Brain Research, 593 (1992) 128-135 ~'~ 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
128
BRES 25398
Amyloid precursor protein accumulates in regions of neurodegeneration following focal cerebral ischemia in the rat D i a n e T. S t e p h e n s o n , K a r e n R a s h , and J a m e s A. C l e m e n s Eli Ldly and Co., CNS Dn'l~:on, Lilly Corporate Center, h~dtanapolis, IN 46285 (USA) (Accepted 21 July 1992)
Key word,~: Focal =schemi'l; fl-Amyloid precursor protein; M=ddle cerebral artery occlusion; Rat; Dystrophic neurite; Stroke
The distribution of/3-amylold precursor protein (APP) was examined immunocytochemically in rats subjected to focal cerebral ischemia by permanent occlusion of the middle cerebral artery. At 4 and 7 days post-occlusion, APP immunoreactivlty was preferentially localized within axonal swellings, dystrophic neurites and neuronal perikarya all along the periphery of the infarct, lmmunolabeling was observed with antibodies generated against N-terminal. midregion, and C-terminal domains of APP. No immunoreactivity was observed with antisera directed against /3-amyloid protein (,'3A4) itself. This pathological accumulation of APP is consistent with alterations of APP recently described in other models of neurodegeneration and implies a role for this protein in the response to CNS injury.
Amyloid precursor protein (APP) is a large, ubiquitously distributed and evolutionarily conserved molecule whose function remains unknown. Three isoforms of APP, two of which contain an insert with significant homology to the Kunitz family of serine protease inhibitors (KPI) are derived from alternative splicing of a sinale gene ~'~'2"~'~'~.Normally, APP undergoes constitutive proteolytic cleavage such that a soluble aminoterminal portion of the molecule is released 41, This normal cleavage takes place within the/3-amyloid peptide (/]A4) region itself "~''n thus precluding/3.amyioid formation. A focus of intense investigation is the identification of the mechanisms responsible for aberrant processing of this protein to yield /]A4 protein, the major component of cerebral amyloid in Alzheimer's disease. Although the precise function of APP in the brain remains unknown, there is some evidence that it plays a role in processes such as cell adhesion 2.=~.29,3s, cell proliferation ~7'2'~and neurite extension =~', Interestingly, recent studies have demonstrated alterations in APP following injury to the CNS. APP is increased following stab lesion z~, kainic acid injection 12.-~4,3~',ibotenic acid injection~'~; head trauma 7, nucleus basalis lesion 4" and colchicine administration 33 in rat brain. The identifica-
tion of mismetabolism, excess synthesis, or overaccumulation of APP in different models of injury may provide insights as to the function of APP in the normal and pathological brain and whether these processes are implicated in aberrant processing of APP to yield ~A4. In the present study, APP was investigated immunocytochemically in rats subjected to local cerebral ischemia. Thirteen malc spontaneously hypertensive rats (SHR) (250-300 g) were subjected to focal cerebral ischemia by permanent occlusion of the middle cerebral artery (MCA) according to the method of Brint et al. 3. The MCA was electrocauterizcd at approximately the level of the rhinal fissure and was occluded immediately distal to the lenticulostriate branches such that the resulting infarction would be restricted to the neocortex. At four (n = 5) and seven (n = 8) days following occlusion, animals were perfused transcardially with phosphate-buffered saline followed by periodate lysine paraformaldehyde (PLP). The brains were excised, postfixed by immersion in the same fixative for 24 h, cryoprotected and snap-frozen in liquid nitrogen-cooled isopentan¢. Serial 10 p.m thick coronal cryosections were taken at the level of the lateral septum and striatum (rostral
Correspondence: J.A. Clemens, Eli Lilly and Co,, Lilly Corporate Center, Indianapolis, IN 46285, USA. Fax: (I)(317) 276-5546.
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Fig. 1. Nissl staining and APP C-terminal immunoreactivity in the region of focal cerebral infarction. A: low magnification micrograph of the infarct at the level used for immunocytochemistry. Arrov,~ indicate regions in which APP immunoreactivity could be observed in adjacent immunostained sections. B: Nissl staining in the dorsomedial aspect of the infarct and the border of the neocortex supplied by the middle cerebral artery and the anterior cerebral artery. C: APP C-terminal immunoreactivity with anti-C7 in the ventrolateral aspect immediately adjacent to the area of infarction. Note immunoreactive neurons (open arrows) and dystrophic neurites (arrows). D: anti-C7 immunoreactivity in the corpus callosum underlying the area of infarction. The above micrographs were taken from an animal at 7 days post-MCA occlusion. Bar = 1000 p,m (A), 250 p,m (B), 50 p,m (C), 16 p,m (D).
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Fig. 2. APP midregion and N-terminal immunoreactivity in the area of cerebral infarction at 4 days post-occlusion. A: neuronal perikaya at the border zone labeled with the midregion antibody, anti-B5. This micrograph was taken from the border of the MCA and the posterior cerebral artery at the ventrolateral ~pect of the infarct. B: anti-B5 immunoreactive axonal swellings within the callosal fibers underlying the infarct. (3:anti-B5 reactivity of a s t ~ at the periphery of the infarct. D: neuronal somata at the ventrolateral border zone stained with the N-terminal antibody, W63N. Note the punctate distribution of stain. E: W63N i m m u n o r ~ t y within macrophages/amoeboid microglia in the zone of infarction. F: lack of staining with R1280, an antiserum directed to ,8A4 itself. This micrograph was taken from the corpus callosum underlying the infarct where nearby sections displayed strong C-terminal and mid-region APP immunoreactivity. Bar = 16 ~tm.
