormone deprivation would be another factor, which may influence the expression of APP in brain. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Adrenalectomy; Alzheimer’s disease; Amyloid protein precursor; Glucocorticoid; Hippocampus; Vascular cell
The expression and metabolism of the amyloid b protein precursor ŽAPP. has gained significant importance in evaluating the pathological mechanisms of Alzheimer’s disease. Recent studies have demonstrated the rapid induction of APP particularly in reactive astrocytes and hippocampal neurons subsequent to neuronal damage caused by intrathecal or intraparenchymal injections of excitotoxins and by physical injury w13–15,20,22x. APP mRNA was also induced after heat shock in cultured lymphoblastoid cells w1x. There have also been studies on the expression of APP in persistent focal ischemia. Abe et al. w2x demonstrated the selective induction of the Kunitz inhibitor domain-containing APP mRNA at 4 days after the ischemic insult. We previously demonstrated the accumulation of APP fragments in reactive astroglia, dystrophic axons and neurons localized in the perifocal regions of infarcted area in rats subjected to cerebral ischemia w9,10x. Enhanced APP immunoreactivity was similarly observed in injured neurons and axons in traumatic head injury ŽM. Buzek, W.D. Lust and R.N. Kalaria, unpublished observations.. Similar findings were also reported in a rat middle cerebral
) Corresponding author. CBV Path Group, MRC Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, UK. Fax: q44-191-272-5291; E-mail: [email protected]
artery occlusion model w25x and in gerbils subjected to global ischemia w16,19x. However, apart from a recent study suggesting that hippocampal APP mRNA expression is modulated by the ovarian sex steroids in old female rats w5x, there is little knowledge on the expression or metabolism of APP during hormonal dysregulation. In an effort to evaluate this, we assessed APP and other markers of AD lesions in rat brains after long-term adrenalectomy ŽADX. which results in chronic corticosteroid deprivation w3,8,23x. Using various antibodies, in this study, we describe the immunocytochemical localization of APP and other antigens in hippocampus of rats subjected to ADX and sham-operated controls. Adult male Sprague–Dawley rats Žmean weights" 220 " 10 g. were bilaterally adrenalectomized essentially as described w3x. We performed adrenalectomy in a total of 30 rats consisting of the experimental group and 10 others were used as sham-operated controls. The rats were let to survive 5 months after surgery during which were given free access to food and water containing 0.9% NaCl. To prevent death after the adrenalectomies, rats were also given graded doses of corticosterone Ž20 to 5 mgrml. in the drinking water for the first three weeks after surgery. At the selected survival time, rats were killed by decapitation and the brains immediately frozen in dry-ice cooled isopentane. Successful adrenalectomies were confirmed by
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 0 7 1 1 - 2
A. Islam et al.r Brain Research 806 (1998) 108–112
visual inspection of the adrenals and corticosterone assay of the plasma w3x. Frozen brain blocks containing the hippocampal formation were cut into coronal sections of 15 mM thickness and were mounted on gelatin subbed slides. The tissue sections were fixed in ice cold 10% formalin for 5 min and then either single or double immunostained using the avidin– biotin complex ŽABC. method essentially as described previously w9,12x. To detect APP, we used the following polyclonal and monoclonal antibodies: mAb 22C11 to amino-terminal ŽAPP51 – 95 ; Boehringher, NJ., aC6 to carboxyl ŽC.-terminal ŽAPP591 – 695 ; Athena, CA., 369W to C-terminal ŽAPP645 – 694 ; courtesy of S. Gandy, NY., 4G8 to Aß17 – 24 ; Drs. Kim and Wisniewski, NYSBRI, NY.. In some experiments, amyloid b associated proteins or cells were immunodetected using antibodies to the following proteins: apolipoprotein E ŽCourtesy of H. Mori, TIP., C3d ŽDako, CA., presenlin 1 ŽH. Mori, TIP., glial acidic fibrillary protein ŽGFAP; Dako, CA. and OX-42 ŽC3bi receptor, Seralab, NY.. The dilutions of all the antibodies were between 1:500 to 1:2000, except 22C11 and OX-42 which were 1:10. The specificity of all the antibodies used in the described experiments has been well-established previously w11,12x. Upon immunoblotting the antibodies recognized the expected bandŽs. of proteins in solubilized preparations of neocortical or cerebral microvascular homogenates. The immunoreactivity was absorbed by the respective peptides. Antibodies to both amino- and C-terminal domains of APP were used to evaluate changes in the distribution of APP reactivity, as well as other markers in the brains of rats subjected to chronic glucorticoid deprivation. Compared to the sham-operated controls, we observed enhanced APP immunoreactivity in the brains of ADX rats killed 5 months after the surgery ŽFig. 1.. The hippocampus and the parietal and piriform cortical areas