Complement C1qB and C4 mRNAs responses to lesioning in rat brain

EX.PJ%&&SE~~AL

118,117-125

rax@%oI.oo~

(1992)

complement ClqB and C4 mRNAs Responses to Lesion~~g in Rat 6rain G. M. PASINE~I,

S. A. JOHNSON, I. ROZOVSKY, M. LAMPERT-EARLY, D. G, MORGAN, M. N. GORDON, T. E. MORGAN, D, WILLOUGHBY, AND C. E. FINCH

Division of Neurogerontobgy, Ethel Percy Andrus Gerontology Center, and Department of Biological Sciences, University of Southern California, hs Angeles, California 90089-0191

cells by the C5b-9 membrane attack complex (MAC), a heteromeric complex with cytolytic activity (29,42). Endogenous inhibitors may block C activation or deactivate C components at several steps along the pathway (14, 16, 38). Cellular responses to C activation are mediated by various receptors (33, 41, 50), e.g., CR3, of which the @-subunit is also an integrin-type receptor subunit (13). A general view is that C would not be expressed in the brain. Because of the blood-brain barrier (BBB), C proteins would not be present except in trace amounts that are observed in cerebrospinal fluid, e.g., C4,0.9-4.0 pg/ ml (39, 56). However, in Alzheimer’s disease brain, neuritic plaques and degenerating neurites are immunopositive for many different C proteins, e.g., Clq and C4, and C3 among other activated C proteolytic fragments (‘7,22, 55). At one level of sensitivity, normal adult human brains did not show definitive immunoreactivity (22). There is also immunocytochemical evidence of C deposits associated with degenerating myelinated fibers in brains of Parkinson’s disease, Pick’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (55). The role of C activation in Alxheimer’s and other slow neurodegenerative conditions is unknown. A major question is whether C components enter the brain due to pre- or postmortem BBB breakdown, as shown for IgG and albumin in human and rodent brain (27). However, mRNAs for putative C inhibitors were also shown in Alzheimer’s disease in association with C deposits during neurodegeneration. The mRNA coding for the complement lysis inhibitor (CLI), the human homologue of the rat sulfated glycoprotein 2 (SGP-2), was increased during Alzheimer’s disease in hippocampal neurons (6, 21, 21a); this suggests that brain CL1 deposits could be locally synthesized and may not occur only as an artifact of BBB breakdown. Moreover, mRNA and protein for the membrane inhibitor of reactive lysis were shown in control and Alzheimer brains (24). This study shows that rat brain cells locally synthesize C component mRNAs (ClqB, C4, SGP-2) and describes their responses to deafferenting and excitotoxin lesions. These C components were selected on the basis

data show the presence of mRNAs for two comcomponents (C) in the adult rat brain and describe their responses to experimental lesions. Cortical deafferentation caused elevations in striatal ClqB and C4 mRNAs that coincided temporally and overlapped anatomically with the course of degeneration of cortieostriatal afferent fibers. By in sitis hybri~zation, ClqB mRNA in the lesioned striatum was colocalized to cells immunoreactive for CR3, a complement receptor found on microglia-macrophages. The mRNA for SGP2, a putative C inhibitor in rat, showed parallel changes. Similarly, in hippocampus and other brain regions, kainic acid lesions increased ClqB mRNA. The data suggest that microglia-macrophages and possibly other cells in rat brain rapidly up-regulate C-mRNAs in response to deafferentation and local neuron injury. These experimental responses provide models to analyze changes in C components during Alzheimer’s disease and other chronic neurodegenerative conditions. These plement

0 1992

Academic

Pm&?.Inc. INTRODUCTION

Little is known about the occwwnce of mRNAs for the complement (C) component proteins in the adult rat

brain. C proteins circulate in the blood and are also synthesized locally by fibroblasts, tissue macrophages, and other cells that participate in inflammatory responses (11,35,42). The C system is a fundamental effector in the natural immunity that appears to have phylogenetitally preceded antigen-specific or acquired immune responses (9). More than 25 proteins compose the C system; many are activated proteolytically from inert precursors as a cascade. Complement activation may be mediated by antigen-antibody complexes (classical pathway) or by other macromolecules (classical and/or alternate pathway). Activated C components have multiple roles in anaphylactic responses that include enhancement of phagocytosis (opsonization); chemotaxis to guide the migration of inflammatory cells (e.g., neutrophils and mononuclear phagocytes) (11); and cytotoxic effects on target 117

