Complement mRNA in the mammalian brain: Responses to Alzheimer's disease and experimental brain lesioning

NeurobiologyofAging,Vol. 13, pp. 641-648, 1992

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Complement mRNA in the Mammalian Brain: Responses to Alzheimer's Disease and Experimental Brain Lesioning S T E V E N A. J O H N S O N , '

M A R T H A L A M P E R T - E T C H E L L S , G I U L I O M. P A S I N E T T I , I R I N A R O Z O V S K Y A N D C A L E B E. F I N C H

Neurogerontology Division, Andrus Gerontology Center, and Department of Biological Sciences University of Southern California, Los Angeles CA 90089-0191 JOHNSON, S. A., M. LAMPERT-ETCHELLS, G. M. PASINETTI, I. ROZOVSKY AND C. E. FINCH. ComplementmRNA in the mammalian brain: Responses to Alzheimer's disease and experimental brain lesioning. NEUROBIOL AGING 13(6) 641-648, 1992.--This study describes evidence in the adult human and rat brain for mRNAs that encode two complement (C) proteins, C IqB and C4. C proteins are important effectors of humoral immunity and inflammation in peripheral tissues but have not been considered as normally present in brain. Previous immunocytochemical studies showed that C proteins are associated with plaques, tangles, and dystrophic neurites in Alzheimer's disease (AD), but their source is unknown. Combined immunocytochemistry and in situ hybridization techniques show C4 mRNA in pyramidal neurons and ClqB mRNA in microglia. Primary rat neuron cultures also show ClqB mRNA. In the cortex from AD brains, there were two- to threefold increases ofClqB mRNA and C4 mRNA, and increased ClqB mRNA prevalence was in part associated with microglia. As a model for AD, we examined entorhinal cortex perforant path transection in the rat brain, which caused rapid increases ofC l q B mRNA in the ipsilateral,but not contralateral, hippocampus and entorhinal cortex. The role of brain-derived acute and chronic C induction during AD and experimental lesions can now be considered in relation to functions of C proteins that pertain to cell degeneration and/or cell preservation and synaptic plasticity. Complement Microglia

Inflammation

Alzheimer's disease

In situ hybridization

THE complement (C) system is a major humoral defense system composed of two main proteolytic cascades and involving more than 25 blood proteins, including regulators. Complement can be activated by antigen-antibody complexes or other effectors, such as bacterial lipopolysaccharides, polyanionic compounds, and myelin (27,38). By-products include anaphylactic peptides that are also chemoattractants and activators of phagocytes. Further, the activated C cascade may culminate in deposition of the membrane attack complex (MAC) at activation sites. The MAC and various inflammatory responses induced by diffusible C activation peptides (C3a, C4a, and C5a) are potentially cytolytic or damaging to many cell types. In affected regions of brains with Alzheimer's disease (AD), immunocytochemical (ICC) studies detected classical C pathway or C regulatory components in neuritic plaques, neurofibrillary tangles, and degenerating neurites (6,24,32). These ICC studies also detected neoepitopes that are only revealed by C activation, including a C9 neoepitope characteristic of the MAC (24). Together, these reports suggest that C proteins are present and the C cascade may be activated in the AD brain, with production and deposition of MACs.

Cortex

Hippocampus

The AD brain also contains m R N A s for the C regulatory proteins complement lysis inhibitor (CLI; clusterin; SP-40,40) (12,13,22) and membrane inhibitor of reactive lysis (MIRL, CD59) (25). Soluble, plasma-derived forms of both proteins inhibit MAC-mediated cytolysis. The rat ortholog of CLI, sulfated glycoprotein-2 (SGP-2), has extensive (78% aa) similarity to CLI (13,22a). SGP-2 m R N A increases in astrocytes during responses to entorhinal cortex perforant path lesions in rats (19) but may also be present in neurons (22). Immunodeposits of SGP-2 in the neuropil of lesioned rats (18,19,26,31) suggest that astrocytes secrete SGP-2 protein, which could regulate local, soluble (but not cell bound) cytotoxic actions of the complement MAC. In view of postmortem extravasation and neuronal uptake of immunoglobulin (Ig)G (28), and because of dogma that the brain normally makes few immune components, it could be postulated that C proteins associated with AD lesions entered the brain after blood-brain barrier breakdown during the disease process or after death. An alternative, however, is local synthesis of C components by the major resident brain cell types. Thus, we focused on localization and quantitation of C I qB chain and C4 mRNAs. C 1q binds to various C activators and initiates the classical pathway; C4 is the substrate for activated C 1 complex and is the source of the C4a chemoattractant

