SYNAPSE 11~105-111(1992)
Possible Functions of a New Genetic Marker in Central Nervous System: The Sulfated Glycoprotein-2 (SGP-2) DENIS MICHEL, JEAN-GUY CHABOT, EMMANUEL MOYSE, MARC DANIK, AND &MI QUIRION Ecole Normale Superieure de Lyon, Laboratoire de Biologie Moleculaire et Cellulaire, Lyon, Cedex, France (D.M., E.M.); Douglas Hospital Research Centre and Department of Psychiatry, Faculty of Medicine, McGill University, Verdun, Quebec, Canada H4H 1R3 (J.-G.C.,M.D., R.Q.); Centre de Recherche de l’H6pital Maisonneuve-Rosemont, University of Montreal, Montreal, Quebec, Canada H1 T 2M4 (M.D.)
KEY WORDS
Clusterin, SP40,40, TRPM-2, Gene expression, Apoptosis, Complement, In situ hybridization
This brief review discusses the recent characterization in the brain of a ABSTRACT gene coding for a protein that may be involved in programmed cell death and/or brain plasticity. We will term it sulfated glycoprotein-2 (SGP-2),the name corresponding to the first cDNA characterized. Recent studies have demonstrated the overexpression of this sulfated glycoprotein in various CNS disorders, such as certain gliomas, Alzheimer’s disease and epilepsy, as well as after experimental brain injury in animals where different cell types were undergoing tissue remodelling or cell death. In peripheral tissues, SGP-2 gene expression has been found to be strikingly increased following experimental manipulations in which cells of injured tissues were undergoing programmed cell death or apoptosis. The results reported thus far are intriguing and suggest the possible involvement of SGP-2 in apoptotic mechanisms as well as its interaction with components of the immune system possibly associated with cell death in neurodegenerative disorders. 8 1992 Wiley-Liss, Inc.
INTRODUCTION A novel gene has recently burst into the field of neurosciences, and more precisely of neuropathology. High levels of mRNA transcribed from this gene are correlated with the occurrence of central nervous system (CNS) disorders such as brain tumors, Alzheimer disease, and scrapie. Moreover, a clear relationship has been drawn between other cases of tissue degeneration and the expression of this gene. Accumulation of the corresponding protein had been reported in Alzheimer’s disease, glomerulonephritis, and various cases of kidney degeneration. The same gene had been shown in rat t o be activated in cells undergoing programmed death, such as in embryonic regressing tissues, and in prostatic cells during the prostate involution that follows castration. Several expression features, as well as the functional and structural relationships between this protein and molecules of the cytolytic membrane attack complex of the complement, suggest a causal involvement in cell death in brain and peripheral tissues. Search for novel genes specifically expressed in pathological brains is a powerful way to investigate the molecular bases of these pathologies. Differential screening of cDNA libraries is the method most widely 0 1992 WILEY-LISS, INC
used for such purposes. This approach permits direct identification of cloned nucleotide sequences corresponding to genes regulated at the level of transcription. Using this technique, four independent groups have isolated the same gene from different species; two of them starting from neuropathological material. Surprisingly this gene had been previously isolated in laboratories interested by distantly related topics, including the biology of reproduction. Since it was discovered independently by many groups, this single gene is now endowed with more than fifteen different names (Sylvester et al., 1991); in this review, we will term it SGP-2 for sulfated glycoprotein-2, the name given to the major secretion product of Sertoli cells and corresponding to the first cDNA characterized (Collard and Griswold, 19871,although it was recently proposed that this protein should be referred to as clusterin in all species (OBryan et al., 1990). STRUCTURE AND PROPERTIES OF THE SGP-2 LIKE PROTEINS SGP-2 cDNAs contain open-reading frames coding for a 449 amino acid long precursor protein in human Received September 6, 1991; accepted in revised form October 21, 1991
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concentration of 35-105 pg/ml (Murphy et al., 1988), which is considerably lower than that found in seminal plasma (OBryan et al., 1990).The widespread distribution of SGP-2 suggests that this protein may possess various roles. From the results recently reported by Choi et al. (19891,we can speculate that the circulating form of SGP-2, probably secreted from liver or shed by cells undergoing apoptosis, modulates the formation of the complement membrane attack complex and the subsequent lysis of target cells. Alternatively, serum SGP-2 may block complement-induced cell lysis, since it has also been shown that this protein is a potent inhibitor of the terminal complement pathway (Jenne and Tschopp, 1989; Murphy et al., 1989b). Circulating form of SGP-2 could also play a role in lipid transport, on the basis of its structural similarity with Apo-J (de Silva et al., 1990a,b). The function of the non circulating SGP-2 found in healthy organs mostly remains to be established. It may reflect the basal level of cell mortality that naturally exists in all tissues, including the CNS. However, the absence of correlation between the high levels of SGP-2 and apoptosis in healthy tissues or cells (e.g., Sertoli cells, chromaffin cells, ependymocytes, motoneurons) indicates that the role of SGP-2 is not exclusively related to cell death. In fact, it is likely that SGP-2 has various physiological functions. For example, the presence in the choroid plexus may suggest a possible involvement in fluid production. A link has also been suggested between SGP-2 and secretion mechanisms (Palmer and Christie, 1990). Indeed, SGP-2 is abundant in chromaffin granules, where it coexists in both soluble and membrane-bound forms. Hypotheses about the biological function(s) of the SGP-2 gene are still unclear. However, an involvement of the SGP-2 protein in membrane fusion processes would be consistent with many observations. Indeed, a role in membrane vesiculation linked to the acrosome reaction would explain the main acrosomal localization of SGP-2 on spermatozoa. Moreover, the coalescing of SGP-2: A SINGLE GENE THAT MAY ENSURE the secretion granules with the inner face of the plasma MULTIPLE FUNCTIONS membrane is probably triggered by specific cellular maThe precise physiological role