0014-4686/92

$5.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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of the following characteristics. ClqB mRNA represents the first C component (40,46); several cellular and subcellular membranes may bind Clq with resultant classical pathway activation in the absence of specific antibody (46). C4 (1) is the source of C4a, an anaphylatoxic proteolytic fragment of the activated C4, which mediates inflammatory responses (11); C4 mRNA was also localized in neurons that were immunopositive for neuron-specific enolase (16a). SGP-2 mRNA is a rat homologue of human CL1 (78% AA sequence identity), which blocked cell lysis from the MAC in a hemolytic assay (16). The schedule of C mRNA’s up-regulation overlaps with the period of afferent fiber degeneration, which suggests a role for C in responses of resident brain cells to various types of injury. MATERIALS

AND

METHODS

Animals. Adult male F344 rats (3 months old, 250300 g) were housed in a temperature-controlled room and maintained on a 12 h light/dark cycle with free access to food and water. Striatal okflerentation. Corticostriatal lesion groups include intact rats (unlesioned controls) or rats sacrificed at 3, 10, and 27 days postlesioning. Replicate groups of six to eight rats were prepared for RNA extraction, for in situ hybridization, and/or for morphological studies. Following pentobarbital anesthesia (50 mg/ kg, ip), rats were placed in a Kopf (Tujunga, CA) stereotaxic frame; frontal cortex and parts of the cingulate cortex were aspirated unilaterally using a Pasteur pipette under a dissecting microscope. Coordinates of removed tissues were, with respect to Bregma: rostrocaudal, +3 to -4 mm; medial-lateral, +2 to +6 mm (34). The lesion was packed with Gelfoam (Upjohn) and the scalp was sutured. Survival of rats was 100%. Kainic acid (KA) lesions. Rats were injected ip with KA (Sigma, St. Louis, MO) (10 mg/kg) or vehicle, and sacrificed at 4 to 96 h after injection. Only rats showing seizures indicative of limbic status epilepticus were used (2, 44). RNA extraction. After sacrifice by decapitation, the dissected tissues were stored at -75°C for subsequent RNA extractions. Total RNA was extracted from each striatum or hippocampus, left or right, individuals, not pooled (3). Briefly, tissues were homogenized for 1 min in 4 A4 guanidinium thiocyanate, 25 mM sodium citrate (pH 7.5), 0.5% sarcosyl, and 0.1 M p-mercaptoethanol, final volume 0.5 ml. After extraction by acidified phenol-chloroform and ethanol precipitation, the RNA pellet was rinsed with ethanol. The purified RNA was then dissolved in 0.5% sodium dodecyl sulfate (SDS) and stored at -75°C. Polyadenylated RNA was isolated from total RNA (rat whole brain) by oligo-dT-cellulose chromatography (43). Poly(A)+ RNA (1 pg) or total

ET AL.

RNA (5 pg) from striatum of each rat (four groups, intact, 3, 10, and 27 days postlesioning) was electrophoresed on denaturing (0.2 M formaldehyde) agarose gels and transferred to nylon membrane (Nylon 66 plus; Hoeffer, San Francisco, CA) in 2 X SSC (43).

