From the Laboratoire d'Oncologie Virale CNRS UPR
9045, IFC1, 94801 Villejuif cedex France and the
Laboratoire de
Neurovirologie CEA, 92265 Fontenay aux Roses cedex, France
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ABSTRACT |
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To define genes associated with or responsible
for the neurodegenerative changes observed in transmissible spongiform
encephalopathies, we analyzed gene expression in scrapie-infected mouse
brain using "mRNA differential display." The RNA transcripts of
eight genes were increased 3-8-fold in the brains of scrapie-infected
animals. Five of these genes have not previously been reported to
exhibit increased expression in this disease: cathepsin S, the
C1q B-chain of complement, apolipoprotein D, and two
previously unidentified genes denominated scrapie-responsive gene
(ScRG)-1 and ScRG-2, which are
preferentially expressed in brain tissue. Increased expression of the
three remaining genes, 2 microglobulin, F4/80, and
metallothionein II, has previously been reported to occur in
experimental scrapie. Kinetic analysis revealed a concomitant increase
in the levels of ScRG-1, cathepsin S, the C1q
B-chain of complement, and
2 microglobulin mRNA as well as
glial fibrillary acidic protein and F4/80 transcripts,
markers of astrocytosis and microglial activation, respectively. In
contrast, the level of ScRG-2, apolipoprotein D, and
metallothionein II mRNA was only increased at the terminal stage of
the disease. ScRG-1 mRNA was found to be preferentially
expressed in glial cells and to code for a short protein of 47 amino
acids with a strong hydrophobic N-terminal region.
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INTRODUCTION |
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Scrapie is a transmissible progressive neurodegenerative disease occurring naturally in sheep and goats. The disease has been adapted to the laboratory mouse to constitute one of the most widely studied models of transmissible spongiform encephalopathies (TSE)1, which include other animal diseases such as bovine spongiform encephalopathy and human pathologies such as Creutzfeldt-Jakob disease, German-Sträussler-Scheinker syndrome, and Kuru (1-5).
The neuropathology of TSE is characterized by the appearance in the brain of an abnormal insoluble and protease-resistant form of a host-encoded protein, the prion protein (PrP) (6). A glial reaction involving both astrocytes and microglia follows the appearance of the modified form of PrP, PrPSc, which is specifically associated with TSE (1, 7-10). The glial reaction precedes the vacuolization of neurons and neuropil, the deposition of amyloid, and the neuronal loss, which are characteristic of TSE diseases.
The histopathological modifications observed in the brain of scrapie-infected animals are associated with changes in the production of certain cytokines and increased levels of a number of enzymes and transport proteins (11-22). The systematic study of the molecular changes that occur in the brain of scrapie-infected animals could facilitate an understanding of the pathogenesis of TSE and in particular the interrelations between the different types of cells implicated in the disease process.
The work presented herein together with certain previous reports (18, 20-23) raises the question of whether the continuous and widespread activation of glial cells, a host response most probably designed for more local, limited, or transitory injuries of the brain, may be more detrimental than beneficial to neuron survival in TSE. Such a hypothesis is supported by in vitro studies of the neurotoxicity of PrP-related peptides, the results of which show that neuronal death is mediated by activated microglial cells (23). Moreover, reactive astrocytosis has been reported to accompany neuronal degeneration in brains of mice with cerebral overexpression of the interleukin-6 gene (24).
To identify those genes the altered expression of which is associated with or may even be responsible for the neurodegenerative changes observed in TSE, we have systematically analyzed modifications of gene expression in scrapie-infected mouse brain using the mRNA differential display screen described by Liang and Pardee (25) and Liang et al. (26). This approach has led to the detection of an increased level of expression of eight cellular genes and the slight decreased expression of one other gene. Five of these genes have not previously been reported to be enhanced in scrapie. Indeed, two are previously unrecognized genes that are specifically expressed in brain tissue. The three others encode cathepsin S, the C1q B-chain of complement, and apolipoprotein D. The increased expression of cathepsin S, a cysteine-protease produced by cells of monocytic/macrophage lineage that is secreted and which retains activity at neutral pH (27), together with the possible activation of a component of the cytotoxic complement pathway suggest that some pathological lesions observed in scrapie may result directly from microglial products.