of SGP-2 has not yet chinery. It is known, for example, that the epinephrine been delineated. SGP-2 is strongly expressed in brain release is controlled by calcium fluxes in chromaffin degenerating tissues and in normal testes, but basal cells, the molecular mechanism governing the last step levels have also been shown in several other organs of exocytosis being not clear yet. Assuming that such a including brain (Collard and Griswold, 1987; Day et al., function is ensured by SGP-2, it is conceivable that high 1990; de Silva et al., 1990a; Palmer and Christie 1990; levels of this protein may have deleterious effects on Mayet al., 1990; Chabot et al., 1991; Danik et al., 1991; membrane integrity, and hence on cell viability. In the Garden et al., 1991; Pasinetti and Finch, 1991). In the nervous system, SGP-2 may then act as an apolipoproCNS, in situ hybridization experiments revealed the tein and ensure lipid recycling from damaged to regenpresence of SGP-2 transcripts, under basal conditions, eratingnerves (Boyle et al., 1989; Goodrum, 1991; Poirin the choroid plexus, hippocampal formation, various ier et al., 1991). Thus, this single gene could play hypothalamic and brainstem nuclei, and the cortex distinct roles depending on its expression rate. The fine (Day et al., 1990; May et al., 1990; Chabot et al., 1991; transcriptional tuning of the SGP-2 gene (Herault et Garden et al., 1991). SGP-2 is present in serum at a al., 1992) agrees with such a possibility. It seems likely
and in bird, 447 in rat and 439 in steer. All of them contain a typical secretion signal peptide. Although limited at the level of the amino acid sequence identity, the SGP-2 protein structures are relatively well conserved between the different species. The regions of highest homology are grouped around two strictly conserved clusters of five cysteines. The less-conserved domain includes an internal proteolytic cleavage site adjacent to an arginine residue, yielding two subunits that remain associated by a disulfide bond. Four potential glycosylation sites are also conserved. Interestingly, a long amino-terminal a-helix motif, thought to promote SGP-2 dimerization via a myosin-like coiledcoil structure (Jenne and Tschopp, 1989; Tsuruta et al., 1990)is present in all species. This dimerization capacity, together with the cell binding ability, would be responsible for the cell-aggregating activity of “clusterin” (Cheng et al., 1988). A consensus sequence located around the first cysteine cluster typical of the terminal components of the complement (Kirszbaum et al., 1989) is also conserved in the four proteins and suggests that SGP-2 is related to this family of cytolytic proteins. A fundamental aspect of SGP-2 is its capacity to interact with lipids of plasma membranes, or circulating origin. Indeed, SGP-2 has been isolated as an apolipoprotein, called apolipoprotein-J (Apo-J) (de Silva et al., 1990b), that shares several features, including the existence of amphipatic a-helices, with other apolipoproteins. Little is known about the different possible post-translational modifications of SGP-2-likeproteins. It has been shown that the glycosylation patterns as well as the proteolytic cleavages are different, depending on the source of SGP-2-like proteins (Cheng et al., 1988; Buttyan et al., 1989). Since Southern analysis clearly indicated that rat, human, and avian haploid genomes contain a single copy of this gene, these maturation differences could explain the heterogeneous behavior of these proteins and perhaps their involvement in different biological processes.
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that, when deregulated, excessive membrane fusion could accelerate cell death, and possibly remodeling, linked to neuronal plasticity. In that regard, it is of interest that in situ hybridization has revealed that SGP-2 signals are most abundant in brain areas demonstrating high plasticity such as the olfactory bulb and the hippocampus (Day et al., 1990; May et al., 1990; Chabot et al., 1991; Laping et al., 1991).
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consequence of genetically programmed cell death. Alternatively, it is possible that this gene is expressed as an attempt to protect targeted cells, SGP-2 acting as an inhibitor of complement-initiated cytolysis.
RELATIONSHIPS BETWEEN SGP-2 AND MOLECULES OF THE IMMUNE SYSTEM The human SGP-2 protein known as SP40,40 was initially identified as a component of immune deposSGP-2 AND PROGRAMMED CELL DEATH its-containing glomerular basement membranes of a A rat mRNA homologous t o SGP-2, called testoster- patient suffering from membranous glomerulonephrione-repressed prostate message-2 (TRPM-2), was iso- tis (Murphy et al., 1988). Immunological studies of lated on the basis of its strong expression in ventral SGP-2-like materials in renal biopsies have shown that prostate after castration (Leger et al., 1987; Bettuzzi it is an invariable component of the terminal compleet al., 1989). This biological situation was chosen as a ment complex SC5b-9 whose vascular and tubular model of programmed cell death in order to search for basement membrane deposits are markedly increased novel RNA involved in this biological process. Actually, subsequent to renal injury. Similar observations situathe lack of androgens leads to a rapid involution of the tions can be made in interstitial nephritis and following prostate mediated by cell death. Furthermore, products allograft rejection (Murphy et al., 1989a). SC5b-9 is of genes expressed during the androgen-programmed known to contain seven proteins, including the termicell death can potentially be used as markers of urogen- nal components of the complement system (C5b, C6, ital injury (Bandyk et al., 1990). Induction of this gene C7, C8, C9), as well as S-protein (vitronectin) and was further generalized to other cases of tissue regres- SGP-2; and to be cytolytically inactive. As mentioned sion naturally occurring during embryonic develop- above, SGP-2 inhibits C5b-6 or C56f-initiated reactive ment or caused by experimental traumatisms. For ex- hemolysis (Jenne and Tschopp, 1989; Choi et al., 1989; ample, the regression of interdigital tissue during rat Murphy et al., 1989b; OBryan et al., 1990). This may limb bud formation, the renal atrophy which follows indicate that in the early stages of cell death, dying cells ureteral obstruction or the chemotherapeutic regres- can express SGP-2 as an inhibitor of complement comsion of a tumor are all