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131 to the point of electrocauterization). Some animals were evaluated at additional levels of the infarct. Sections were stained with Cresyl violet for Nissi substance, with hematoxylin and eosin (H&E), by modified Bielschowsky silver (after post-fixing in 10% buffered formalin for 4 h) and with antibodies directed to various regions of APP. Five different antisera directed to synthetic peptides of APP were used: aminoterminal 21 residues [APP(18-38); W63Nli], midregion, extramembranous portion of APP [APP(444-592), anti-Bx52m'22] and C-terminal inclusive to f l a p region [APP(676-695); anti-Bx6 ~4'22,anti-C724 and anti-c8a2]. In addition, antibodies directed to human synthetic /3-amyloid (/3A4)were also used: anti-/3A4(1-38)(antibody y)10 anti-/3A4(1-40) (R1280) aa and a monoclonal antibody (10D5) 9. Several of these antibodies have been shown to bind rat APP or synthetic tat /3A4 by different biochemical criteria. Immunocytochemistry employed the avidin-biotin peroxidase system (ABC system, Vector Labs) with 0.05% diaminobenzidine as a chromogen. The specificity of each antiserum was confirmed by immunostaining cryosections of Alzheimer's disease cortex in parallel with each experiment. Negative controls included incubating sections with rabbit serum as a substitute for the primary antiserum or by immunostaining sections with antisera directed against other, non-APP related proteins. Other antibodies included anti-glial fibriilary acidic protein (GFAP) (Biogenex), anti-synaptophysin (Boeringer Mannheim Biochemicals), and anti-rat macrophage monoclonal antibody EDI (Serotec). Permanent occlusion of the MCA produced a large, well-demarcated zone of infarction extending throughout the rostrocaudal extent of the neocortex. The infarcted tissue lacked Nissl stained neurons (Fig. 1A and B) and showed evidence of necrosis and monocyte/r~acrophage infiltration in H & E stained sections. Infiltration was more extensive at 4 than at 7 days post-occlusion. A clearly defined zone, which may have corresponded to a penumbral zone, could be observed at the border of the tissue supplied by the MCA and that supplied by either the anterior cerebral artery at the dorsomedial aspect of the infarct (Fig. 1B) or the posterior cerebral artery along the ventrolateral aspect. Astrogliosis, identified with anti-GFAP immunostaining, was observed at the periphery of the infarct (not shown). Within the core of the infarct, however, no GFAP immunoreactivity was observed. The observation of degeneration of this very plastic cell type in the brain reiterates the severity of the insult. Modified Bielschowsl~ silver staining demonstrated argentophilic neurites, axonal spheroids and neuronal cell bodies particularly concentrated at the periphery
of the infarct. Within the core of the infarct, large patches of punctate granular silver deposits were observed in addition to a variable number of silver stained neurons. These pathological changes were more pronounced at 4 days post-occlusion than after 7 days. There was evidence for degeneration of commissural fibers since the contralateral cortex showed increased silver staining of axons and dendrites in the motor and somatosensory areas and not in the piriform or cingulate cortices. APP immunoreactivity was strongly associated with structures surrounding or bordering the region of infarction at 4 and 7 days post-occlusion. No APP immunolabeling was observed within the core of the infarct. In regions distant to the region of infarction, labeling was observed with some of the antibodies that is consistent with published accounts of endogenous levels of APP in rat brain 4. With other antibodies, endogenous APP was beyond the level of detection. Staining along the periphery of the infarct was more prominent after 4 days than 7 days. With the C-terminal domain directed antibodies (anti-C7, anti-C8, and anti-Bx6), enlarged axonal swellings and dystrophic-appearing neurites were intensely immunoreactive (Fig. 1C). Beaded-like structures were often observed, particularly in the callosal fibers underlying the infarct (Fig. 1D). Fine labeled threads were particularly prominent in the corpus callosum immediately ventral to the anterior cingulate cortex at the midsagittal level. in the cortical lamina of the border zone an increased intracellular labeling of neuronal cell bodies was observed. Sometimes, immunoreactive cell bodies were found adjacent to labeled pathological neurites (Fig. 1C). These structures could be detected in all brains although they were much more prominent in the 4-5 day post.occlusion animals. Glial cells were not stained with C-terminal directed antibodies. The midregion antibody (anti-B5) stained similar structures as those observed with C-terminal antibodies but with weaker intensity. Neuronal perikarya at the periphery of the infarct (Fig. 2A) and axonal swellings in the corpus callosum were lightly stained with anti-B5 antiserum (Fig. 2B). In addition, reactive astrocytes m these regions were immunolabeled (Fig. 2C). Anti-B5 did not recognize endogenous levels of APP, i.e. APP expressed in non-lesioned tissue. At 7 days postocclusion, anti-B5 immunoreactivity was associated with astrocytes and neurons at the border zone with only weak labeling of dystrophic neurites and axonal swellings. The N-terminal APP antibody W63N stained neurons at the periphery of the infarct (Fig. 2D). Axons and dystrophic neurites were not immunostained. At both 4 and 7 days post-occlusion, prominent staining
132 rat macrophages (ED1) (data not shown). N-Terminal APP immunoreactivity of both macrophages and neurons was punctate. The contralateral hemisphere showed no specific alterations in APP immunoreactiv-
was found in association with macrophages and vascular elements within the infarct itself (Fig. 2E). Labeled cells were identified as macrophages by staining adjacent sections with a monoclonal antibody directed to
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Fig. 3. Comparison of Biehchowsky silver staining (A,C) and C-terminal APP immunoreactivity (B,D) in focal ischemia, A and B: low magnification micrographs at the level of the corpus callosum immediately underlying the cerebral infarct, Caliosal fibers (arrows) and axonal ~pheroid~ (open arrows) can be observed with both silver staining and APP immunostaining. C: a high magnification micrograph illustrates punctate silver labchng at Ihe ventrolateral border zone. D: the same area as shown in C in a nearby section reveals anti-C7 immunoreactivity associated with neuronal somata (arrow), dystrophic neurites and axonal swellings (arrowheads). Bar = 50 ~m (A,B), 16/~m (C,D).