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EXPERIMENTAL PROCEDURES |
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Inoculation of Animals--
Fifty-three C57Bl/6 mice (Charles
River, France) were inoculated intracerebrally with 20 µl of a 1%
brain homogenate of C506M3 seventh passage scrapie agent (a gift from
Dr. D. C. Gajdusek, NIH, Bethesda, MD) (19). Eighteen control mice
were inoculated with the same volume of 1% normal brain homogenate.
Scrapie-inoculated and control animals were killed by cervical column
disruption on days 2, 45, 88, 120, 150 and in the late clinical stages
of the disease at days 165, 171, 175, or 184 post-infection. The brains
were split, and each hemisphere was frozen directly in liquid nitrogen
and stored at 80 °C until use. Only the hemispheres opposite the
site of inoculation were further used for RNA extraction.
Cell Culture-- The C6 rat glioma cell line was a gift from Dr. J. J. Hauw (Pitié-Salpêtrière Hospital, Paris, France). The murine neuroblastoma cells N2A and NIE-115 were a gift from Dr. G. Barbin (Pitié-Salpêtrière Hospital, Paris, France). All the cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum, except for the PC12 cell line derived from a rat pheochromocytoma, which was supplemented with 5% horse serum and 10% fetal calf serum.
RNA Extraction, Northern Blot, and Hybridization-- Total cellular RNA was isolated from (a) control and scrapie-infected mouse brains, (b) various normal mouse organs, and (c) the C6, N2A, NlE-115, and PC12 cell lines using the method described by Chomczynski and Sacchi (28). Poly(A)+ RNA from animals in late clinical stages of disease was obtained after one cycle of affinity chromatography of total brain RNA on oligo(dT) cellulose columns. Northern blots were performed using glyoxal denaturation according to standard protocols, and the blots were hybridized as described by Church and Gilbert (29) using probes radiolabeled to a specific activity of at least 1 × 109 cpm/µg using the Megaprime DNA labeling systems kit from Amersham Pharmacia Biotech. The blots were first exposed to autoradiography and then submitted to quantification using PhosphorImager.
mRNA Differential Display Analysis--
The procedure
employed was based on the use of the "MessageClean" and the
"RNAimage" kits of GenHunter Corp. and was essentially the same
as that described by the manufacturer, with minor modifications. Briefly, RNA was treated with a RNase-free DNase, and 1 µg was reverse-transcribed in 100 µl of reaction buffer using either one or
the other of the three one-base-anchored oligo(dT) primers, (HT11) A,
C, or G. All the samples to be compared were reverse-transcribed in the
same experiment, separated into aliquots, and frozen. The amplification
was performed with only 1 µl of the reverse transcription sample/reaction in 10 µl of amplification mixture containing the Taq DNA polymerase and [-33P]dATP (3000 Ci/mmole). Eighty 5' end (HAP) primers were used in combination with
each of the three (HT11) A, C, or G primers. Samples were then run on
7% denaturing polyacrylamide gels and exposed to autoradiography.
Bands of interest were cut out, reamplified according to the
instructions of the supplier, and further used as probes to hybridize
Northern blots.
Cloning and Sequencing-- Reamplified bands from the differential display screen were cloned in the Sfr1 site of the pCR-Script SK(+) plasmid (Stratagene), and cDNA amplified from the rapid amplification of cDNA ends were isolated by TA cloning in the pCR3 plasmid (Invitrogen). DNA was sequenced using the Thermo Sequenase cycle sequencing kit (Amersham).