cRNA probes for hybridization. The ClqB cDNA clone used in these studies was produced by polymerase chain reaction (PCR) from human brain frontal cortex cDNA using oligonucleotide primers based on the human ClqB sequence (40): sense primer sequence, 5’GATCGAATTC CCCAGAAAAT CGCCTTCTCT GC 3’; antisense primer sequence, 5’ATGCGGATCC g GGAAAAGAT GCTGTTGGCA CC 3’. Underlining represents the ClqB sequence; nonunderlined bases are restriction sites used for subcloning, EcoRl (sense) or BamHl (antisense). The 375 nt PCR product (nt 282 to 656 of ClqB mRNA) (40) is specific for the globular domain of ClqB (54). The fragment was gel purified and ligated into an EcoRl/BamHl linearized Bluescript SK+ transcription vector (Stratagene, San Diego, CA). Dideoxy sequencing confirmed accurate cloning of the human ClqB cDNA, which showed the 82% nt sequence similarity to mouse ClqB cDNA, as reported (54). Human C4 cDNA (clone pC4ALl) (51) was obtained from American Type Culture Collection (ATCC, Rockville, MD); a 603 nt PST I fragment, 418 nt of coding sequence and representing C4 nt 4847 to 3’ end (l), was subcloned into Bluescript SK+. Rat SGP-2 cDNA in pGEM vector was a gift from Dr. Michael Griswold (4), as used by us (31). The sequences of C4 and SGP-2 cDNAs were verified by dideoxy sequencing. Antisense [32P]cRNA probes were transcribed from linearized subcloned cDNA transcription vectors. Blot hybridization was carried out with lo6 cpm/ml in 50% formamide, 1.5 X SSPE, 1% SDS, 0.5% Carnation dried milk, 100 pg/ml yeast total RNA, and 500 fig/ml salmon sperm DNA; 53°C for 15 h. Blots were washed to a final stringency at 72°C in 0.2 X SSC, 0.2% SDS, and exposed to Kodak X-ray film (XAR-5, Eastman Kodak, Rochester, NY) with intensifying screens at -70°C. Optical densities were integrated by computerized video densitometry (Image Measure, Microscience, Inc., Federal Way, WA). Data were analyzed by ANOVA. Morphological studies. For in situ hybridization and/or immunocytochemistry (ICC) and silver impregnation histochemistry, brains were quickly rinsed in cold phosphate buffer (PBS, 10 mM, pH 7.4) and immersed in methylbutane at -25°C for 3 min. Brains were sliced (10 pm) and frozen sections mounted on polylysine coated slides, and stored at -70°C. For ICC and/or in situ hybridization, frozen sections were postfixed in PBS containing 4% paraformaldehyde (30 min, room temperature) and rinsed in PBS. Tissue sections for ICC were pretreated with normal serum and incubated overnight at 4°C with a monoclonal antibody for

COMPLEMENT

ClqB mRNA I

GENE

EXPRESSION

ADULT

119

BRAIN

RESULTS

c4 mRNA I

Striatal

-5.6 kb

FIG. 1. ClqB and C4 mRNA Northern blot hybridization from total rat brain poly(A)+ RNA, probed with human cRNA probes from coding regions.

signal antisense

CR3 complement receptor (47) (MRC 0X-42, Serotec; Kidlington, Oxford, U.K.). Vectastain ABC kit (Vector, Burlingame, CA) was used in subsequent steps to complete the diaminobenzidine staining (30). Control sections which were treated with PBS as substitutes for the primary antibody gave negative staining. Following ICC, tissue sections were rinsed in PBS and counterstained with cresyl violet or processed for in situ hybridization (30). Briefly, tissue sections were rinsed in 0.1 IM triethanolamine (TEA), pH 8.0, incubated in acetic anhydride (AA, 0.25% v/v in TEA, 10 min), and rinsed in TEA and PBS. Following AA treatment, tissue sections were hybridized with [“‘S]cRNA probes (0.3 ,ug/ml, 2 X ld dpm pg-‘) made from linearized transcription vectors. Sense strand hybridization was used as control. Following hybridization (3 h, 5O”C), stringency washes (0.1 X SSC, 60°C), and dehydration, slides were exposed to X-ray film, coated with NTB-2 emulsion (Kodak, Rochester, NY), and exposed for 7 days. Developed slides were counterstained with cresyl violet. Degenerating fibers and synaptic end structures were identified by using a silver impregnation, the FinkHeimer technique (10) which was modified for frozen tissue sections. BriefIy, tissue sections were postfixed for 1 week in PBS containing 4% paraformaldehyde, pH 7.0, rinsed in PBS, and incubated in a solution containing KMnO,. Sections were bleached, rinsed in PBS, and incubated in a solution containing AgNOB and UO&NO&. Following incubation in AgNO, solution cont~~ng NaOH and N&OH, sections were incubated in ethanol containing paraformaldehyde and citric acid. After incubation in (Na&&O, 5H,O), tissue sections were rinsed in PBS and dehydrated. l