~To whom requests for reprints should be addressed. 641

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peptide. We show by combined ICC/in situ hybridization (ISH) that these C mRNAs are present in several inherent brain cell types, including neurons, indicating the potential for local inflammatory responses in the brain.

centrifugation method (4). Poly(A) + RNA was isolated from total RNA by two cycles of oligo-d(T)-cellulose chromatography (15), capillary blotted onto nylon membrane (Hoeffer, San Francisco, CA), and hybridized and washed at high stringency to C 1qB or C4 antisense cRNA transcripts as described (16)

METHOD A utopsy Tissue

Human brain autopsy material was obtained from Dr. C. Miller of the USC Alzheimer's Disease Research Center, Dr. J. Rogers of the Institute of Biogerontology Research (Sun City, AZ), Dr H. Chui of the Rancho Los Amigos Medical Center (Downey, CA), and Dr. W. W. Tourtellotte of the National Neurological Research Bank, VA Wadsworth Medical Center (Los Angeles, CA). Brain specimens were evaluated histopathologically according to standard criteria for plaques and tangles. Seven AD (four males and three females; mean age = 86 years) and 6 non-AD (three males and three females; mean age = 82 years) cases were used in this study; cause of death for non-AD cases: lung cancer (two males), heart failure (three females), and Parkinson's disease (one male). None of the AD cases had notable secondary lesions. cDNA Clones

The C I qB cDNA clone was produced by polymerase chain reaction (PCR) from human brain frontal cortex cDNA using oligonucleotide primers based upon the human ClqB chain mRNA sequence (33) [sense primer sequence, 5' GATCGAATTC CCCA GAAAA T CGCCTTCTCT GC 3'; antisense primer sequence, 5'ATGCGGATCC CGGAAAAGA T GCTGTTGGCA CC3"; the italicized letters represent C lqB sequence while other letters represent extra nucleotides and EcoR 1 (sense) or BamH l (antisense) restriction sites for cloning]. The 375 bp PCR product [nt 282 to 656 ofClqB mRNA (33), completely specific for the globular domain ofC 1qB (41 )] was gel purified and directly ligated into an EcoR l/BamH 1 cut Bluescript S K + transcription vector (Stratagene, San Diego, CA). Dideoxy sequence analysis confirmed the human ClqB sequence was cloned and showed the expected 82% nt sequence similarity to mouse ClqB cDNA (41). Human C4 cDNA was obtained from ATCC [Rockville, MD; ATCC 59502; clone pC4ALI (39) and a 603 nt PST l fragment [containing 418 nt of coding sequence and representing C4 nt 4847 to 3' end (3)] was subcloned into the Bluescript S K + . This clone contains ca. 40 nt of GC sequence at the 3' end due to the use of the GC tailing method for production of the original cDNA library from which pC4AL 1 was derived (39). However, the lack of appreciable signal with sense strand C4 probes after ISH employing posthybridization ribonuclease treatment shows the GC tract has not compromised probe specificity. Cell Culture

Pure astrocyte cultures were derived from 2- to 5-day-old neonatal entorhinal cortex. Astrocytes were purified from the mixed cell culture by a shaking procedure after 1 week (23) and grown for 2-3 weeks with change of media at 2-day intervals. All mature astrocyte cultures were GFAP positive by ICC. Neurons from E 16 embryos were cultured for 6 days in serumfree medium and contained less than 3% GFAP-immunopositive cells. RNA Extraction and Blot Hybridization

RNA was extracted from frozen human frontal cortex (area 10) or whole rat brain by the guanidinium thiocyanate/CsCl

IStt and 1CC

Ten-micron paraffin or 40-t~m frozen sections were fixed lightly in 4% buffered paraformaldehyde and pretreated with proteinase K (paraffin sections only). After hybridization with cRNA probes, sections were RNAse treated and washed at high stringency (14). When ISH was combined with ICC on the same sections, the ICC was performed first (30). All 1CC used Vectastain ABC kits (Vector Laboratories, Burlingame, CA) with diaminobenzidine as chromagen. Sense strand probe labeled to equivalent specific radioactivity of antisense probe was used in all experiments on adjacent sections to control for background hybridization. Antibodies