5'-Rapid Amplification of cDNA Ends-- One µg of poly(A)+ RNA from scrapie-infected mice (171 days) was reverse-transcribed, processed according to the instruction of the marathon amplification procedure (CLONTECH), and amplified with a reverse primer specific for extension of the differential display cDNA fragment numbered 7322 (5'-GTGAAGGCCTTCAGGACCATGTTCTCC-3'). The amplified cDNA was run on a 1% agarose gel; longer molecules were isolated by electroelution and cloned into plasmid pCR3 (Invitrogen). Relevant cDNA molecules were then targeted by colony filter hybridization with a radiolabeled primer (5'-AGTGCAAGGCAGATCCTCAG-3') designed from the sequence of the 7322 band upstream of the primer used for the amplification (see Fig. 4).
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RESULTS |
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mRNA Differential Display Screen-- To identify mRNA species, the expression of which was increased in scrapie-infected mouse brain, we used the mRNA differential display (DD) strategy (25, 26). C57Bl/6 mice were inoculated intracerebrally with scrapie strain C506M3, and brains were harvested at different times post-inoculation corresponding to different stages of the disease: 45 days, PrPSc is barely detectable; 88 days, PrPSc is clearly detectable; 120 days, gliosis had occurred, and increased GFAP mRNA was observed in astrocytes; 150 days, the first clinical signs of the disease appeared; and between 165-184 days, the terminal stage of the disease, with neurodegeneration and spongiosis, occurred (19, 30). Brain RNA from scrapie-infected and control mice were submitted to reverse transcription with either one or the other of the three (HT11) A-, C-, or G-anchored primers. The cDNA obtained was amplified by PCR with the oligo(dT) used previously and a second primer arbitrary in sequence (HAP), and size-fractionated on a denaturing polyacrylamide gel (Fig. 1A). To minimize false positives, one sample of control brain RNA (lane C) and four separate samples of scrapie-infected brain RNA from mice sacrificed on days 2, 120, 150, and 171 (lanes 2, 120, 150, and 171) were simultaneously compared in the DD screen. For each primer combination used, about 100 cDNA bands can be visualized per RNA sample. We performed 240 PCR amplifications using different primer combinations, and we examined the intensity of some 24,000 bands obtained for each sample. As expected, the majority of the cDNA bands from scrapie-infected samples were similar in intensity to those of the control, whereas some bands exhibited an increased abundance either at the late clinical stage of the disease (Fig. 1A, star) or simultaneously at days 120, 150, and 171 post-inoculation (Fig. 1A, arrow). After confirmation of the putative difference by displaying the samples for a second time, candidate cDNA was recovered from the polyacrylamide gel, PCR-amplified, and used to probe a Northern blot containing total control and scrapie-infected brain RNA. For example, the two cDNA indicated with a star in Fig. 1A were found to represent the same mRNA species, the abundance of which is markedly increased in the brain of mice with clinical signs of scrapie (Fig. 1B, lane 171). This RNA was shown to code for metallothionein II, a gene previously identified as overexpressed in scrapie (11). In contrast, the other cDNA selected (indicated by the arrow in Fig. 1A) revealed a mRNA that was present in greater amounts from 120 to 171 days post-inoculation (Fig. 1B, lanes 120, 150, and 171). Seventy-two differently expressed candidate bands were isolated using the screen and further studied using Northern blot analysis. Twelve of the bands hybridized more strongly with RNA from scrapie-infected mouse brain and correspond to eight distinct genes whose transcripts are significantly increased during scrapie (one of these genes was represented three times in the screen, and two other genes were represented twice).
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Expression of ScRG-1, ScRG-2, and ScRG-3 Genes in Various Mouse Tissues-- We characterized the pattern of tissue expression of the three previously unidentified genes, ScRG-1, ScRG-2, and ScRG-3, by examining the relative levels of the different mRNAs present in different adult mouse tissues (Fig. 3A).