IN

Responses to Deufferentation

Because the rat is rarely used for molecular studies of C components (42), we first showed that the human C cRNAs probes detected similar mRNAs in the rat brain. Northern blot hybridization of rat brain poly(A)+ RNA with human cRNA probes detected single mRNA hybridization bands for ClqB (1.1 kb) and C4 (5.6 kb) (Fig. l), which resemble those in peripheral C-secreting cells of human (40) and guinea pig (51). As assessed by Northern blot hybridization of striatal total RNA, frontal cortex ablation induced ClqB mRNA in the ipsilateral deafferented striatum by eightfold at 3 days and fivefold at 10 days postlesion (Fig. 2). C4 mRNA was also induced fourfold by 10 days. SGP-2 mRNA was similarly elevated by 10 days postlesion. By 27 days after lesion, ClqB, C4, and SGP-2 mRNAs approached control levels. For comparison, striatal neurofilament (NF-68)-mRNA did not significantly change at any time postlesioning. In situ hybridization showed shifting regional location of these mRNAs. At 3 days, ClqB mRNA hybridization was increased in the cortical wound site (aspiration cavity) which is separated from the striatum (caudateputamen) by the corpus callosum (Fig. 3A). Then, at 10 days, the main ClqB mRNA signal shifted to the dorsal half of the deafferented striatum (Fig. 3B). At a cellular level, in situ hyb~~zation showed that striatal ClqB mRNA was distributed in large clusters of densely packed cells (Figs. 4A-4D). These cell clusters overlapped with the distribution of degenerating afferent terminals as assessed by silver impregnation histochemis-

10

27

7i?ME POSTLEs/oNIING

(DiWS)

FIG. 2. Time course of ClqB, C4, SGP-2, and NF-68 mRNAs changes in the ipsilateral striatum following unilateral frontal cortex ablation. Ordinates represent the integrated optical density of the respective bands from Northern blot autoradiogram. Means +: SEM, n = 4, *P < 0.01 relative to intact (nonlesioned) group.

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ClqB mRNA

3 Days

10 Days

ET AL.

C4 mRNA

COMPLEMENT

GENE EXPRESSION

IN ADULT

BRAIN

121

FIG. 3, Autoradiographic pseudocolor image of ClqB (A,B) and C4 (C,D) in striatum at 3 fA,Cf and 10 days (B,D) after unilateral frontal cortex (FRl) ablation as assessed by in situ hybridization assay. Abbreviations: CGl, cingulate cortex area 1; CG2, cingulate cortex area 2; FRl, frontal cortex area 1; FR2, frontal cortex area 2; FL, forelimb area of cortex; CC, corpus callosum; PAR, parietal cortex area 1; CPU, caudate putamen (striatum); LV, lateral ventricle; aca, anterior commissure. Atlas coordinates in mm (+1.20) relative to bregma; redrawn from (34). Images are representative of n = 4 per group. FIG. 4. In situ hybridization emulsion autoradiography of the ClqB mRNA distribution in the deafferented striatum 10 days after frontal cortex ablation, as visualized at low-power (A,B) and high-power magnification (C,D). Arrows indicate the same cluster of ClqB mRNA hybridization shown in dark (A,C) and bright (B,D) field illumination. Bar length, 100 pm. FIG. 5. Striatal ClqB mRNA (A), degenerating afferent terminals (B), and CR3 (0X-42) C receptor immuno~activity (C) ~stribution in the deafferented striatum, 10 days after cortical ablation as assessed by in situ hyb~~zation, silver impregnation histochemist~ (FinkHeimer stain), and ICC on neighboring tissue sections. In D, striatal ClqB mRNA in situ hybridization combined with ICC for OX-42 (CR3 C receptor) on the same tissue section. In B, arrows indicate the areas of silver impregnation over the background signal; in C, arrows indicate a cluster of OX-42 immunopositive microglia-macrophage. Bar length: A, B, C, 20 pm; D, 10 pm.