Antihuman neuron-specific enolase (17437, Polysciences, Inc., Warrington, PA) was used at 1:3,000 dilution for ICC. Antihuman B-lymphocyte LN3 (69-303, ICN Immunobiologicals, Costa Mesa, CA) was prediluted by manufacturer and used without further dilution. LN3 detects a nonpolymorphic, class lla, HLA-DR surface antigen characteristic of macrophages and activated microglia. Perforant Path Lesion

Unilateral entorhinal cortex perforant path transection lesions were modified from Gibbs et al. (11); the authors thank B. Cummings and C. Cotman for teaching them the knife cut technique (5). A Scouten retractable wire knife (K6pf Instruments, Topanga, CA) was placed stereotaxically to cut the perforant path; stereotaxic coordinates, relative to lambda, were: 1 mm anterior, 6.3 mm lateral, and 5 m m ventral from dura, with a dorsal excursion of 4 mm with the extended blade (2 mm extension). RNA was extracted from the hippocampi of 3month-old Fischer 344 males at 2, 4, and 10 days postlesion. RESULTS C mRNA Localization in the Brain

Analysis of human and rat brain RNA used cRNA probes constructed from human C mRNA coding sequences. Single mRNA species were detected for ClqB and C4 on northern blots of human frontal cortex poly(A)+RNA and corresponded to the expected sizes of mRNAs found in complement-producing peripheral tissues (Fig. 1). Cell type specificity of CIqB mRNA was determined by ISH and ICC. Combined LN3 ICC/ClqB ISH on the same tissue section showed the colocalization ofC lqB mRNA in LN3immunopositive (HLA-DR antigen presenting) microglia in human neocortical (Brodman area 10) gray matter (Fig. 2A). Low-power, dark-field photomicrography of C IqB mRNA ISH on frontal cortex (Fig. 2B) demonstrated the distribution o f C l q B mRNA-containing cells in all cortical layers of both AD (shown here) and non-AD specimens (not shown). The molecular layer (layer 1), which has few neurons, was the most strongly labeled in the majority of AD cases. Other AD and some non-AD cases had a more uniform CIqB mRNA distribution among the six cortical layers. In most AD cases, neuron-rich cortical layers II-VI did not differ much in ClqB mRNA hybridization signal. A few cases, however, appeared

C O M P L E M E N T MRNA IN ALZHEIMER'S DISEASE

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FIG. 1. RNA gel blot analysis of ClqB and C4 mRNAs in the human frontal cortex. Northern blots of the human frontal cortex poly(A) + RNA hybridized to [32p]ClqB or -C4 antisense cRNA probes. Each lane contained 0.5 ug A + RNA; the blot was exposed to Kodak XAR film for 16 h. The single species detected in each case represents the same-sized RNA found in peripheral C-producing tissues. to have fewer C I qB mRNA-containing cells in deeper layers V and VI (e.g., Fig. 2B). White matter in general contained few ClqB mRNA-expressing cells in AD or non-AD cases (not shown). At higher magnification, Figs. 2C-2E show the abundance of LN3-immunopositive microglia and CIqB mRNAcontaining cells in cortical layer 1 of an AD specimen, suggesting that L N 3 + microglia are the predominant ClqB expressing cell in the outermost cortical layer (layer 1) in this AD case. Note the absence of meninges, which rules out an adventitious contribution of blood-borne, C l q-producing macrophages or monocytes to the ClqB m R N A labeling pattern seen here. In general, there were no gross anatomic (qualitative) differences in C lqB expression in the frontal cortex between AD and non-AD cases. Both AD and non-AD cortical specimens show only quantitative differences in cellular CIqB mRNA prevalence (cellular grain density) (Fig. 3) and the number of ClqB mRNA-positive cells (not shown; Lampert-Etchells et al., in preparation). However, typical AD cases have much stronger HLA-DR expression throughout the neocortex and more HLA-DR-immunopositive cells in layer 1. Thus, ClqB m R N A is probably expressed in most microglia, including HLA-DR-immunopositive, activated microglia that predominate in the AD brain. In addition, throughout neocortical layers II-VI ClqB m R N A was found in numerous cells stained by cresyl violet or neutral red that are not stained by the LN3 antibody (not shown). These are unlikely to be astrocytes because we have been unable to colocalize ClqB m R N A with GFAP-immunopositive astrocytes in the AD cortex (not shown) or detect CIqB m R N A in GFAP-positive astrocyte cell culture RNA (see below). Thus, these C 1qB mRNA-positive cells that do not colocalize with LN3 may be neurons or possibly LN3 immu-