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Characterization of ScRG-1-- We have identified two unknown genes, ScRG-1 and ScRG-2, the mRNA of which are increased in scrapie-infected mouse brain and that are preferentially expressed in brain tissue (Fig. 2 and Fig. 3A). We decided to further investigate ScRG-1 because the change in the mRNA content of this gene occurred the earliest throughout the course of scrapie infection (Fig. 2).
The ScRG-1 cDNA fragment isolated with the DD screen was only 197 bp in length and represented the 3' end of the mRNA. To generate cDNA molecules extending to the 5' end of the message, we performed a 5'-rapid amplification of cDNA ends-PCR with a specific reverse primer derived from within the 5' half of the sequence of the 7322 DD band. The amplified cDNA was cloned and screened by colony hybridization using a 20-mer probe corresponding to the 5' end of the 7322 band (Fig. 4A). We sequenced the ScRG-1 cDNA present in three positive clones numbered 15, 16, and 24. The inserts of clones 16 and 24 were identical with one exception, a nucleotide that extends the insert of clone 24 at the 5' terminus. The sequence of clone 15 cDNA was shorter by 145 nucleotides at its 5' end than that of the two other cDNA and was found to be identical to them for the remaining region with one exception, a T (clones 16 and 24) change to a C (clone 15) substitution at position 481 of clone 24 cDNA. The full sequence of ScRG-1 cDNA given in Fig. 4B is the sequence of the insert of clone 24 extended in 3' by the additional sequence determined from the DD band. The size of the the full-length cDNA is in perfect agreement with our previous determination of the ScRG-1 mRNA size on Northern blot (700 bp), and two polyadenylation signals are present at its 3' end, one at position 639 and the other at position 681.
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DISCUSSION |
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To investigate the molecular modifications that occur in brain
after scrapie infection, we have screened up to 24,000 bands of
randomly amplified 3' end cDNA synthesized from control and scrapie-infected mouse brain using the method of Liang and Pardee (25)
and Liang et al. (26). We have detected nine mRNA that are differentially expressed in scrapie-infected brain. Three of them,
the mRNA encoding the 2 microglobulin, the macrophage-restricted cell surface glycoprotein F4/80, and the metallothionein II, have previously been reported to be increased by scrapie infection (11, 13,
35). We have described six additional mRNA transcripts, the
expression of which is modulated in the brains of scrapie-infected mice. Three of them were identified by their nucleotide sequence to
encode cathepsin S, complement C1q B-chain, and apolipoprotein D. The
three other mRNAs previously unknown in the nucleic acid sequence
data bases were denominated ScRG-1 to -3 (Table
I, Figs. 1B and 2). We also determined the 0.7 bp sequence
of ScRG-1 mRNA (Fig. 4).
The gliosis or glial activation that follows the appearance in brain of
PrPSc, the disease-specific and proteinase K-resistant form
of the prion protein, consists of both an astrocytosis and a microglial activation. In the C506M3 murine scrapie model, the gliosis starts between 90 and 120 days post-inoculation. Indeed, at 120 days, this
activation can be detected by increased levels of both GFAP and F4/80 transcripts, which are expressed by astrocytes and
macrophages/microglia, respectively (Fig. 2A, Table I, and
Refs. 14 and 19). The results of the kinetic data presented in this
study show an increased expression of ScRG-1, cathepsin S,
complement C1q-B chain, and 2 microglobulin mRNAs at the same
time as gliosis occurs (Fig. 2).
Marked differences in the tissue distribution of ScRG-1 and cathepsin S transcripts were observed for the two genes (Fig. 3A). Cathepsin S is expressed in high levels in lung, thymus, and spleen, similar to that in brain. In contrast, the expression of ScRG-1 is confined to brain (Fig. 3A). Moreover, cathepsin S mRNA is not detected in various cell lines of glial (C6) or neuronal (PC12, N2A, NIE-115) origin (Ref. 27 and data not shown) when ScRG-1 transcripts are expressed in C6 cells (Fig. 3B). The expression of GFAP that in brain is restricted to astrocytes is also detected in the rat glioma C6 cell line (Fig. 3B).