133 ity with any of the five antibodies. Antibodies against /3A4 itself (R1280, antibody Y and 10D5) showed no specific immunoreactivity at either time point (Fig. 2F). The region that demonstrated the most APP immunoreactivity was found to correspond to areas where silver positive staining was concentrated. This could be determined by comparing the APP immunostained sections to near-adjacent sections stained with modified Bielschowsky silver (Fig. 3). APP immunoreactive structures appeared to correspond to a subset of argentophilic structures. These results demonstrate that APP accumulates in dystrophic axons and neuronal perikarya following permanent focal cerebral ischemia. This result is consistent with the fate of APP following stab lesion 23 and kainate infusion n'34 in rat brain. Indeed, structures morphologically similar to those shown in the present study were observed in these studies. Most notable, localization of APP in axonal swellings has been recently described in human brain lesions, including cases of cerebral infarction 2°. The observation in the present study that antisera generated to N- and C-terminal domains of APP itself labeled neurons at the periphery of the infarct suggests that some full-length APP accumulates following the ischemia. The decreased intensity of labeling observed with the midregion directed antibody relative to that observed with C-terminal directed antibodies as well as the lack of staining of neuritic elements with the N-terminal antibody indicates that either the antibodies are of different affinities, that C-terminal epitopes are more exposed following the injury, or that putative amyloidogenic C-terminal fragments may be generated, as has been most recently observed in experimental head trauma ~, by some as yet to be determined proteolytic event(s). Western blot analyses of the MCA occlusion animals will be critical in addressing this issue directly. The lack of detectable/3A4 immunoreactivity indicates that fibrillar/3-amyloid protein is not being formed. Although regions of APP staining following focal ischemia correlate with argentophilic areas, it is possible that APP is associated with areas of regeneration as well. The finding of APP overexpression following peripheral nerve degeneration and regeneration 3m, the enhanced levels of expression of APP early on during embryogenesis6, the preferential localization of APP at synaptic sites 3° and the detection of growth associated protein GAP-43 in sprouting neurites and synapses within senile plaques t7 indicate that APP may be involved in regenerative processes. Our own findings in the MCA brains with anti-synaptophysin antibody shows increased synaptic staining in the vicinity of the infarct (unpublished observations), suggesting sprout-
ing or synaptic remodeling is occurring following focal ischemia in the areas where APP increases. Appearance of increased APP immunoreactivity could be explained by either overexpression of the protein at the genomic level or by accumulation of existing APP due to the injury itself. There is some evidence to suggest that both processes may be occurring after infarction. Damage to the nervous system elicits inflammatory responses, gliosis, and neuronal responses each of which can potentially amplify the expression of APP at the mRNA level. For example, interleukin-1 which has been shown to be overexpressed early on following ischemia18, is capable of increasing APP mRNA expression in vitro s. More directly, a selective induction of KPI domain-containing APP mRNA has been reported in the same model of focal ischemia as in this study ~. This induction was found to occur maximally at 4 days post-occlusion, the time point at which we observed maximal APP immunoreactivity. Finally, the increased immunoreactivity of neurons adjacent to the necrotic area supports this hypothesis. Our observation of APP immunoreactivity within dystrophic neurites and axonal swellings supports the idea that there is overaccumulation of APP following this insult. Of particular re,levance to this issue are the reports that neuronal APP is associated with the cytoskeleton 2~ and undergoes fast axonal transport 15. Thus, interruption of axonal transport, whether it be due to trauma- (e.g. stab lesion, experimental head trauma) or chemical-induced injury (e.g. kainate) can lead to accumulation of APP. Strong evidence for this idea is the recent report of Shigcmatsu and McGeer '~'~ in which administration of an agent that selectively disrupts axoplasmic transport leads to accumulation of APP in swollen proximal axons and cell bodies. Thus, focal ischemia may lead to accumulation of APP at the periphery of the infarct due to disturbances in axoplasmic flow to and from the large region of necrotic tissue. Further studies of APP following CNS insults are certain to lead to a greater understanding into the mnction of this protein and its role in neurodegenerative disease. The authors thank Dr. Dennis Selkoe for providing the APP antisera and the Alzheimer's disease brain tissue. We are grateful to Dr. Haruyasu Yamaguchi for providing the N-terminal antiserum W63N. We thank Cindy Lemere, Dora Games and Dennis Selkoe for neuropathological consultation and for numerous helpful discussions. 1 Abe, K., Tanzi, R.E. and Kogure, K., Selective induction of Kunitz-type protease inhibitor domain-containing amyloid precursor protein mRNA after persistent focal ischemia in rat cerebral cortex, Neurosci. Lett., 125 (199I) 172-174. 2 Breen, K.C., Bruce, M. and Anterton, B.H., Beta amylmd precursor protein mediates neuronal cell-cell and cell-surface adhesion, J. Neurosci. Res., 28 (1991) 90-100.