were particularly striking apparent with antibodies to the Cterminal of APP. In the hippocampus, APP immunoreactivity to the C-terminal antibodies Ž aC6 and 369W., was prominent in neurons in the CA fields ŽFig. 1A.. The CA1, CA3 and CA4 fields were readily immunostained. In contrast, there was relatively sparse APP reactivity in the granule cells of the dentate gyrus ŽFig. 1A–C.. However, a number of the surviving cells showed accumulation of the C-terminal fragments of APP ŽFig. 1C.. The general absence of APP reactivity in the dentate gyrus presumably reflected the resultant ADX induced cell loss. The antibody to the amino-terminal of APP, 22C11 was only weakly reactive and did not show the same distribution as the C-terminal antibodies Žnot shown.. Occasionally, we observed intracellular granular accumulations of APP in the CA3 field that were not apparent elsewhere. However, we also noted numerous APP positive extracellular filaments in the CA3 field ŽFig. 1E.. These immunocytochemical observations on APP reactivity were not apparent with
109
any of the antibodies tested in any of the sham-operated control rats ŽFig. 1D.. Similarly, control experiments showed that tissue sections incubated with preimmune rabbit serum and antibodies absorbed with APP Ž aC6. gave only diffuse background staining. Antibodies to the C-terminal of APP also revealed accumulations of APP in pyramidal neurons of the parietal and piriform cortices Žcf. Fig. 1C, inset.. Some APP reactivity was also apparent in the neurons within the thalamus and in three rats in the medial hypothalamus Žnot shown.. The amino-terminal APP antibody revealed similar distribution of staining but it was considerably weaker. Remarkably, none of the APP antibodies showed APP immunoreactivity in astrocytes or microglial cells ŽFig. 2A.. There was no staining in the white matter of the ADX rats. However, we did observe strong staining in the smooth muscle layers of the large meningeal and intraparenchymal vessels ŽFig. 2B.. Antibody 4G8 to the amyloid ß peptide was negative and revealed no differences between the ADX and control rats Žnot shown.. In further experiments, we immunostained sections for certain cellular markers. In the majority of the ADX rats, sections stained with antibodies to GFAP did not reveal a profound astrocytic response that was different from the sham controls. However, in two ADX animals there were more astrocytic fibres in the molecular layer of the dentate gyrus. The lack of active astrogliosis in the hippocampus or elsewhere in the cortex was also studied by double immunostaining ŽFig. 2A.. These observations confirmed APP reactivity was largely restricted to neurons and that there was a lack of localization of APP in astrocytes dissimilar to that found after acute ischemic or traumatic brain injury w10x. In further experiments, we noted intense OX-42 reactivity, as a mircoglial marker, in the dentate gyrus compared to other regions of the hippocampus, piriform or parietal cortex ŽFig. 2C.. This was predominantly present in the molecular layer of the dentate and absent in the sham controls ŽFig. 2D.. Interestingly, antibodies to apolipoprotein E showed diffuse immunostaining in the pyramidal cells of the CA4 in hippocampus and neocortex that was not apparent in the controls ŽFig. 1F.. Neither astrocytes nor other cells were stained by apolipoprotein E. Antiserum to complement C3d only revealed vascular staining and was not remarkable Žresults not shown.. PS1 ŽS182. antibodies only weakly stained some neurons and periventricular astrocytes in both ADX and the control rats. There were no apparent differences in the distribution of the PS1 reactivity in the two groups of animals. Consistent with previous observations showing that APP reactivity is enhanced after a variety of acute insults w6,9,20,24,25x, we observed enhanced reactivity of APP in existing pyramidal cells of the CA fields in the hippocampus, as well as in the parietal and piriform cortices of long-term ADX rats. APP reactivity in the dentate gyrus was, however, sparse and mainly associated with surviving
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Fig. 1. Immunocytochemical localization of APP in hippocampus of rats after long-term adrenalectomy ŽADX.. ŽA–C. Immunostaining of pyramidal cells with aC6 antibody in ADX rats. ŽA. Low power view showing CA fields and lack of staining in dentate gyrus Žarrow.. ŽB. High power view of the depleted dentate gyrus and CA4 neurons. ŽC. Higher power view of CA4 and some dentate neurons. Inset shows C-terminal APP reactive surviving cells in the granular layer Žinset bar s 15 mm.. ŽD. Similar field as in ŽC. from a control rat showing lack of APP immunoreactivity. ŽE. The CA3 region exhibiting degenerative ‘extracellular filamentous’ structures with APP antibodies Ž369W. in an ADX rat. ŽF. Diffuse apolipoprotein E immunoreactivity in CA4 cells and surviving cells in dentate gyrus from a long-term ADX rat. Magnification bar: A s 330 mm, B and E s 150 mm, C, D, and F s 55 mm.