643 nonegative microglia. In support of neuronal localization of C l qB mRNA, the anatomic distribution of C l qB m R N A in the human (AD or non-AD) hippocampus was isomorphous with the pyramidal and dentate granule neuron layers (Fig. 2F). Moreover, C 1qB hybridization was also seen outside these well-defined neuronal layers of hippocampus, suggesting expression of CIqB in nonneuronal cell types, such as the microglia seen in frontal cortex. To further investigate the cell types containing CIqB mRNA, we examined primary brain cell cultures. RNA from embryonic rat brain (E 16) cultures highly enriched in neurons, and lacking microglia due to serum-free culture, gave strong signals for ClqB m R N A by northern blots (not shown). Consistent with the absence of in vivo astrocytic labeling for C 1qB mRNA, northern blots (not shown) gave negative CIqB hybridization signals with total RNA from several independent cultures of mature (GFAP positive) astrocytes. These blots also contained whole-brain poly(A) + RNA and cultured neuron RNA that each showed positive C IqB signals. C4 m R N A was localized to neurons in AD hippocampal pyramidal and dentate granule layers; the C4 ISH signal colocalized with cells that were also immunoreactive for neuronspecific enolase (Fig. 4A). The distribution of C4-containing cells corresponded to neuronal layers in the hippocampus and adjacent temporal cortex (Figs. 4B-3E). However, note the labeling of smaller cells in layer 1 of the adjacent cortex that is devoid of neurons (Fig. 4E). There may be subtle anatomic differences in ClqB and C4 m R N A expression between the human frontal and temporal cortex. While CIqB and C4 antisense probes both strongly label cortical layer I in frontal cortex, only scattered layer I cells in temporal cortex adjacent to the hippocampus are labeled by these C probes (compare Figs. 2B and 4D and 4E). Examination of additional temporal cortex specimens is needed to confirm this possibility. AD and Lesioned Rat Brain

Pathologic correlates of C m R N A prevalence with affected regions of AD brain were examined by ISH for ClqB and C4 mRNAs in the frontal cortex. By comparison with agematched non-AD cases, AD cortex showed a twofold increase of cellular grain density for ClqB mRNA (Fig. 3). The antisense cRNA signal was predominately in the cortical gray matter (see Fig. 2B). Similarly, C4 mRNA prevalence was increased threefold in the AD vs. non-AD frontal cortex (Fig. 3) and showed an anatomic distribution in the frontal cortex similar to ClqB (not shown). This study did not resolve whether CIqB and C4 mRNAs are coexpressed in the same cells or which cell types (neurons and/or microglia) provide the increased C IqB or C4 m R N A signals seen in AD specimens. Lesioned rat brains were also investigated for effects on C I qB m R N A prevalence (Fig. 5). As a model for hippocampal deafferentation seen in AD (10), we unilaterally damaged cortical afferents to the hippocampus by perforant path transection. This lesion increased ipsilateral, but not contralateral, hippocampal C lqB m R N A prevalence at 2 and 4 days postlesion, with declines to control levels by 10 days (Fig. 5A). As shown in Fig. 5B, the lesion caused a much greater CIqB m R N A prevalence increase in the ipsilateral entorhinal cortex. There was no CIqB m R N A induction in the contralateral entorhinal cortex. Different C I qB m R N A responses between the entorhinal cortex and hippocampus may reflect the fact that layer II entorhinal cortex neurons degenerate (5), while synaptic reorganization without noticable cell death occurs in the hippocampus after perforant path transection.

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A

C

F

FIG. 2.