134 3 Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J. and Pulsinelli, W., Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsdateral common carotid arteries, J. Cerebral Blood Flow Metab., 8 (1988) 474-485. 4 Card, J.P., Meade, R.P. and Davis, L.G., Immunocytochemical localization of the precursor protein for ~-amylold in the rat central nervous system, Neuron, 1 (1988) 835-846. 5 Esch, F.S., Keim, P.S., Beattie, E.C., Blacher, R.W., Culwell, A.R., Oltersdorf, T., McClure, D. and Ward, P.J., Cleavage of amylmd/3 peptide during constitutive processing of its precursor, Science, 248 (1990) 1122-1124. 6 Fisher, S., Gearhart, J.D. and ester-Granite, M.L., Expression of the amyloid precursor protein gene in mouse oocytes and embryos, Prec. Natl. Acad. Sci. USA, 88 (1991) 1779-1782. 7 Games, D., Khan, K.H., Soriano, F.G., Lieberburg, I. and Smha, S., Evidence for altered APP processing following acute head trauma in the ro~'ent, Soc. Neurosci. Abstr., 22 (1992) 1437. 8 Goldgaber, D., Harris, H.W., 141a, T., Maciag, T., Donnelly, R.J., Jacobsen, J.S., Vitek, M.P and Gadjdusek, D.C., Interleukin 1 regulates synthesis of amyloid /3-protein precursor mRNA in human endothelial cells, Prec. Natl Acad. Set. USA, 86 (1989) 7606-7610. 9 Hyman, B.T., Tanzi, R.B., Marzloff, K., Barbour, R. and Schenk, D B., Kunitz protease inhibitor containing amyio~d beta protein precursor ~mmunoreact~vlty in AIzheimer's disease, J. Neuropathol. E~p. Neurol , 51 (1991) 76-83. 10 Joachim, C., Morris, J.H. and Selkoe, D.J., Diffuse amyloid plaques occur commonly in the cerebellum in AIzheimer's disease, Am. J. PathoL, 138 (1989) 373-384. Ii Kawarabayashi, T., Shnji, M., Harigaya, Y., Yamaguchi, H. and Hirai, S., Amyloid /3/A4 protein precursor is widely distributed in I,oth the central and peripheral nervous systems of the mouse, Brain Res., (1991) I-7. 12 Kawarabayashi, T., Shoji, M., Iiarigaya, Y., Yamaguchi, H. and Hirai, S., Expression of APP in the early stage of brain damage, Braitl Rt,s., 563 (1991) 334-338. 13 Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and lip, !!., Novel precursor of Al~heimer's disease amyloid protein shows protease inhibitory activity, Nature, 331 (1988) 530=532. 14 Knops, J., Johnson.Wood, K., Schenk, D.B., Sinha, S,, Lieberbur~, I. and McConlogue, L., Isolation of baculovirusoderived secreted and full-length heta.:lmyloid precursor protein, J. Biol, ('l.,m., 266 ( 1991 ) 7285-72~). 15 Koo, E.H., Sisodia, S.S., Archer, D.R., Martin, L,J,, Weidemann, A., Beyreuther, K,, Fischer, P., Masters, C.L. and Price, D.L., Precursor of amyloid protein in Alzheimer's disease undergoes fast anterograde axonai transport, Prec. Natl. Acad. Sci. USA, 87 (1990) 1561- 1565. 16 LeBlanc, A.D., Kovacs, P.M., Chen, H.Y., Villare, F., Tykocinski, M., Autilio-Gambetti and Gambetti, P., Role of amyloid precursor protein (APP): study with antisense transfection of human neuroblastoma cells, J. Net~ro.s'cL Rcs., 31 (1992)635-645. 17 Masliah, E., Mallory, M,, Hansen, L,, Alford, M., DeTeresa, R,, Terry, R., Baudier, J. and Saitoh, T., Localization of amyloid precursor protein in GAP43-immunoreactive aberrant sprouting neurites in AIzheimer's disease, Brain Res., 574 (1992) 312-316. 18 Minaret, M., Kuraishi, Y., Yabuuchi, K., Yamazaki, A. and Satoh, M., Induction of interleukin-l~ mRNA in rat brain after transient forebrain ischemia, J. Neurochem., 58 (1992) 390-392. 10 Nakamura, Yu, Takeda, M., Niigawa, H.. llariguchi, S. and Nishimura, T., Amyloid ~-protein precuisor deposition in rat h~ppocampus lesioned by ibotenic acid injection, Neuroses. Lett, 136 (1992) 95-98. 20 Ohgami, T., Kitamoto, T. and Tateishi, J., AIzheimer's amyloid precursor protein accumulates within axonal swellings in human brain lesions, NeuroscL Lett., 136 (1992)75-78. 21 OItersdorf, T., Fritz, L.C, Schenk, D.B., Liebcrburg, !. JohnsonWood, K.L., Beattie, E.C., Ward, P.J., Blacher, R.W., Dovey, H F. and Sinha, S., The secre'ed form of the Alzheimer's amyloid