Previous reports have shown that in rat brain and in peripheral
tissues, cathepsin S is preferentially expressed in cells of the
mononuclear phagocytic lineage; cathepsin S mRNA was found in
different populations of brain macrophages, microglia in the parenchyma, and perivascular and leptomeningeal macrophages (27, 36).
Our results are generally in agreement with the tissue-specific expression of cathepsin S observed in the rat as well as in humans (37). Furthermore, our results and the results reported for cathepsin S
expression (27, 32, 33) indicate that although cathepsin S and
ScRG-1 mRNA changes in mouse brain appear at the same
period during the scrapie course (Fig. 2A), the messages of
the two genes are expressed and probably up-regulated in different populations of cells, macrophages for cathepsin S and astrocytes for
ScRG-1. Similarly, the increased level of complement C1q
B-chain mRNA observed in scrapie-infected mouse brain (Fig.
2B) probably correlates with the microglial reaction, as C1q
complement genes are essentially expressed in cells of the
monocytic/macrophage lineage (38, 39). The overexpression of cathepsin
S and C1q complement is also reported as a characteristic feature of
the preinflammatory response of monocytic cells (27, 40). In contrast, reactive astrocytes may account for the increased levels of 2 microglobulin transcripts (Fig. 2B), as indicated by
immunocytochemistry in mice infected with scrapie strain 22L (16).
Certain factors up-regulated in activated glia during scrapie infection
are, however, simultaneously produced by astrocytes and microglia; for
example, both cells secrete interleukin-1 and tumor necrosis factor
, and increased expression of these cytokines is observed in
scrapie-infected mouse brain (18, 20-22). Moreover, an increase of
cathepsin S and complement C1q B-chain immunoreactivity in neurons of
Alzheimer's disease patients was recently reported (40, 41). Whether
such an increase in neurons may occur during scrapie remains to be
determined, but the 3-8-fold increase of cathepsin S and complement
C1q B-chain mRNA that we detected in brain by Northern
blot analysis (Table I) could be more readily explained by microglial
activation. Thus, to explore the different hypotheses concerning the
expression of these newly identified genes, we are currently
determining the cellular localization of increased expression of these
genes by in situ hybridization and/or
immunocytochemistry.
Glial activation results in the increased expression of a panel of genes as well as an increase in the number and size of activated cells (14). The key question is to determine whether changes in the different mRNA identified are due to increased expression within individual cells or to an increase in the number of cells expressing a particular transcript. Furthermore, within a population of cells expressing a mRNA modulated by scrapie infection, only a subset of cells is really activated. For example, the major increase in mRNA abundance of apolipoprotein E occurs in the gray matter of scrapie-infected mouse brain (3-6-fold increase of apolipoprotein E mRNA in individual astrocytes and 2-fold increase in the number of astrocytes concerned) (14). However, when the mRNA change is determined from Northern blot analysis, only a 2-3-fold increase is observed because both white and gray matter contributed mRNA.
Disease-specific prion protein (PrPSc) accumulates before
the development of gliosis, and glial activation with altered gene expression precedes the spongiosis and the neuronal death in scrapie. It is not presently clear whether the PrPSc protein is
neurotoxic by itself in vivo or, rather, induces a neurodegenerative pathway (23, 42, 43). Recent evidence supports the
possibility that glia and factors produced by activated glia may play a
role in neuronal loss and other pathological lesions observed in
scrapie. For example, mice with cerebral overexpression of
interleukin-6 were shown to exhibit neuronal degeneration and spongiosis (24). Intraocular inoculation of tumor necrosis factor produces Creutzfeldt-Jakob disease-like lesions in mouse optic nerves
(44). Furthermore, the toxic effect of the human prion protein peptide,
PrP106-126, on cultured neurons requires the presence of
microglia (23). In this context it is of interest to consider that the
increased levels of cathepsin S, ScRG-1, and complement
C1q B-chain mRNA observed in scrapie-infected mouse
brain probably result from glial activation.