associated with SGP-2 message plex formation, in order t o protect themselves against accumulation (Buttyan et al., 1989; Connor et al., autoimmune attack. In contrast, it has also been proposed that SGP-2, by acting as a cytolytic enhancer, can 1991). At present, SGP-2 is the only satisfactory probe for promote complement-mediated cytolysis. For example, programmed cell death in a variety of tissues and cell SGP-2 has been shown to stimulate hemolysis strongly types, including the CNS (Buttyan et al., 1989; Connor when in contact with cells before or at the same time as et al., 1991; Ucker, 1991). SGP-2 induction has been the C56f, C7, C8, and C9 components of complement reported in all cases of cell or tissue autolysis tested so (Choi et al., 1989). In that regard, it is of interest that far. Its involvement was recently extended to the well an activation of the classical complement pathway has defined glucocorticoid-induced thymocyte apoptosis been described in nephropathy (Murphy et al., 1989a; Cybulski et al., 1986) and in Alzheimer’s disease (Mc(Bettuzzi et al., 1991). Several phenotypic manifestations of programmed Geer et al., 1989a).An increased frequency of autoanticell death appear to be common to all types of tissues, bodies directed against cells of the nervous system has such as loss of adhesion to adherent cells and break- often been observed in Alzheimer’s brains, suggesting down of the nuclear envelope. Since these features are that associated neuronal death could be mediated by also characteristic of mitosis, it was proposed that cell the immunological activation of various components of suicide proceeds by an abortive mitosis mechanism the complement. The presence of numerous immuno(Ucker, 1991). This hypothesis is strikingly in keeping globulins in amyloid plaques (Ishii et al., 1988) supwith the fact that the SGP-2 avian gene transcription ports this hypothesis. SGP-2 is capable of physically interacting with the strictly requires the presence of classical mediators of the mitogenic stimuli, such as the junlfos Ap-1 complex cytolytic complement components but also shows struc(Herault et al., 1991). Consistently, brain lesions, heat tural relationships with this protein family. A consenstress and seizures were shown to produce induction of sus amino acid sequence typical of the terminal compoc-fos immunoreactivity (Morgan and Curran, 1991) in nents of the complement is conserved in the SGP-2 various brain areas enriched with SGP-2 mRNA proteins from mammalian as well as avian origin (Fig. (Chabot et al., 1991). Although SGP-2 has been pro- 1).When interacting with the complement system, the posed as a putative marker of cell death, it is still un- SGP-2 protein is regularly associated with the S protein clear whether SGP-2 overexpression is the cause or the (vitronectin),containing the tripeptide RGD that is spe-
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Fig. 1. Related cysteine-rich motifs in SP-40,40 and components of the complement terminal components incorporated in the membrane attack complexes. (Modified from firszbaum et al.. 1989.)
cifically recognized by a vitronectin receptor belonging to the integrin family (Jenne and Stanley, 1985). Interestingly, the vitronectin receptor is implicated in the macrophage recognition of cells undergoing programmed cell death that leads t o their phagocytosis (Savill et al., 1990). This situation is likely to occur in Alzheimer brain tissue, in which some areas contain both extracellular vitronectin and microglial cells strongly positive for a vitronectin receptor: these two immunoreactivities being present in senile plaques (Akiyama et al., 1991). Recently, an additional activity of SGP-2 has been reported in relation with the immune system, namely its ability to bind immunoglobulins. This property makes possible to retain circulating SGP-2 from plasma by immunoglobulin affinity (Wilson et al., 1991) and may have several implications potentially important in the physiological context. First, this can account for the inhibiting activity of SGP-2 on complement-mediated hemolysis (Jenne and Tschopp, 1989; Murphy et al., 1989b) through a competitive interaction with a single domain of the constant fraction (Fc) of immunoglobulins, as suggested by the ability of SGP-2 to prevent the antagonizing effect of the complement on immunoprecipitation (Wilson et al., 1991). Second, because of its dual ability to bind cells and immunoglobulins, SGP-2 is a good candidate for the antibody-dependent cellmediated cytotoxicity (ADCC), including the natural killer cells and macrophages. The abundance of immunoglobulins (Ishii et al., 1988) and phagocytic cells (McGeer et al., 198913) in Alzheimer’s brains could permit such an activity of SGP-2. It remains to be established whether the complement system is required for SGP-2 activity. Indeed, the presence of complement molecules have been established in ovarian follicular fluid (D’Cruz et al., 1990) and can perhaps be functionally associated to sperm SGP-2. Thus, structural similarities between SGP-2 and complement proteins are certainly most intriguing and should be further investigated. The hypothesis that SGP-2 expressed at high levels in various experimentally induced injury and pathological conditions, is a cell death inducer, can also be reconciled with observations about transformed cells. The
overexpression of SGP-2 by transformed cells (Michel et al., 1989) and in gliomas (Danik et al., 1991) can constitute a suicide attempt aimed to preserve the organism integrity. In this respect, it is interesting to note that the RSV-transformed cells that express high levels of SGP-2 (Michel et al., 19891, are easily rejected in vivo, the maintenance of tumor being only the consequence of the viral recruitment of novel infected cells (Halpern et al., 1991). CORRELATION BETWEEN SGP-2 OVEREXPRESSION AND CERTAIN BRAIN DISORDERS If one assumes that the scrapie agent possesses a nucleotidic genome, the molecular cloning of such sequences would be achieved by adapting the differential screening procedure to the comparison of genes expressed between infected and non infected brains. Unexpectedly, such a strategy have so far failed to point to any viral sequence, but have shown that several cellular genes are clearly up-regulated upon scrapie infection. One of them is the glial fibrillary acidic protein, already known to be highly expressed in infected brain (Duguid et al., 1989). SGP-2 was also identified as a gene newly overexpressed