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precursor protein with the Kunitz domain is protease nexin-ll, Nature, 341 (1989) 144-147. OItersdorf, T., Ward, P.J., Henriksson, T., Neve, R., Lieberburg, l. and Fritz, L.C., The Alzheimer amyloid precursor protein: identification of a stable intermediate in the biosynthetic/ degradative pathway, J. Btol Chem., 265 (1990) 4492-4497. Otsuka, N., Tomonaga, M. and ikeda, K., Rapid appearance of /3-amyloid precursor protein Immunoreactwity in damaged axons and reactive glial cells in rat brain following needle stab injury, Brain Res., 568 (1991) 335-338. Podlisny, M.B., Tolan, D.R. and Selkoe, D.J., Homology of the amyloid beta protein precursor in monkey and human supports a primate model for beta amyloidosis in Alzheimer's disease, Am. J. PathoL, 138 (1991) 1423-1435. Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hus, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller F. and Cordell, B., A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors, Nature 331 (1988) 525 -527. Refolo, L.M., Wittenberg, I.S., Friedrich, V.L. and Robakis, N.K., The AIzheimer amyloid precursor is associated with the detergent-insoluble cytoskeleton, J. Neuroso., 11 (1991)38883897. Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T. and Schenk, D.B., Secreted form of amyloid fl protein precursor is involved in the growth regulation of fibroblasts, Cell, 58 (1989) 615-622. Schubert, D., Cole, G., Saitoh, T. and OItersdorf, T., Amyloid beta protein precursor is a mitogen, Biochem. Biophys. Res. Commun., 86 (1989) 83-88. Schubert, D., Jin, L.-W., Saitoh, T. and Cole, G., The regulation of amyloid/3 protein precursol secretion and its modulatory role in cell adhesion, Neuron, 3 (1989) 689-694. Schubert, W., Prior, R., Weldemann, A., Dircksen, H., Multhaup, G., Masters, C.L. and Beyreuther, K., Localization of AIzheimer /3A4 amyloid precursor protein at central and peripheral synaptic sites, Brain R£:~., 563 (1991) 184-194. Scott, J.M., Parhad, I,M, and Clark, A.W., #-Amyloid precursor protein gene is differentially expressed in axotomized sensory and motor systems, Mol. Brain Res,, 10 (1991) 315-325, Selkoe, D J,, Podlisn?, M,B,, Cork, L,D. and Price, D.L., OAmyloid precursor protein of AIzheimer disease occurs as ! 10- to 135-kilodtdton membrane-associated proteins in neural and nonneural lissues, Pro('. Natl. Acad. Sci. USA, 85 (1988) 7341-7345. Shigematsu, K. and McGeer, P.L,, Accumulation of amyloid precursor protein in neurons after intraventricular injection of colchicine, Am. J, Pathol,, 140 (1992) 787-794, Shigematsu, K., McGeer, P.L., Walker, D.G., lshii, T. and McGeer, E.G., Reactive microglia/macrophages phagocytose amyloid precursor protein produced by neurons following neural damage, J. Neurosci. Res., 31 (1992) 443-453. Shivers, B.D., Hilbich, D., Multhaup, G., Salbaum, M., Beyreuther, K, and Seeburg, P.H,, AIzheimer's disease amyloidogenie glycoprotein expression pattern in rat brain suggests a role in cell contact, EMBO l., 7 (1988) 1365-1370, Siman, R., Card, J,P., Nelson, R,B. and Davis, L.G., Expression of beta-amyloid precursor protein in reactive astrocytes following neuronal damage, Neuron, 3 (1989) 275-285. Sisodia, S.S., Koo, E,H., Beyreuther, K., Unterbeck, A. and Price, D.L., Evidence that ~-amyloid protein in AIzheimer's disease is not derived by normal processing, Science, 243 (1990) 492-495. Tamaoka, A., Kalaria, R.N., Lieberburg, l. and Selkoe, DJ., Identification of a stable fragment of the Alzheimer amyloid precursor containing the /3-protein in brain microvessels, Prec. Natl. Acad. Sci. USA, 89 (1992) 1345-1349. Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa-Komaroff, L.L., Gusella, J.F. and Neve, R.L., Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with AIzheimer's disease, Nature, 331 (1988) 528-530. Wallace, W.C., Bragin, V., Robakis, N.K., Sambamurti, K., Van-
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41 Weidemann, A., Konig, G., Bunke, D., Fischer, P., S,dbaum, J.M., Masters, C.L. and Beyreuther, K., Identification, biogen~:sis, and localization of precursors of Alzhelmer's disease A4 drnylotd protein, Ce//, 57 (1989) 115-126.