Cathepsin S, which is distinct from cathepsin D, aspartyl protease, previously identified as enhanced in scrapie (14), is a member of the family of cysteine-lysosomal proteases. These enzymes are essential for the turnover of intracellular proteins and are implicated in pathological processes involving tissue destruction. Based on the findings that cathepsin S can retain activity at neutral pH, can be actively secreted, and can degrade various extracellular matrix in vitro, it has recently been argued that this protease could play an important role in the clearance of debris and in the extracellular matrix remodeling in degenerative disorders (27). Our results, indicating an increased expression of cathepsin S in scrapie, confirm an involvement of this enzyme in neurodegenerative conditions and suggest that some lesions observed in the scrapie model we studied might be due to the action of cathepsin S.
C1q complement which consists of 18 protein chains, six A, six B, and
six C chains, is a part of C1, the first element of the complement
cascade. The genes encoding each C1q chain are closely clustered, and
their expression appears to be coordinately regulated (45). For this
reason, an increase in the level of the B-chain mRNA suggests an
increased expression for the other C1q chains and perhaps also for the
other components of the complement cascade in the brains of mice
infected with scrapie. This question is currently under investigation.
Interestingly, most of the senile plaques in Alzheimer's disease are
associated with different elements of the complement, including C1q
(38, 40). C1q complement has also been shown to bind fibrillar
-amyloid (38, 40). It is noteworthy that a number of genes whose RNA
is increased in brain during scrapie, such as apolipoprotein E,
cathepsin D, GFAP, sulfated glycoprotein 2, and transferrin
(12, 14), also exhibit increased cerebral expression in Alzheimer's
disease.
ScRG-1, which is largely expressed in cells of glial origin, encodes a small 47-amino acid sequence of unknown function, with no clear and significant homology with any other known protein even if the C-terminal region of this protein exhibited weak homology with some neurotoxins. The N-terminal region is strongly hydrophobic and might be a signal peptide, suggesting that this molecule is either partly included in the membrane or secreted by the cells, this last hypothesis fitting well with a possible role for ScRG-1 in the pathogenesis of scrapie.
We have also identified three additional genes, the transcripts of which are increased in the late clinical stage of the disease, when neuronal degeneration occurs. They are apolipoprotein D, ScRG-2, and metallothionein II (Table I). Apolipoprotein D, which is distinct from apolipoproteins E and J previously implicated in scrapie (14, 46, 47), is a member of the superfamily of carrier proteins called lipocalins (48). The increased expression of this protein might be correlated with the enhanced mRNA synthesis of some other transport molecules such as sulfated glycoprotein 2 (also called clusterin or apolipoprotein J) and transferrin, previously reported in late clinical scrapie (12) and probably due to the stress response observed at this stage of the disease associated with an increase of expression of the heat shock protein HSP 70 and of the metallothionein II (11, 17).
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FOOTNOTES |
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* This work was supported by grants from CNRS, INSERM, and the Association Nouvelles Recherches Biomédicales (ANRB).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ223206 (for ScRG-1), AJ223207 (for ScRG-3), and AJ223208 (for mouse cathepsin S).
§ A student of l'Ecole Supérieure des Techniques de Biologie Appliquée (ESTBA), Paris, France.
¶ A recipient of a fellowship from the Association Recherche et Partage, Paris, France.
** To whom correspondence should be addressed: Laboratoire d'Oncologie virale, 7 rue guy Moquet, BP8, 94801 Villejuif, France. Tel.: 1-49 58 34 22; Fax: 1-49 58 34 44; E-mail: mdron{at}infobiogen.fr.
1 The abbreviations used are: TSE, transmissible spongiform encephalopathies; PrP, prion protein; DD, differential display; GFAP, glial fibrillary acidic protein; ScRG, scrapie-responsive gene; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s).
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REFERENCES |
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