after scrapie infection (Duguid et al., 1989). Two groups subsequently reported the overexpression of the same gene in Alzheimer disease by probing mRNA in postmortem hippocampi (Duguid et al., 1989; May et al., 1990).Moreover, there is a good correlation between the occurrence of SGP-2 like immunoreactive material and plaque densities in cortical and hippocampal areas in normal aged and Alzheimer brain tissues (Moyse et al., 1991). However, overexpression of SGP-2 mRNA and protein is not restricted to Alzheimer disease. We recently observed that brain gliomas (Fig. 2A) but not primitive neurectodermal tumor overexpressed SGP-2 (Danik et al., 1991). Similarly, overexpression of the message was observed in epileptic foci (Danik et al., 1991) (Fig. 2B). Moreover, expression of SGP-2 transcripts is also detected in normal aged brain tissues. For example, in the temporal cortex of a 81-year-old patient devoid of major neuropathological deficits, we observed hybridization signals throughout the gray matter (Danik et
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Fig. 2. Low-power dark-field photomicrographs and autoradiographs demonstrating the results of in situ hybridization with SGP-2 riboprobe. A Note the high labeling density in malignant glioma. B: Some clusters of silver grains were found in epileptic focis. C: Darkfield autoradiograph at the level of the hippocampal formation and choroid plexus of young adult rat. A very strong hybridization signal is detected over the choroid plexus. In the hippocampus, moderate labeling is observed over the pyramidal cell layer (py)and the granular cell layer of the dentate gyrus (GrDG). Note the prominent hybridization signal over the hippocampal fissure (HiF). D: Dark-field autoradiograph a t the level of the hippocampus in kainate-lesioned rat. Unilateral injection (1 PI) of kainic acid (2 kg/kl) was made into the dorsal hippocampus of adult rat. The contralateral hippocampus was used as
control. Ten days later, rats were sacrificed, and brains were prepared for in situ hybridization histochemistry. Note the difference of labeling between the lesioned and intact hippocampus. E: Dark-field montage photomicrograph of emulsion-coated sections from control human temporal cortex. Numerous clusters of silver grains representing SGP-2 mRNA-positive cells are detected throughout gray matter areas (a).Note the absence of signal on a consecutive section incubated with the sense probe (b). F Dark-field autoradiograph at the level of the cerebellum and brainstem. Note the important labeling seen over certain brainstem nuclei. G: Low-power dark-field photomicrograph in the gigantocellular reticular nucleus. Intense clusters of silver grains are seen over motoneurons. For more details on SGP-2 in situ hybridization histochemistry, see Danik et al. (1991).
al., 1991)(Fig. 2E). In normal rat brain tissues, we have also observed a widespread distribution of SGP-2 transcripts (Figs. BC,F,G) (Chabot et al., 1991).The highest level of expression was found in the choroid plexus and ependyma. Prominent labeling was detected over the pyramidal cell layer of the hippocampus, the granular
cell layer of the dentate gyrus, and the hippocampal fissure. In a few hypothalamic nuclei, some neurons were SGP-2 mRNA positive. In the brainstem, certain populations of motoneurons also displayed intense labelling (Fig. 2F,G). In all cases, the sense probe gave little or no hybridization signal demonstrating the spec-
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ificity of the observed labelling (data not shown). Similar results are recently been reported in adult rat brain (Garden et al., 1991). The expression of SGP-2 mRNA in gliomas and the hippocampal fissure suggests that astrocytes are likely associated to the basal expression of SGP-2 in normal brain tissues. On the other hand, neurons are also likely to express this mRNA, since some were labeled in epileptic foci, human temporal cortex and certain regions of the rat brain. Although the precise role of this protein in the CNS remains to be established, we recently suggested that SGP-2 overexpression is a general characteristic of neural cell deathlor plasticity on the basis of data obtained in brain tumors, epileptic foci and Alzheimer hippocampi (May et al., 1990; Danik et al., 1991). However, its overexpression in healthy brain tissues, such as ependymocytes and motoneurons (Chabot et al., 1991; Garden et al., 1991), suggests that SGP-2 may play additional roles, such as an involvement in lipid transport, fluid production, and neurotransmitter secretion process. In addition, it was recently suggested that SGP-2 may be a marker of neuronal differentiation (Garden et al., 1991),a hypothesis that certainly deserves further consideration. Concerning its possible implication in cell death (or brain plasticity), it has recently been shown that manipulations that are accelerating brain aging and cell death such as experimental neuronal injuries (Day et al., 1990;May et al., 1990; Pasinetti and Finch, 1991) or the withdrawal of testicular and adrenal hormones (Day et al., 1990; Laping et al., 1991), enhanced the expression of SGP-2 mRNA. We also recently observed a marked increase of the SGP-2 hybridization signals in kainate-lesioned rat striatum and hippocampus (Chabot et al., 1991)(Fig. 2D). A great number of astrocytes in the vicinity of the lesion sites as well as degenerative neurons were SGP-2 mRNA positive (data not shown). Such a response of neuronal cells to major physiological disturbances had already been established from a differential screening experiment performed with avian neuroretinal cells (Michel et al., 1989).Actually, the most abundant mRNA synthesized in these cells after in vitro infection by the Rous sarcoma retrovirus (RSV) carrying the v-src oncogene is the avian counterpart of SGP-2, termed T64 (Michel et al., 1989).This gene expression is the consequence of an increase of its transcription rate and experiments are now in progress to determine whether the induction of this gene is directly linked to the intense cell death associated with RSV infection. Finally, a fourth differential screening experiment, aimed a t the search for genes involved in programmed cell death has identified a SGP-2 homologue as a gene induced in rat ventral prostate regressing after castration (Bandyk et al., 1990).Taken together, these data strongly suggest that SGP-2 is a key gene associated with cell response to various biological stressors.