128
BRES 25398
Amyloid precursor protein accumulates in regions of neurodegeneration following focal cerebral ischemia in the rat D i a n e T. S t e p h e n s o n , K a r e n R a s h , and J a m e s A. C l e m e n s Eli Ldly and Co., CNS Dn'l~:on, Lilly Corporate Center, h~dtanapolis, IN 46285 (USA) (Accepted 21 July 1992)
Key word,~: Focal =schemi'l; fl-Amyloid precursor protein; M=ddle cerebral artery occlusion; Rat; Dystrophic neurite; Stroke
The distribution of/3-amylold precursor protein (APP) was examined immunocytochemically in rats subjected to focal cerebral ischemia by permanent occlusion of the middle cerebral artery. At 4 and 7 days post-occlusion, APP immunoreactivlty was preferentially localized within axonal swellings, dystrophic neurites and neuronal perikarya all along the periphery of the infarct, lmmunolabeling was observed with antibodies generated against N-terminal. midregion, and C-terminal domains of APP. No immunoreactivity was observed with antisera directed against /3-amyloid protein (,'3A4) itself. This pathological accumulation of APP is consistent with alterations of APP recently described in other models of neurodegeneration and implies a role for this protein in the response to CNS injury.
Amyloid precursor protein (APP) is a large, ubiquitously distributed and evolutionarily conserved molecule whose function remains unknown. Three isoforms of APP, two of which contain an insert with significant homology to the Kunitz family of serine protease inhibitors (KPI) are derived from alternative splicing of a sinale gene ~'~'2"~'~'~.Normally, APP undergoes constitutive proteolytic cleavage such that a soluble aminoterminal portion of the molecule is released 41, This normal cleavage takes place within the/3-amyloid peptide (/]A4) region itself "~''n thus precluding/3.amyioid formation. A focus of intense investigation is the identification of the mechanisms responsible for aberrant processing of this protein to yield /]A4 protein, the major component of cerebral amyloid in Alzheimer's disease. Although the precise function of APP in the brain remains unknown, there is some evidence that it plays a role in processes such as cell adhesion 2.=~.29,3s, cell proliferation ~7'2'~and neurite extension =~', Interestingly, recent studies have demonstrated alterations in APP following injury to the CNS. APP is increased following stab lesion z~, kainic acid injection 12.-~4,3~',ibotenic acid injection~'~; head trauma 7, nucleus basalis lesion 4" and colchicine administration 33 in rat brain. The identifica-
tion of mismetabolism, excess synthesis, or overaccumulation of APP in different models of injury may provide insights as to the function of APP in the normal and pathological brain and whether these processes are implicated in aberrant processing of APP to yield ~A4. In the present study, APP was investigated immunocytochemically in rats subjected to local cerebral ischemia. Thirteen malc spontaneously hypertensive rats (SHR) (250-300 g) were subjected to focal cerebral ischemia by permanent occlusion of the middle cerebral artery (MCA) according to the method of Brint et al. 3. The MCA was electrocauterizcd at approximately the level of the rhinal fissure and was occluded immediately distal to the lenticulostriate branches such that the resulting infarction would be restricted to the neocortex. At four (n = 5) and seven (n = 8) days following occlusion, animals were perfused transcardially with phosphate-buffered saline followed by periodate lysine paraformaldehyde (PLP). The brains were excised, postfixed by immersion in the same fixative for 24 h, cryoprotected and snap-frozen in liquid nitrogen-cooled isopentan¢. Serial 10 p.m thick coronal cryosections were taken at the level of the lateral septum and striatum (rostral
Correspondence: J.A. Clemens, Eli Lilly and Co,, Lilly Corporate Center, Indianapolis, IN 46285, USA. Fax: (I)(317) 276-5546.
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Fig. 1. Nissl staining and APP C-terminal immunoreactivity in the region of focal cerebral infarction. A: low magnification micrograph of the infarct at the level used for immunocytochemistry. Arrov,~ indicate regions in which APP immunoreactivity could be observed in adjacent immunostained sections. B: Nissl staining in the dorsomedial aspect of the infarct and the border of the neocortex supplied by the middle cerebral artery and the anterior cerebral artery. C: APP C-terminal immunoreactivity with anti-C7 in the ventrolateral aspect immediately adjacent to the area of infarction. Note immunoreactive neurons (open arrows) and dystrophic neurites (arrows). D: anti-C7 immunoreactivity in the corpus callosum underlying the area of infarction. The above micrographs were taken from an animal at 7 days post-MCA occlusion. Bar = 1000 p,m (A), 250 p,m (B), 50 p,m (C), 16 p,m (D).
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Fig. 2. APP midregion and N-terminal immunoreactivity in the area of cerebral infarction at 4 days post-occlusion. A: neuronal perikaya at the border zone labeled with the midregion antibody, anti-B5. This micrograph was taken from the border of the MCA and the posterior cerebral artery at the ventrolateral ~pect of the infarct. B: anti-B5 immunoreactive axonal swellings within the callosal fibers underlying the infarct. (3:anti-B5 reactivity of a s t ~ at the periphery of the infarct. D: neuronal somata at the ventrolateral border zone stained with the N-terminal antibody, W63N. Note the punctate distribution of stain. E: W63N i m m u n o r ~ t y within macrophages/amoeboid microglia in the zone of infarction. F: lack of staining with R1280, an antiserum directed to ,8A4 itself. This micrograph was taken from the corpus callosum underlying the infarct where nearby sections displayed strong C-terminal and mid-region APP immunoreactivity. Bar = 16 ~tm.