COMPLEMENT

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FIG. 2. (Facing page) Expression of C mRNAs in microglia and neuronal lamina of the human cortex and hippocampus. (A). Colocalization of C 1qB mRNA in LN 3 + (HLA-DR + ) microglia by immunocytochemistry (ICC)/in situ hybridization (ISH). The LN3-immunostained microglia in this panel are necessarily slightly out of focus to enable visualization of silver grains that lie in the photographic emulsion coating the tissue section. Scale bar = l0 urn. (B). Low-power dark-field photomicrograph ofISH of ClqB in the human frontal cortex. Scale bar = 0.5 mm. (C). Bright-field photo (100 × field) of adjacent section to that seen in (B) after staining with prediluted LN3 antibody (ICN, Costa Mesa, CA). Scale bar = 100 zm. (D). Same microscopic field as in (C) of adjacent section after staining with neutral red and counterstaining with luxol blue. Scale bar = 100 um. (E). Same field as (C) and (D) of additional adjacent section after ISH with antisense C1qB cRNA shown as a dark-field image, where silver grain clusters glow brightly. Scale bar -- 100 um. (F). Dark-field picture ofAD hippocampal formation after ISH to ClqB antisense cRNA showing signal in neuron-rich dentate granule cell and pyramidal cell layers. Scale bar = 1.0 mm. The specificity o f C l q B iSH was established in all cases by hybridization of adjacent sections with sense strand probes, which uniformly gave negative hybridization signals over the neuron-rich pyramidal and dentate granule cell layers (not shown). Roman numerals I-VI indicate cortical layer: CA 1, CA3, cornu ammonis layers 1 and 3: DG, dentate granule cell layer; S, subiculum.

FIG. 3. Combination of C4 antisense cRNA in situ hybridization (ISH) with neuron-specific enolase (NSE) immunohistochemistry on same section of the human hippocampus. (A). High-power magnification of hilar pyramidal neurons demonstrating colocalization of C4 mRNA to NSE + neurons. Scale bar = 50 ~m. (B). Colocalization of C4 mRNA to NSE + dentate granule neuron layer of the human hippocampal formation. Scale bar = 100 um. (C). Dark-field photomicrograph of same field in (B) showing C4 mRNA ISH signal over dentate granule layer and hilar pyramidal neurons. (D). Low-power dark-field photo of C4 mRNA ISH to human hippocampal formation and adjacent cortex. Box in upper right corner defines area shown in (E). Scale bar = I mm. (E). Dark-field picture of temporal cortex adjacent to hippocampal formation showing C4 mRNA in all cortical layers. Scale bar = 500 urn. Inset (defined by boxed area) shows positive signal over smaller cells (indicated by arrowhead) in layer 1 of temporal cortex, which in general is devoid of neurons. Inset scale bar = 100 urn. See Fig. 2 legend for explanation of abbreviations. TC, temporal cortex.

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Cellular C1 qB & C4 mRNA in AD vs CTL Cortex O

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400 0n,Z

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300-

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C4 mRNA

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FIG. 4. C 1q B and C4 cellular mRNA prevalence is increased in the Alzheimer's disease (AD) frontal cortex, Cellular mRNA prevalence after in situ hybridization was determined using a computer-based grain counting system (Southern Micro Systems, Atlanta, GA) as dcscflbed (14). Grain density from approximately 150 randomly chosen ceils throughout the cortical gray matter was determined for each of seven AD and six non-AD cases for ClqB and 4 AD and three non-AD cases for C4. AD vs. non-AD significance by t-test; ClqB, p = 0.004; C4, p = 0.04.

DISCUSSION These data show by a combination o f l S H and immunohistochemistry that resident brain cells contain m R N A s for several proteins in the C system: C I qB m R N A was found in HLADR-immunopositive microglia and C4 m R N A was found in NSE-immunopositive neurons. These data do not preclude the coexpression of C l q B and/or C4 m R N A in neurons, microglia, and other cell types. Although we did not find C l q B m R N A in astrocytes, another main classical pathway component, C3 m R N A , was reported in cultured primary astrocytes (20). And, in northern blot analyses not reported here we detected m R N A s for C2, C3. C5, and C9 in human cortical poly(A) -F RNA. Further, C 1qB and C4 m R N A s are increased in the A D frontal cortex (Fig. 3). Previously, this lab showed increased CLI (clusterin; SP-40,40), m R N A in the A D hippocampus a n d entorhinal cortex (22). It is therefore plausible that the C proteins associated with A D lesions (6,241 are made by local brain cells that include microglia and neurons. C components remain to be demonstrated in astrocytes in vivo. These findings support the hypothesis that complement factors contribute to plaque genesis during A D (6). Moreover. recent studies that show ~-amyloid peptide (81-40) binds to C lq further support this idea (34). Amyloid is implicated as a potential neurotoxin (42); while controversial (40), these mechanisms o f amyloid-induced neurotoxicity could now include roles for C components and other mediators of inflammation. Rapid responses of C expression to brain lesions that mimic select aspects o f A D indicate the potential for rapid inflammatory episodes during the prolonged and slow degeneration of AD. The schedule of postlesion C m R N A changes implicates C not only in the dendritic clearance phase of the synaptic remodelling process (21,36) but also possibly in the pefforant path lesion-induced entorhinal cortex layer II neurodegeneratire cascade. In addition, we recently showed elevations of C l q B m R N A in the striatum, in OX-42-positive microglia

after frontoparietal cortical ablation, and in the hippocampus after systemic kainate administration (29). OX-42 is an antibody to rat complement receptor 3 (CR3). Both lesions also increased the m R N A and protein levels o f SGP-2 (l 8,19,31), the rat ortholog o f CLI (clusterin, SP-40,40), and a potential inhibitor of the MAC. Thus, our data suggest the involvement