CONCLUDING REMARKS Cell death belongs to the critical biological processes necessary to both the development and the survival of pluricellular organisms. The cell death genetic program remains active in most tissues of adult organisms but constitutes an important aspect of morphogenesis. Autolysis of embryonic temporary structures, often reminiscent of ancestral schemes, permits edification of the definitive organization. It is also postulated that the development of the nervous system by “epigenesis” is based upon the spontaneous regression of synapses (Changeux et al., 1973). The massive neuronal death occurring during development may represent selective losses of neurons having erroneous or in adapted projections (Oppenheim, 1991). Among the complex and aleatory connections of the brain at birth, a few networks should be stabilized by their activation, while others are definitively lost, for lack of adequate stimuli occurring during a critical period of apprenticeship. Neuronal cell death should thus allow conservation of only those neuronal circuits adapted to environment. It is conceivable that an abnormal reactivation of genes involved in the cell death program among the selected neurons leads to degenerative processes and diseases. Such a reactivation can result either from an exogenous stimulus like a viral infection, or from an endogenous, yet undetermined defect. The elucidation of mechanisms which regulate the SGP-2 gene expression could allow for the identification of genetic deregulations occurring in neural tissues under pathophysiological conditions. REFERENCES Akiyama, H., Kawamata, T., Dedhar, S., and McGeer, P.L. (1991) Immunohistochemical localization of vitronectin, its receptor and beta-3 integrin in Alzheimer brain tissue. J. Neuroimmunol., 32:1928. Bandyk, M.G., Sawczuck, I S . , Olsson, C.A., Katz, A.E., and Buttyan, R. (1990) Characterization of the products of a gene expressed during androgen-programmed cell death and their potential use as a marker of urogenital injury. J . Urol., 143:407413. Bettuzzi, S., Hiipakka, R.A., Gilna, P., and Liao, S. (1989) Identification of an androgen-repressed mRNA in rat ventral prostate as coding for sulphated glycoprotein 2 by cDNA cloning and sequence analysis. Biochem. J., 257:293-296. Bettuzzi, S., Troiano, L., Davalli, P., Tropea, F., Ingletti, M.C., Grassilli, E., Monti, D., Corti, A,, and Franceschi, C. (1991) In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death. Biochem. Biophys. Res. Commun., 175:810-815. Boyle, J.K., Zoellner, C.D., Anderson, L.J., Kosick, L.M., Pitas, R.E., Weismaber. K.H., Hui. D.Y.. Mahlev. R.W.. Gebicke-Haeter. P.J.. Ignagus, M.J., and Shooter, E.M. (1989)A role for apolipoprotein E; apolipoprotein A-1 and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve. J . Clin. Invest., 83:1015-1031. Buttyan, R., Olsson, C.A., Pintar, J., Chang, C., Bandyk, M., Ng, P.-U., and Sawczuk, I.S. (1989) Induction of the TRPM-2 gene in cells undergoing programmed death. Mol. Cell. Biol., 9:3473-3481. Chabot, J.-G., Danik, M., Mercier, C., Benabid, A.-L., Quirion, R., and Suh, M. (1991) Expression of sulfated glycoprotein-2 (SGP-2) in human gliomas, epileptic foci and rat brain tissues. Presented a t the Twenty-first Annual Meeting of the Society for Neuroscience, 271.11,692. Changeux, J.P., Courrege, P., and Danchin, A. (1973) A theory of the epigenesis of neuronal networks by selective stabilization of synapses. Proc. Natl. Acad. Sci. USA, 70:2974-2978.
SGP-2 EXPRESSION IN THE CNS Cheng, C.Y., Mathur, P.P., and Grima, J . (1988) Structural analysis of clusterin and its subunits in ram rete testis fluid. Biochemistry, 27:4079-4088. Choi, N.-H., Madza, T., and Tomita, M. (1989) A serum protein SP40,40 modulates the formation of membrane attack complex of complement on erythrocytes. Mol. Immunol., 262435-840. Collard, M.W., and Griswold, M.D. (1987) Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells. Biochemistry, 26:3297-3303. Connor, J., Buttyan, R., Olsson, C.A., DAgati, V., OToole, K., and Sawczuk, I S . (1991) SGP-2 expression as a genetic marker of progressive cellular pathology in experimental hydronephrosis. Kidney Int., 39:1098-1103. Cybulski, A.V., Rennke, H.J., Feinzeig, I.D., and Salant, D.J. (1986) Complement-induced glomerular epithelial cell injury. Role of the membrane attack complex in rat membranous nephropathy. J. Clin. Invest., 77:1096-1107. Danik, M., Chabot, J.-G., Mercier, C., Benabid, A.-L., Chauvin, C., Quirion, R., and Suh, M. (1991) Human gliomas and epileptic foci express high levels of a mRNA related to rat testicular sulfated glycoprotein 2, a purported marker of cell death. Proc. Natl. Acad. Sci. USA, 88:8577-8581. Day, J.R., Laping, N.J., McNeill, T.H., Screiber, S.S., Pasinetti, G., and Finch, C.E. (1990) Castration enhances expression of glial fibrillary protein and sulfated glycoprotein-2 in the intact and lesion-altered hippocampus of the adult male rat. Mol. Endocrinol., 4:1995-2002. DCruz, O.J., Haas, G.G., and Lambert, H. (1990) Evaluation of antisperm complement-dependent immune mediators in human ovarian follicular fluid. J. Immunol., 144:3841-3848. de Silva, H.V., Harmony, J.A.K., Stuart, W.D., Gil, C.M., and Robbins, J . (1990a) Apolipoprotein J : Structure and tissue distribution. Biochemistry, 295380-5389. de Silva, H.V., Stuart, W.D., Park, Y.B., Mao, S.J.T., Gil, C.M., Wetterau, J.R., Busch, S.J., and Harmony, J.A.K. (1990b) Purification and characterization of apolipoprotein J . J . Biol. Chem., 265:1429214297. Duguid, J.R., Bohmont, C.W., Liu, N., and Tourtellotte, W.W. (1989) Changes in brain gene expression shared by scrapie and