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131 to the point of electrocauterization). Some animals were evaluated at additional levels of the infarct. Sections were stained with Cresyl violet for Nissi substance, with hematoxylin and eosin (H&E), by modified Bielschowsky silver (after post-fixing in 10% buffered formalin for 4 h) and with antibodies directed to various regions of APP. Five different antisera directed to synthetic peptides of APP were used: aminoterminal 21 residues [APP(18-38); W63Nli], midregion, extramembranous portion of APP [APP(444-592), anti-Bx52m'22] and C-terminal inclusive to f l a p region [APP(676-695); anti-Bx6 ~4'22,anti-C724 and anti-c8a2]. In addition, antibodies directed to human synthetic /3-amyloid (/3A4)were also used: anti-/3A4(1-38)(antibody y)10 anti-/3A4(1-40) (R1280) aa and a monoclonal antibody (10D5) 9. Several of these antibodies have been shown to bind rat APP or synthetic tat /3A4 by different biochemical criteria. Immunocytochemistry employed the avidin-biotin peroxidase system (ABC system, Vector Labs) with 0.05% diaminobenzidine as a chromogen. The specificity of each antiserum was confirmed by immunostaining cryosections of Alzheimer's disease cortex in parallel with each experiment. Negative controls included incubating sections with rabbit serum as a substitute for the primary antiserum or by immunostaining sections with antisera directed against other, non-APP related proteins. Other antibodies included anti-glial fibriilary acidic protein (GFAP) (Biogenex), anti-synaptophysin (Boeringer Mannheim Biochemicals), and anti-rat macrophage monoclonal antibody EDI (Serotec). Permanent occlusion of the MCA produced a large, well-demarcated zone of infarction extending throughout the rostrocaudal extent of the neocortex. The infarcted tissue lacked Nissl stained neurons (Fig. 1A and B) and showed evidence of necrosis and monocyte/r~acrophage infiltration in H & E stained sections. Infiltration was more extensive at 4 than at 7 days post-occlusion. A clearly defined zone, which may have corresponded to a penumbral zone, could be observed at the border of the tissue supplied by the MCA and that supplied by either the anterior cerebral artery at the dorsomedial aspect of the infarct (Fig. 1B) or the posterior cerebral artery along the ventrolateral aspect. Astrogliosis, identified with anti-GFAP immunostaining, was observed at the periphery of the infarct (not shown). Within the core of the infarct, however, no GFAP immunoreactivity was observed. The observation of degeneration of this very plastic cell type in the brain reiterates the severity of the insult. Modified Bielschowsl~ silver staining demonstrated argentophilic neurites, axonal spheroids and neuronal cell bodies particularly concentrated at the periphery
of the infarct. Within the core of the infarct, large patches of punctate granular silver deposits were observed in addition to a variable number of silver stained neurons. These pathological changes were more pronounced at 4 days post-occlusion than after 7 days. There was evidence for degeneration of commissural fibers since the contralateral cortex showed increased silver staining of axons and dendrites in the motor and somatosensory areas and not in the piriform or cingulate cortices. APP immunoreactivity was strongly associated with structures surrounding or bordering the region of infarction at 4 and 7 days post-occlusion. No APP immunolabeling was observed within the core of the infarct. In regions distant to the region of infarction, labeling was observed with some of the antibodies that is consistent with published accounts of endogenous levels of APP in rat brain 4. With other antibodies, endogenous APP was beyond the level of detection. Staining along the periphery of the infarct was more prominent after 4 days than 7 days. With the C-terminal domain directed antibodies (anti-C7, anti-C8, and anti-Bx6), enlarged axonal swellings and dystrophic-appearing neurites were intensely immunoreactive (Fig. 1C). Beaded-like structures were often observed, particularly in the callosal fibers underlying the infarct (Fig. 1D). Fine labeled threads were particularly prominent in the corpus callosum immediately ventral to the anterior cingulate cortex at the midsagittal level. in the cortical lamina of the border zone an increased intracellular labeling of neuronal cell bodies was observed. Sometimes, immunoreactive cell bodies were found adjacent to labeled pathological neurites (Fig. 1C). These structures could be detected in all brains although they were much more prominent in the 4-5 day post.occlusion animals. Glial cells were not stained with C-terminal directed antibodies. The midregion antibody (anti-B5) stained similar structures as those observed with C-terminal antibodies but with weaker intensity. Neuronal perikarya at the periphery of the infarct (Fig. 2A) and axonal swellings in the corpus callosum were lightly stained with anti-B5 antiserum (Fig. 2B). In addition, reactive astrocytes m these regions were immunolabeled (Fig. 2C). Anti-B5 did not recognize endogenous levels of APP, i.e. APP expressed in non-lesioned tissue. At 7 days postocclusion, anti-B5 immunoreactivity was associated with astrocytes and neurons at the border zone with only weak labeling of dystrophic neurites and axonal swellings. The N-terminal APP antibody W63N stained neurons at the periphery of the infarct (Fig. 2D). Axons and dystrophic neurites were not immunostained. At both 4 and 7 days post-occlusion, prominent staining
132 rat macrophages (ED1) (data not shown). N-Terminal APP immunoreactivity of both macrophages and neurons was punctate. The contralateral hemisphere showed no specific alterations in APP immunoreactiv-
was found in association with macrophages and vascular elements within the infarct itself (Fig. 2E). Labeled cells were identified as macrophages by staining adjacent sections with a monoclonal antibody directed to
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Fig. 3. Comparison of Biehchowsky silver staining (A,C) and C-terminal APP immunoreactivity (B,D) in focal ischemia, A and B: low magnification micrographs at the level of the corpus callosum immediately underlying the cerebral infarct, Caliosal fibers (arrows) and axonal ~pheroid~ (open arrows) can be observed with both silver staining and APP immunostaining. C: a high magnification micrograph illustrates punctate silver labchng at Ihe ventrolateral border zone. D: the same area as shown in C in a nearby section reveals anti-C7 immunoreactivity associated with neuronal somata (arrow), dystrophic neurites and axonal swellings (arrowheads). Bar = 50 ~m (A,B), 16/~m (C,D).