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Antisense

Sense

ET

AL.

CA3 pyramidal neuron layer, as visualized 48 h after KA treatment (Fig. 7B). ClqB mRNA was also elevated in the parietal cortex, amygdaloid complex, and hypothalamus (Fig. 7B). DISCUSSION

FIG. 6. Antisense (A) and sense (B) ClqB mRNA in situ hybridization signal on adjacent tissue sections from intact adult rat brain (horizontal plane) as visualized by dark field illumination. Abbreviations: C, cerebral cortex; CPU, caudate-putamen (striatum); CRL, cerebellum. Large arrowhead, pyramidal layers of hippocampus. Small arrowhead, granule cell layer of dentate gyrus.

try (Fink-Heimer stain) on adjacent sections (Figs. 5A and 5B). ICC for CR3 complement receptor (0X-42) (47), a marker for microglia-macrophages, showed a pattern similar to ClqB mRNA in situ hybridization and Fink-Heimer histochemistry (Fig. 5C). In situ hybridization combined with OX-42 ICC confirmed the colocalization of ClqB mRNA with CR3 immunopositive microglia-macrophages (Fig. 5D). C4 mRNA was also localized to the dorsal half of the deafferented striatum at 10 days postlesion (Fig. 3D). However, C4 mRNA was not induced in the wound cavity at 3 days postlesion (Fig. 3C), in contrast to ClqB mRNA. Brain ClqB mRNA Distribution Responsesto Systemic KA

and Hippocampal

In control (intact) adult rat brain, the ClqB mRNA signal was mostly localized to the pyramidal and granule (dentate gyrus) cell layers of the hippocampus, as assessedby in situ hybridization (Fig. 6A). ClqB mRNA hybridization was also observed in cerebral cortex, striatum (caudate-putamen), and cerebellum with signals suggesting concentrations in neuronal layers or other laminate structures. In contrast, hybridization with ClqB sense strand gave a weak and relatively homogeneous signal (Fig. 6B). Systemic KA (ip) induced neuron degeneration by a different approach that does not cause mechanical trauma to the brain. Hippocampal ClqB mRNA increased threefold by 48 h and fourfold by 96 h, as assessed by Northern blot (Fig. 7A). No change was detected at 4, 12, and 24 h after KA. By in situ hybridization, hippocampal ClqB mRNA increased mostly in the

Two experimental lesions induced increases of several C-related mRNAs in adult rat brain. Deafferenting lesions caused rapid changes in the striatum. The postlesion schedule of striatal ClqB-, C4-, and SGP-2 mRNA induction corresponded to the degenerating phase of the corticostriatal afferents and was associated with increased glial responses (32). ClqB mRNA was identified in OX-42 (CR3 receptor) immunopositive microglia-macrophage cell clusters that colocalized with degenerating corticostriatal afferents identified by the Fink-Heimer silver stain. The CR3 receptor is a marker for resident microglia-macrophages (37, 47), but also identifies circulating macrophages, neutrophils, and natural killer cells (48), which may infiltrate the brain at the injury site (12). Cells which contained ClqB mRNA

A 6

60

_ ClqBmRNA

B 40 Q g 20 8 0

CO 4 12 24 it3 96 TIME POSTLESIONING

(HOURS)

B

FIG. 7. (A) Time course of hippocampal ClqB mRNA changes following single injections of kainic acid (KA) as assessed by Northern blot hybridization (CO, control; 4, 12,24,48,96 h after KA treatment). Means -+ SEM, n = 4, *P < 0.01 relative to control. (B) Distribution of ClqB mRNA changes 48 h after KA lesions as visualized by in situ hybridization autoradiography. CO, control; KA, kainic acid. Abbreviations: CC, corpus callosum; PC, parietal cortex; CA3, pyramidal layer of hippocampus CA3; DG, dentate gyrus; AC, amygdaloid complex; VMH, ventral hypothalamic region.