granule cells. This presumably reflects the characteristic preferential destruction of the dentate granule cells after long-term deprivation of glucocorticoids w3,23x. The mechanism of this ADX induced cell loss is not currently understood but apotosis has been previously implicated w24x. Our findings on APP in other hippocampal fields relate to previously described changes in certain pyramidal cells of the CA fields w3x. The intense localization of APP in surviving cells of the hippocampal CA1, CA3, and CA4 fields and weakly in CA2 indicates the accumulation of
APP or its fragments in cells including vascular myocytes that are vulnerable as a result of chronic deprivation of the glucocorticoids w3,8x. Our observations also suggest that the accumulations of APP within neurons presumably contain more C-terminal fragments since mAb 22C11, which recognises the amino-terminal of APP only weakly stained the product in the cells. This finding is consistent with previous observations indicating enhanced accumulation of C-terminal fragments in vulnerable or degenerating neurons w9,22x. We do not know whether the observed changes in protein in long-term ADX are accompanied by alter-
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Fig. 2. Glial and vascular immunoreactivity in the hippocampus of ADX rats. ŽA. High power view of double-stained section for APP and GFAP. Intense APP reactivity observed in CA4 cells and some cells in the dentate gyrus but there was no profound astrocytic reaction. ŽB. Vascular APP reactivity in an ADX rat. Smooth muscle cells within the media were clearly stained. ŽC. Section immunostained with OX-42, a microglial marker. Intense reactivity was limited to the dentate gyrus. ŽD. Section from a control rat stained with OX-42. Magnification bar: A and B s 55 mm, C and D s 380 mm.
ations in APP mRNA. However, a previous report showed that hippocampal APP mRNA was not altered at 7 days survival period after ADX w5x. It is plausible that three mechanisms may be involved in the observed change in the localization and distribution of APP reactivity analogous to that seen in neurons subjected to acute insults. First, APP is expressed and accumulates in cells, which become vulnerable w18,21x because of secondary events related to hormone deprivation. Second, the glucocorticoids may have a direct effect on APP regulation although this has not, thus far, been studied in vitro. Third, APP may be increased indirectly via inflammatory mechanisms by the action of cytokines or other inflammatory agents. Previous studies have shown that APP is regulated by certain inflammatory cytokines w7x and that cerebral APP expression is enhanced after lipopolysaccharide treatments w4x. Therefore, it is possible that some of the increased APP reactivity we observed in cortical areas may arise from the inflammatory reactions associated with ADX w8x. We did not observe APP reactivity in astrocytes or microglial cells. This is in contrast to previous studies where acute insults induced both by excitotoxic lesions and cerebral ischemia w16,19,20,23x result in striking APP
reactivity within astrocyte cell bodies and cellular processes besides neurons. The reason for lack of astrocytic localization of APP in the ADX model could be two fold. One, that ADX induces an astrocytic reaction, which may have been protracted from the changes in APP expression and two, unlike the other models, hormonal effect is perhaps limited to APP modulation in neurons. However, we are not certain that long-term ADX induces a profound astrocytic proliferation in the hippocampus although a microglial reaction was apparent. The investigation of the brains at earlier survival times would be necessary to evaluate whether astrogliosis is concomitant with APP expression in the ADX model. We also noted diffuse apolipoprotein E immunoreactivity that was localized to CA neurons in the hippocampus. While it is suggested that astrocytes and macrophages may synthesize and release apolipoprotein E within damaged areas to scavenge cholesterol and phospholipids from cellular debris, it is possible that the neuronal localization of protein reflects lipid mobilization in vulnerable or at risk cells in the hippocampus w17x. In summary, we observed enhanced APP reactivity in the CA fields and remaining granule cells of the dentate gyrus in rats subjected to ADX. Cerebrovascular myocytes
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and certain pyramidal neurons in the parietal and piriform cortices were also strongly reactive to antibodies to the C-terminal of APP. These cellular changes were not associated with a profound astrocytic response as shown by GFAP immunocytochemistry. While our observations are related to enhanced APP accumulation in other experimental conditions, long-term hormonal deprivation might be another factor that may directly or indirectly modulate the expression of APP in brain.
Acknowledgements We thank Dawn Cohen, Dr. Isam Suliman and Dr. Adlan Elhassan for technical assistance. We also thank Dr. Hiroshi Mori, Tokyo Institute of Psychiatry ŽTIP. for the antibodies. We acknowledge financial support from Loo och Hans Ostermans fond, Swedish Society for Medical Research, Stiftelsen Sigurd och Elsa Goljes Minne, KI fonder, Greta Lindenau Hansells Minnesfond, Gamla Tjanarinnor Foundation, the Alzheimer’s Association ŽChicago. and the USPHS for grants ŽRNK. AG08992 and AG10030.
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