133 ity with any of the five antibodies. Antibodies against /3A4 itself (R1280, antibody Y and 10D5) showed no specific immunoreactivity at either time point (Fig. 2F). The region that demonstrated the most APP immunoreactivity was found to correspond to areas where silver positive staining was concentrated. This could be determined by comparing the APP immunostained sections to near-adjacent sections stained with modified Bielschowsky silver (Fig. 3). APP immunoreactive structures appeared to correspond to a subset of argentophilic structures. These results demonstrate that APP accumulates in dystrophic axons and neuronal perikarya following permanent focal cerebral ischemia. This result is consistent with the fate of APP following stab lesion 23 and kainate infusion n'34 in rat brain. Indeed, structures morphologically similar to those shown in the present study were observed in these studies. Most notable, localization of APP in axonal swellings has been recently described in human brain lesions, including cases of cerebral infarction 2°. The observation in the present study that antisera generated to N- and C-terminal domains of APP itself labeled neurons at the periphery of the infarct suggests that some full-length APP accumulates following the ischemia. The decreased intensity of labeling observed with the midregion directed antibody relative to that observed with C-terminal directed antibodies as well as the lack of staining of neuritic elements with the N-terminal antibody indicates that either the antibodies are of different affinities, that C-terminal epitopes are more exposed following the injury, or that putative amyloidogenic C-terminal fragments may be generated, as has been most recently observed in experimental head trauma ~, by some as yet to be determined proteolytic event(s). Western blot analyses of the MCA occlusion animals will be critical in addressing this issue directly. The lack of detectable/3A4 immunoreactivity indicates that fibrillar/3-amyloid protein is not being formed. Although regions of APP staining following focal ischemia correlate with argentophilic areas, it is possible that APP is associated with areas of regeneration as well. The finding of APP overexpression following peripheral nerve degeneration and regeneration 3m, the enhanced levels of expression of APP early on during embryogenesis6, the preferential localization of APP at synaptic sites 3° and the detection of growth associated protein GAP-43 in sprouting neurites and synapses within senile plaques t7 indicate that APP may be involved in regenerative processes. Our own findings in the MCA brains with anti-synaptophysin antibody shows increased synaptic staining in the vicinity of the infarct (unpublished observations), suggesting sprout-
ing or synaptic remodeling is occurring following focal ischemia in the areas where APP increases. Appearance of increased APP immunoreactivity could be explained by either overexpression of the protein at the genomic level or by accumulation of existing APP due to the injury itself. There is some evidence to suggest that both processes may be occurring after infarction. Damage to the nervous system elicits inflammatory responses, gliosis, and neuronal responses each of which can potentially amplify the expression of APP at the mRNA level. For example, interleukin-1 which has been shown to be overexpressed early on following ischemia18, is capable of increasing APP mRNA expression in vitro s. More directly, a selective induction of KPI domain-containing APP mRNA has been reported in the same model of focal ischemia as in this study ~. This induction was found to occur maximally at 4 days post-occlusion, the time point at which we observed maximal APP immunoreactivity. Finally, the increased immunoreactivity of neurons adjacent to the necrotic area supports this hypothesis. Our observation of APP immunoreactivity within dystrophic neurites and axonal swellings supports the idea that there is overaccumulation of APP following this insult. Of particular re,levance to this issue are the reports that neuronal APP is associated with the cytoskeleton 2~ and undergoes fast axonal transport 15. Thus, interruption of axonal transport, whether it be due to trauma- (e.g. stab lesion, experimental head trauma) or chemical-induced injury (e.g. kainate) can lead to accumulation of APP. Strong evidence for this idea is the recent report of Shigcmatsu and McGeer '~'~ in which administration of an agent that selectively disrupts axoplasmic transport leads to accumulation of APP in swollen proximal axons and cell bodies. Thus, focal ischemia may lead to accumulation of APP at the periphery of the infarct due to disturbances in axoplasmic flow to and from the large region of necrotic tissue. Further studies of APP following CNS insults are certain to lead to a greater understanding into the mnction of this protein and its role in neurodegenerative disease. The authors thank Dr. Dennis Selkoe for providing the APP antisera and the Alzheimer's disease brain tissue. We are grateful to Dr. Haruyasu Yamaguchi for providing the N-terminal antiserum W63N. We thank Cindy Lemere, Dora Games and Dennis Selkoe for neuropathological consultation and for numerous helpful discussions. 1 Abe, K., Tanzi, R.E. and Kogure, K., Selective induction of Kunitz-type protease inhibitor domain-containing amyloid precursor protein mRNA after persistent focal ischemia in rat cerebral cortex, Neurosci. Lett., 125 (199I) 172-174. 2 Breen, K.C., Bruce, M. and Anterton, B.H., Beta amylmd precursor protein mediates neuronal cell-cell and cell-surface adhesion, J. Neurosci. Res., 28 (1991) 90-100.
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