COMPLEMENT

GENE

EXPRESSION

and were immunopositive for CR3 (0X-42) in the deafferented striatum at 10 days postlesion could also have been infiltrating blood-derived macrophages which migrated along degenerating corticostriatal afferent fibers for distances of several millimeters. However, the ramified shape of these cells is more characteristic of resident mieroglia (Fig. 5C) (26, 47). ClqB mRNA is normally expressed in normal human neocortical microglia, as assessed by combined LN3 (HLA-DR) ICC and ClqB in situ hybridization (16a). An interesting anatomical shift of ClqB mRNA distribution was found after lesioning. Both ClqB and C4 mRNA peak elevation in the deafferented striatum showed a similar topographic distribution by 10 days postlesioning as assessed by in situ hybridization (Figs. 3B and 3D). However at 3 days postlesioning, ClqB mRNA signal was mostly localized in cells immediately adjacent to the cortical wound, C4 mRNA showed no signal at this time or place (Figs. 3A and 3C). Although C4 mRNA showed no definitive increase in the deafferented striatum by in situ hybridization 3 days postlesioning (Fig. 3C), Northern blot assay indicated a significant increase (Fig. 2). An important technical consideration from this study is that the peak elevation of ClqB mRNA from microdissected striata 3 days postlesioning, assayed by Northern blot, is possibly due to contamination of the tissue adjacent to the wound cavity which lay dorsal to the deafferented striatum. The failure to detect C4 mRNA induction in the wound cavity 3 days postlesioning is intriguing and suggests the possibility of independent regulation of these C mRNAs in microglia or by other cells. ClqB mRNA may be also expressed in neurons, as suggested by the dense in situ labeling over neuronal layers in control (intact) adult rat hippocampus (Fig. 6A). While KA induced ClqB mRNA in the pyramidal layer of hippocampus, further in situ hybridization studies combined with neuronal or glial immunodetection are needed to establish the hippocampal cell type expressing ClqB mRNA. ClqB mRNA also showed major increases after KA in the cerebral cortex, amygdaloid complex, and hypothalamus; KA induces neurodegeneration in these regions (2, 44). The reason for higher ClqB mRNA induction in striatum following deafferentation compared to hippocampal responses to kainate treatment is unknown and could merely reflect differences in the ma~itude of lesion and/or repair schedule between the two lesion paradigms. Further studies of extended times in response to KA treatment may resolve this question. Hippocampal deafferentation by perforant pathway transection induces ClqB mRNA (Pasinetti et al., in prep~ation), concurrently with the degeneration of hippocampal afferents (15, 45) in coincidence with microglia-macrophage activation (8): the cell location is under investigation.