FIG. 5. C lqB mRNA prevalence is increased in the hippocampus and entorhinal cortex after pcfforant pahtway transection lesion. RNA was extracted from the ipsilateral hipptx~mpus and entorhinal cortex at indicated times after pefforant path transection. Northern blots containing total RNA (5 #g/lane) were hybridized to human ClqB antisense cRNA ([riP]labeled; 2 × 109dpm//zg) and washed at stringent criteflon (16). C indicates nonlesioned control animals; numbers indicate postlesion times in days. Blots were exposed to Kodak X-AR X-ray film with an intensifying screen for 5 days at -70"C. Data presented are representative of several animals/time point. The weak band above the authentic C lqB signal represents background probe adherance to 18-s rRNA ( 1.85 kb) and shows equivalent loading of total RNA in all lanes. This 18-s band is considered spurious because it is not present when poly(A) ~- RNA is hybridized with CIqB mRNA on northern blots. In general, spurious background is removed from blots by mild RNAse digestion: a much stronger RNAse digestion is used in our in situ hybridization experiments.

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of C - m e d i a t e d processes in acute responses to lesions that include activation o f microglia and astrocytes during the removal o f debris and the onset o f synaptic remodelling (7,37). The presence of several acute-phase i n f l a m m a t o r y proteins in senile plaques, for example, a r a n t i c h y m o t r y p s i n (l), which is m a d e by astrocytes (17), interleukin 6 and a2-macroglobulin (2), and elevated serum levels of t u m o r necrosis factor (8), suggests the presence o f an ongoing inflammatory response in the A D brain. However, the contributions from acute vs. chronic i n f l a m m a t o r y m e c h a n i s m s in neurodegeneration during A D c a n n o t yet be estimated. The role of brain-derived, acute, and chronic increases of C during A D can now be considered in relation to the n u m e r o u s functions o f the C system in peripheral tissues during inflammatory processes that include e n h a n c e d phagocytosis of cellular debris, cytotoxicity, and c h e m o t a x i s o f phagocytic (9) and extracellular matrix-produc-

ing cells, such as fibroblasts (35). The presence of C c o m p o n e n t m R N A s in the n o r m a l brain suggests that the functionally c o m p l e x C proteins m a y have roles in the normal brain or during brain d e v e l o p m e n t i n d e p e n d e n t o f those functions normally associated with i n f l a m m a t o r y responses and cytolysis.

ACKNOWLEDGEMENTS We thank Nick Laping and Chris Zarow for help with perforant path lesioning and discussions, Dr. Marcia Gordon for help with immunocytochemistry, and Chris Anderson for help with graphics. They also thank Drs. Franz Hefti and Thomas McNeill for critical reading of the manuscript. This work was supported by NIA LEAD Award AG 07909 to C.E.F.; NIA AG 10673 to S.A.J.; the Turken Scholarship (Alzheimer Association) and a grant from the United Parkinson Foundation to G.M.P.