Alzheimer disease. Proc. Natl. Acad. Sci. USA, 86:7260-7264. Garden, G.A., Bothwell, M., and Rubel, E.W. (1991)Lack of correspondence between mRNA expression for a putative cell death molecule (SGP-2) and neuronal cell death in the central nervous system. J. Neurobiol., 22:590-604. Goodrum, J.F. (1991) Cholesterol from degenerating nerve myelin becomes associated with lipoproteins containing apolipoprotein E. J. Neurochem., 56:2082-2086. Halpern, M.S., England, J.M., Coates, L., Stolzfus, C.M., and Mason, W.S. (1991)Regression of u-src DNA-induced sarcomas is under host genetic control? Virology, 180:857-860. Herault, Y., Chatelain, G., Brun, G., and Michel, D. (1992) v-src induced expression of a programmed cell death marker through a n M-1dependent mechanism. Oncogene Res., submitted. Ishii, T., Haga, S., and Kametroi, F. (1988) Presence of immunoglobulins and complements in the amyloid plaques in the brain of patients with Alzheimer’s disease. In: Immunology and Alzheimer’s Disease. A. Pouplard-Barthelaix, J. Emile, and Y. Christen, eds. Springer, Berlin, pp. 17-29. Jenne, D., and Stanley, K.K. (1985) Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion. EMBO J., 4:3153-3157. Jenne, D.E., and Tschopp, J . (1989) Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: Identity to sulfated glycoprotein 2, a constituent of rat testis fluid. Proc. Natl. Acad. Sci. USA, 86:7123-7127. Kirszbaum, L., Sharpe, J.A., Murphy, B., d’Apice, A.J.F., Classon, B., Hudson, P., and Walker, I.D. (1989) Molecular cloning and characterization of the novel human complement-associated protein, SP40,40: A link between the complement and reproductive systems. EMBO J., 8:711-718.
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Laping, N.J., Nichols, N.R., Day, J.R., and Finch, C.E. (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. Leger, J.G., Montpetit, M.L., and Tenniswood, M. (1987) Characterization and cloning of androgen repressed mRNAs from rat ventral prostate. Biochem. Biophys. Res. Commun., 147:196-203. May, P.C., Lampert-Etchells, M., Johnson, S.A., Poirier, J., Masters, J.N., and Finch, C.E. (1990) Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer’s disease and in response to experimental lesions in rat. Neuron, 5831-839. McGeer, P.L., Akiyama, H., Itagaki, S., and McGeer, E.G. (1989a) Activation of the classical complement pathway in brain tissue of Alzheimer patients. Neurosci. Lett., 107:341-346. McGeer, P.L., Akiyama, H., Itagaki, S., and McGeer, E.G. (1989b) Immune system response in Alzheimer’s disease. Can. J . Neurol. Sci., 16(suppl):516-527. Michel, D., Gillet, G., Volovitch, M., Pessac, B., Calothv. G.. and Brun. G. (1989) Expression of a novel gene encoding a 51:5 kD precursor protein is induced by different retroviral oncogenes in quail neuroretinal cells. Oncogene Res., 4:127-136. Morgan, J.I., and Curran, T. (1991) Stimulus-transcription coupling in the nervous system: Involvement of the inducible proto-oncogenes fos andjun. Annu. Rev. Neurosci., 14:421-451. Moyse, E., Michel, D., Choi, N., Robitaille, Y., Brun, G., Quirion, R., and Chabot, J.-G. (1991) Selective expression of sulfated glycoprotein-2 (SGP-2) by hippocampal neuron and ependyma in human brain enriched in P-amyloid senile plaques of the Alzheimer-type. Presented at the Twenty-first Annual Meeting of the Society for Neuroscience, 271.12,692. Murphy, B.F., Kirszbaum, L., Walker, I.D., and d’Apice, A.J.F. (1988) SP40,40, a newly identified normal human serum protein found in the SC5b-9 complex of complement and in the immune deposits of glomerulonephritis. J . Clin. Invest., 81:1858-1864. Murphy, B.F., Davies, D.J., Morrow, W., and d’Apice, A.J.F. (1989a) Localization of terminal complement components, S-protein and SP40,40 in renal biopsies. Pathology, 21:275-278. Murphy, B.F., Saunders, J.R., OBryan, M.K., Kirszbaum, L., Walker, I.D., and d’apice, A.J.F. (198913) SP-40,40 is a n inhibitor of C5b-6 initiated hemolysis. Int. Immunol., 1:551-554. OBryan, M.K., Baker, H.G.W., Saunders, J.R., Kirszbaum, L., Walker, I.D., Hudson, P., Liu, D.Y., Glew, M.D., d’Apice, A.J.F., and Murphy, B.F. (1990) Human seminal clusterin (SP40,40). Isolation and characterization. J. Clin. Invest., 85:1477-1486. Oppenheim, R.W. (1991) Cell death during development of the nervous system. Annu. Rev. Neurosci., 14:453-501. Palmer, D.J., and Christie, D.L. (1990) The primary structure ofglycoprotein 111 from bovine adrenal medullary chromaffin granules. J. Biol. Chem., 265:6617-6623. Pasinetti, G.M., and Finch, C.E. (1991) Sulfated glycoprotein-2 (SGP-2) mRNA is expressed in rat striatal astrocytes following ibotenic acid lesions. Neurosci. Lett., 130:14. Poirier, J., Baccichet, A., Dea, D., and Gauthier, S. (1991) Selective alteration of the cholesterol levels in the hippocampus during reactive synaptogenesis. J . Neurochem., submitted. Savill, J., Dransfield, I., HOEE.N.. and Haslett. C. (1990) Vitronectin receptor-mediated phagocytosis of cell undergoing apoptosis. Nature. 343:170-173. Sylvester, S.R., Morales, C., Oko, R., and Griswold, M.D. (1991)Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat. Biol. Reprod., 45,195-207. Tsuruta, J.K., Wong, K., Fritz, I.B., and Griswold, M.D. (1990) Structural analysis of sulphated glycoprotein 2 from amino acid sequence. Relationship to clusterin and serum protein SP40,40. Biochem. J., 268:571-578. Ucker, D.S. (1991) Death by suicide: One way to go in mammalian cellular development. New Biol., 3:103-109. Wilson, M.R., Roeth, P.J., and Easterhrook-Smith, S.B. (1991) Clusterin enhances the formation of insoluble immune complexes. Biochem. Biophys. Res. Commun., 177:985-990.