IN

ADULT

BRAIN

123

C components may play key roles in the initiation and control of local inflammation and phagocytosis in brain that are fundamental in responses to lesions. For example, inflammatory responses may enhance axonal regeneration in peripheral nerves (20). Proteolysis of particular C components, e.g., C3, C4, and C5, can generate anaphylatoxic C residues (C3a, C4a, C5a) that are chemotactically active in inflammatory responses (11,42). For example, direct infusion of C5a into hypothalamus caused infiltration of polymorphonuclear leukocytes within 6 h (52). Also, there are receptors for C3a in the normal rat h~othalamus (53). Moreover, anaphylactic C residues may promote release of lysosomal enzymes and degranulation of mast cells (11) which are resident in normal rat brain (17, 36). Antibody-independent C activation may be triggered by the binding of dystrophic cellular components to Clq (46). This mechanism, which is distinct from the alternate complement pathway, could also be pertinent to several neurodegenerative events, whereby C may be initially responsible for the production of several mediators of acute inflammation, as speculated for certain brain demyelinating diseases (49). Phagocytosis, which involves the binding of a targeted degenerating structure to the surface of a phagocytic cell, is also a C-mediated response (41). This process is mediated by the interaction of C residues with specific C receptors on phagocytic cells. For example the C3-derived C3b proteolytic fragment may bind to the CR3 C receptor that is also expressed in brain microglia-macrophages (47). The C3 cleavage residues, iC3b, C3c, C3d, and C3dg (28) were immunolocalized to senile plaques in Alzheimer brains (7,22). Provisional evidence that patients receiving antiinflammatory treatments for rheumatoid arthritis show less dementia than predicted for the age group (23), might implicate C-mediated mechanisms in cytotoxic aspects of neurodegeneration. The present studies show the involvement of C-component mRNAs in acute phases of response to lesions. An open question is whether the Alzheimer-related C responses had occured just before death, or were part of a slow inflammatory process lasting many years which could be either an effect or a cause of neurodegeneration. Concurrently with the lesion-induced increases of ClqB and C4 mRNAs, we observed increased mRNA for SGP-2. A human homolo~e of SGP-2 is a plasma protein, complement lysis inhibitor (CLI) with 78% AA identity which shows activity in blocking the cytolytic membrane attack complex (MAC) C5b-9 (16,21b). The potential CL1 activity of brain SGP-2 remains to be investigated. In view of the increases of C4 and ClqB mRNAs during responses to lesions, it is therefore pertinent that SGP-2 mRNA is induced in hippocampal astrocytes by deafferenting cortical lesions (5,19). SGP-2 immunoreactivity also increases after KA lesions in

124

PASINETTI

hippocampal pyramidal neurons (21). SGP-2 may be secreted, as suggested by immunoreactive deposits of SGP-2 in the neuropil after three different lesions: excitotoxin lesions in the striatum (31), corticosteroid-dependent neuron death in the hippocampus (25), and deafferenting lesions in the hippocampus (18). Thus, the presence of SGP-2 immunoreactivity in hippocampal pyramidal neurons after excitotoxin lesions (21) could represent the uptake or binding of SGP-2 protein or fragments that were secreted by astrocytes. Blockade of the potentially cytolytic MAC (C5b-9) of the C pathways by SGP-2 could protect brain cells from harm by locally activated C components. Together, these early observations indicate that the brain makes mRNA’s that encode representatives of molecules used in the C-mediated aspects of inflammatory responses. Several C-component proteins have multiple functions, any of which could participate in neural responses to lesions: chemotaxis for recruiting phagocytic cells, opsonization and phagocytosis to remove debris, possibly directing the sprouting of afferent fibers, and cytotoxic mechanisms. These acute responses to several brain lesions could be pertinent to various phases of Alzheimer’s disease that include neurodegeneration, as well as protection against further injury during this prolonged disease.

ET

STAM. 1989. Complement heimer’s disease. Virchows

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ACKNOWLEDGMENTS The study was supported by the United Parkinson Foundation and by the Turken scholarship from the Alzheimer Association to G.M.P.; NIA AG-7909 and AG-9793 and the MacArthur Foundation Research Program on Successful Aging to C.E.F.; and AG-10673 to S.A.J. We thank Dr. Nicholas Laping, Dr. Jonathan Day, and Ms. Chris Zarow for help in experimental lesioning.

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R. FREDRIK, N. J. BERNSHAW, L. A. WILCOX, 1989. Isolation and characterization of a from normal human erythrocytes that inof the erythrocytes of paroxysmal nocturnal Clin. Znoest. 84: 7-17.

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LAMBRACHT-HALL, M., V. DIMITRIADOU, AND T. C. THEOHARIDES. 1990. Migration of mast cells in the developing rat brain. Dew. Brain Res. 56: 151-159.

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LAPING, N. J., N. R. NICHOLS, J. R. DAY, AND C. E. FINCH. 1991. Corticosterone differentially regulates the bilateral response of astrocyte mRNAs in the hippocampus to entorhinal cortex lesions in male rats. Mol. Brain Res. 10: 291-297.

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Lu, X., AND P. M. RICHARDSON. 1991. Inflammation cell body enhances axonal regeneration. J. Neurosci. 978.

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