REFERENCES 1. Abraham, C. R.; Selkoe, D. J.; Potter, H. Immunocytochemical identification of the serine protease inhibitor, alphal-antichymotrypsin, in the brain amyloid deposits of Alzheimer's disease. Cell 52:487-501; 1988. 2. Bauer, J.; Strauss, S.: Schreiter-Gasser, U.: Ganter, U.: Schlegel, P.; Witt, l.: Yolk, B.; Berger, M. Interleukin-6 and alpha-2-macroglobulin indicate an acute phase state in Alzheimer's disease cortices. FEBS Lett. 285:111-114; 1991. 3. Belt, K. T.; Carroll, M. C.; Porter, R. R. The structural basis of the multiple forms of human complement C4. Cell 36:907-914: 1984. 4. Chirgwin, J. M.; Przybyla, A. E.: MacDonald, R. J.: Rutter, W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299; 1979. 5. Cummings, B. J.; Cotman, C. W. Basic fibroblast growth factor (bFGF) promotes entorhinal layer !1 cell survival after perforant path axotomy. Society for Neuroscience Annual Meeting, St Louis, MO: 1990. 6. Eikelenboom, P.; Hack, C. E.: Rozemuller, J. M.: Stam, F. C. Complement activation in amyloid plaques in Alzheimer's dementia. Virchows Arch. B Cell Pathol. 56:259-262; 1989. 7. Fagan, A. M.: Gage, F. H. Cholinergic sprouting in the hippocampus: A proposed role for IL-I. Exp. Neurol. 110:105-120; 1990. 8. Fillit, H.: Ding, W.: Buee, L.: Kalman, J.: Alystiel, L.: Lawlor, B.: Wolf-Klein, G. Elevated circulating tumor necrosis factor levels in Alzheimer's disease. Neurosci. Lett. 129:318-320:1991. 9. Frank, M. M.: Fries, L. F. The role of complement in inflammation and phagocytosis. Immunol. Today 12:322-326:1991. 10. Geddes, J. W.; Monaghan, D. T.; Cotman, C. W.; Lott, i. T.: Kim, R. C.: Chui, H. C. Plasticity of hippocampal circuitry in Alzheimer's disease. Science 230:1179-1181 ; 1985. 11. Gibbs, R. B.: Harris, E. W.: Cotman, C. W. Replacement of damaged conical projections by homotypic transplants of entorhinal cortex. J. Cornp. Neurol. 237:47-64: 1985. 12. Jenne, D. E.; Tschopp, J. Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: Identity to sulfated blycoprotein 2, a constituent of rat testis fluid. Proc. Natl. Acad. Sci. USA 86:7123-7127; 1989. 13. Jenne, D. E.: Tschopp, J. Clusterin: The intriguing guises of a widely expressed glycoprotein. Trends in biological sciences. 17:154-159: 1992. 14. Johnson, S. A.: McNeill, T.; Cordell, B.; Finch, C. E. Relation of neuronal APP-751/APP-695 mRNA ratio and neuritic plaque density in Alzheimer's disease. Science 248:854-857: 1990. 15. Johnson, S. A.; Morgan, D. G.; Finch, C, E. Extensive postmortem stability of RNA from rat and human brain. J. Neurosci. Res. 16:267-280: 1986.

16. Johnson, S. A.; Rogers, J.; Finch, C. E. APP-695 transcript prevalence is selectively reduced during Alzheimer's disease in cortex and hippocampus but not in cerebellum. Neurobiol. Aging 10:267-272; 1989. 17. Koo, E. H.; Abraham, C. R.; Potter, H.; Cork, L. C.; Price, D. L. Developmental expression of alpha I-antichymotrypsin in brain may be related to astrogliosis. Neurobiol. Aging 12:495-501: 1991. 18. Lampert-Etchells, M.; McNeill, T. H.; Laping, N. J.; Zarow, C.; Finch, C. E.; May, P. C. Sulfated glycoprotein-2 is increased in rat hippocampus following entorhinal cortex lesioning. Brain Res. 563:101-106; 1991. 19. Laping, N. J.: Nichols, N. R.; Day, J. R.; Finch, C. E. 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: 1991. 20. Levi-Strauss, M.; Mallet, M. Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation. J. Immunol. 139:23612366; 1987. 21. Lynch, G. S.; Gall, C.; Cotman, C. W. Temporal parameters of axon "sprouting" in the brain of the adult rat. Exp. Neurol. 54:179-183; 1977. 22. May, P. C.: Lampert, E. M.; Johnson, S. A.; Poirier, J.; Masters, J. N.; Finch, C. E. Dynamics ofgene expression for a hippocampal glycoprotein elevated in Alzheimer's disease and in response to experimental lesions in rat. Neuron 5:831-839; 1990. 22a. May, P. C.; Finch, C. E. Sulfated glycoprotein 2: New relationships of this multifunctional protein to neurodegeneration. Trends in neuroscience 15:391-396; 1992. 23. McCarthy, K. D.; deVellis, J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85:890-902: 1980. 24. McGeer, P. L.; Akiyama, H.; ltagaki, S.; McGeer, E. G. Activation of the classical complement pathway in brain tissue of Alzheimer patients. Neurosci. Lett. 107:341-346; 1989. i5. McGeer, P. L.; Walker, D. G.; Akiyama, H.; Kawamata, T.; Guan, A. L.: Parker, C. J.; Okada, N.; McGeer, E. G. Detection of the membrane inhibitor of reactive lysis (CD59) in diseased neurons of Alzheimer brain. Brain Res. 544:315-319; 1991. 26. McNeill, T. H.; Masters, J. N.; Finch, C. E. Effects of chronic adrenylectomy on neuron loss and distribution of sulfated glycoprotein-2 in the dentate gyrus of prepubertal rats. Exp. Neurol. 111:140-144: 1991. 27. Morgan, B. P. Complement. Clinical aspects and relevance to disease. San Diego, CA: Academic Press: 215; 1990. 28. Moil, S.; Sternberger, N. H.; Herman, M. M.: Sternberger, L. A. Leakage and neuronal uptake of serum protein in aged and Alzheimer brains. Lab. Invest. 64:345-351:199 I. 29. Pasinetti, G.: Johnson, S.: Rozovsky, I.; Lampert-Etchells, M.:

648

30.

31. 32.

33.

34.

35.

.1OHNSON ET AI.. Morgan, D.; Gordon, M.; Morgan, T.; Willoughby, D.; Finch, C. Complement ClqB and C4 mRNA responses to lesioning in rat brain. Exp. Neurol. (in press). Pasinetti, G. M.; Morgan, D. G.; Johnson, S. A.: Millar, S. k.: Finch, C. E. Tyrosine hydroxylase mRNA concentration in midbrain dopaminergic neurons is differentially regulated by reserpine. J. Neurochem. 55:1793- t 799; 1990. Pasinetti, G. P.; Finch, C. E. Sulfated glycoprotein-2 (SGP-2) mRNA is expressed in rat striatal astrocytes following ibotenic acid lesions. Neurosci. Lett, 130:1-4; 1991. Pouplard-Barthelaix, A. Immunological markers and neuropathological lesions in Alzheimer's disease. In: Pouplard-Barthelaix, A.; Emile, J.; E. J.; Christen, Y., eds. Immunology and Alzheimer's disease. Berlin: Springer: 7-16; 1988. Reid, K. B. M. Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C lq of the human complement system. Biochem. J. 231:729-735: 1985. Rogers, J.; Schultz, J.; Webster, S.; Brachova, L.; Ward, P.; Lieberberg, I. C Iq binding to b-amyloid and amyloid precursor protein (APP). Society for Neuroscience, New Orleans, LA, p. 912: 1991. Senior, R. M.: Gritfin, G. L.: Perez, H. D.; Webster, R. O. Human C5a and C5a Des Arg exhibit chemotactic activity for fibroblasts. J. Immunol. 141:3570-3574: 1988.

3~. Steward, O.; Vinsant, S. L. The process of reinnervation in the dentata gyrus of the adult rat: A quantitative electron microscopic analysis of terminal proliferation and reactive synaptogenesis. J. Comp. Neurol. 214:370-386; 1983. 37. Streit, W. J.; Graeber, M. B.: Kreutzberg, G. W. Functional plasticity of microglia: A review. Glia 1:301-307; 1988. 38. Vanguri, P.; Koski, C. L.; Silverman, B.; Shin, M. L. Complement activation by isolated myelin: Activation of the classical pathway in the absence of myelin-specificantibodies. Proc. Natl. Acad. Sci. USA 79:3290-3294; 1982. 39. Whitehead, A. S.; Goldberger, G.; Woods, D. E.; Markham, A. F.; Colten, H. R. Use of a clone for the fourth component of human complement (C4) for analysis of a genetic deficiency of C4 in guinea pig. Proc. Natl. Acad. Sci. USA 80:5387-5391; 1983.

40. Whitson, J. S.; Selkoe, D. J.; Cotman, C. W. Amyloid beta pro, tein enhances the survival of hippocampal neurons in vitro. Science 243:1488-1490; 1989. 41. Wood, L.; Pulaski, S.; Vogeli, G. DNA clones coding for the complete murine B chain of complement Clq: Nucleotide and derived amino acid sequences. Immunol. Lett. 17:115-120; 1988. 42. Yankner, B. A.; Duffy, L. K.; Kirschner, D. A. Neurotrophic and neurotoxic effects of amyloid beta protein: Reversal by tachykinin neuropeptides. Science 250:279-282:1990.