Possible Functions of a New Genetic Marker in Central Nervous System: The Sulfated Glycoprotein-2 (SGP-2) DENIS MICHEL, JEAN-GUY CHABOT, EMMANUEL MOYSE, MARC DANIK, AND &MI QUIRION Ecole Normale Superieure de Lyon, Laboratoire de Biologie Moleculaire et Cellulaire, Lyon, Cedex, France (D.M., E.M.); Douglas Hospital Research Centre and Department of Psychiatry, Faculty of Medicine, McGill University, Verdun, Quebec, Canada H4H 1R3 (J.-G.C.,M.D., R.Q.); Centre de Recherche de l’H6pital Maisonneuve-Rosemont, University of Montreal, Montreal, Quebec, Canada H1 T 2M4 (M.D.)
KEY WORDS
Clusterin, SP40,40, TRPM-2, Gene expression, Apoptosis, Complement, In situ hybridization
This brief review discusses the recent characterization in the brain of a ABSTRACT gene coding for a protein that may be involved in programmed cell death and/or brain plasticity. We will term it sulfated glycoprotein-2 (SGP-2),the name corresponding to the first cDNA characterized. Recent studies have demonstrated the overexpression of this sulfated glycoprotein in various CNS disorders, such as certain gliomas, Alzheimer’s disease and epilepsy, as well as after experimental brain injury in animals where different cell types were undergoing tissue remodelling or cell death. In peripheral tissues, SGP-2 gene expression has been found to be strikingly increased following experimental manipulations in which cells of injured tissues were undergoing programmed cell death or apoptosis. The results reported thus far are intriguing and suggest the possible involvement of SGP-2 in apoptotic mechanisms as well as its interaction with components of the immune system possibly associated with cell death in neurodegenerative disorders. 8 1992 Wiley-Liss, Inc.
INTRODUCTION A novel gene has recently burst into the field of neurosciences, and more precisely of neuropathology. High levels of mRNA transcribed from this gene are correlated with the occurrence of central nervous system (CNS) disorders such as brain tumors, Alzheimer disease, and scrapie. Moreover, a clear relationship has been drawn between other cases of tissue degeneration and the expression of this gene. Accumulation of the corresponding protein had been reported in Alzheimer’s disease, glomerulonephritis, and various cases of kidney degeneration. The same gene had been shown in rat t o be activated in cells undergoing programmed death, such as in embryonic regressing tissues, and in prostatic cells during the prostate involution that follows castration. Several expression features, as well as the functional and structural relationships between this protein and molecules of the cytolytic membrane attack complex of the complement, suggest a causal involvement in cell death in brain and peripheral tissues. Search for novel genes specifically expressed in pathological brains is a powerful way to investigate the molecular bases of these pathologies. Differential screening of cDNA libraries is the method most widely 0 1992 WILEY-LISS, INC
used for such purposes. This approach permits direct identification of cloned nucleotide sequences corresponding to genes regulated at the level of transcription. Using this technique, four independent groups have isolated the same gene from different species; two of them starting from neuropathological material. Surprisingly this gene had been previously isolated in laboratories interested by distantly related topics, including the biology of reproduction. Since it was discovered independently by many groups, this single gene is now endowed with more than fifteen different names (Sylvester et al., 1991); in this review, we will term it SGP-2 for sulfated glycoprotein-2, the name given to the major secretion product of Sertoli cells and corresponding to the first cDNA characterized (Collard and Griswold, 19871,although it was recently proposed that this protein should be referred to as clusterin in all species (OBryan et al., 1990). STRUCTURE AND PROPERTIES OF THE SGP-2 LIKE PROTEINS SGP-2 cDNAs contain open-reading frames coding for a 449 amino acid long precursor protein in human Received September 6, 1991; accepted in revised form October 21, 1991
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concentration of 35-105 pg/ml (Murphy et al., 1988), which is considerably lower than that found in seminal plasma (OBryan et al., 1990).The widespread distribution of SGP-2 suggests that this protein may possess various roles. From the results recently reported by Choi et al. (19891,we can speculate that the circulating form of SGP-2, probably secreted from liver or shed by cells undergoing apoptosis, modulates the formation of the complement membrane attack complex and the subsequent lysis of target cells. Alternatively, serum SGP-2 may block complement-induced cell lysis, since it has also been shown that this protein is a potent inhibitor of the terminal complement pathway (Jenne and Tschopp, 1989; Murphy et al., 1989b). Circulating form of SGP-2 could also play a role in lipid transport, on the basis of its structural similarity with Apo-J (de Silva et al., 1990a,b). The function of the non circulating SGP-2 found in healthy organs mostly remains to be established. It may reflect the basal level of cell mortality that naturally exists in all tissues, including the CNS. However, the absence of correlation between the high levels of SGP-2 and apoptosis in healthy tissues or cells (e.g., Sertoli cells, chromaffin cells, ependymocytes, motoneurons) indicates that the role of SGP-2 is not exclusively related to cell death. In fact, it is likely that SGP-2 has various physiological functions. For example, the presence in the choroid plexus may suggest a possible involvement in fluid production. A link has also been suggested between SGP-2 and secretion mechanisms (Palmer and Christie, 1990). Indeed, SGP-2 is abundant in chromaffin granules, where it coexists in both soluble and membrane-bound forms. Hypotheses about the biological function(s) of the SGP-2 gene are still unclear. However, an involvement of the SGP-2 protein in membrane fusion processes would be consistent with many observations. Indeed, a role in membrane vesiculation linked to the acrosome reaction would explain the main acrosomal localization of SGP-2 on spermatozoa. Moreover, the coalescing of SGP-2: A SINGLE GENE THAT MAY ENSURE the secretion granules with the inner face of the plasma MULTIPLE FUNCTIONS membrane is probably triggered by specific cellular maThe precise physiological role of SGP-2 has not yet chinery. It is known, for example, that the epinephrine been delineated. SGP-2 is strongly expressed in brain release is controlled by calcium fluxes in chromaffin degenerating tissues and in normal testes, but basal cells, the molecular mechanism governing the last step levels have also been shown in several other organs of exocytosis being not clear yet. Assuming that such a including brain (Collard and Griswold, 1987; Day et al., function is ensured by SGP-2, it is conceivable that high 1990; de Silva et al., 1990a; Palmer and Christie 1990; levels of this protein may have deleterious effects on Mayet al., 1990; Chabot et al., 1991; Danik et al., 1991; membrane integrity, and hence on cell viability. In the Garden et al., 1991; Pasinetti and Finch, 1991). In the nervous system, SGP-2 may then act as an apolipoproCNS, in situ hybridization experiments revealed the tein and ensure lipid recycling from damaged to regenpresence of SGP-2 transcripts, under basal conditions, eratingnerves (Boyle et al., 1989; Goodrum, 1991; Poirin the choroid plexus, hippocampal formation, various ier et al., 1991). Thus, this single gene could play hypothalamic and brainstem nuclei, and the cortex distinct roles depending on its expression rate. The fine (Day et al., 1990; May et al., 1990; Chabot et al., 1991; transcriptional tuning of the SGP-2 gene (Herault et Garden et al., 1991). SGP-2 is present in serum at a al., 1992) agrees with such a possibility. It seems likely
and in bird, 447 in rat and 439 in steer. All of them contain a typical secretion signal peptide. Although limited at the level of the amino acid sequence identity, the SGP-2 protein structures are relatively well conserved between the different species. The regions of highest homology are grouped around two strictly conserved clusters of five cysteines. The less-conserved domain includes an internal proteolytic cleavage site adjacent to an arginine residue, yielding two subunits that remain associated by a disulfide bond. Four potential glycosylation sites are also conserved. Interestingly, a long amino-terminal a-helix motif, thought to promote SGP-2 dimerization via a myosin-like coiledcoil structure (Jenne and Tschopp, 1989; Tsuruta et al., 1990)is present in all species. This dimerization capacity, together with the cell binding ability, would be responsible for the cell-aggregating activity of “clusterin” (Cheng et al., 1988). A consensus sequence located around the first cysteine cluster typical of the terminal components of the complement (Kirszbaum et al., 1989) is also conserved in the four proteins and suggests that SGP-2 is related to this family of cytolytic proteins. A fundamental aspect of SGP-2 is its capacity to interact with lipids of plasma membranes, or circulating origin. Indeed, SGP-2 has been isolated as an apolipoprotein, called apolipoprotein-J (Apo-J) (de Silva et al., 1990b), that shares several features, including the existence of amphipatic a-helices, with other apolipoproteins. Little is known about the different possible post-translational modifications of SGP-2-likeproteins. It has been shown that the glycosylation patterns as well as the proteolytic cleavages are different, depending on the source of SGP-2-like proteins (Cheng et al., 1988; Buttyan et al., 1989). Since Southern analysis clearly indicated that rat, human, and avian haploid genomes contain a single copy of this gene, these maturation differences could explain the heterogeneous behavior of these proteins and perhaps their involvement in different biological processes.
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that, when deregulated, excessive membrane fusion could accelerate cell death, and possibly remodeling, linked to neuronal plasticity. In that regard, it is of interest that in situ hybridization has revealed that SGP-2 signals are most abundant in brain areas demonstrating high plasticity such as the olfactory bulb and the hippocampus (Day et al., 1990; May et al., 1990; Chabot et al., 1991; Laping et al., 1991).
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consequence of genetically programmed cell death. Alternatively, it is possible that this gene is expressed as an attempt to protect targeted cells, SGP-2 acting as an inhibitor of complement-initiated cytolysis.
RELATIONSHIPS BETWEEN SGP-2 AND MOLECULES OF THE IMMUNE SYSTEM The human SGP-2 protein known as SP40,40 was initially identified as a component of immune deposSGP-2 AND PROGRAMMED CELL DEATH its-containing glomerular basement membranes of a A rat mRNA homologous t o SGP-2, called testoster- patient suffering from membranous glomerulonephrione-repressed prostate message-2 (TRPM-2), was iso- tis (Murphy et al., 1988). Immunological studies of lated on the basis of its strong expression in ventral SGP-2-like materials in renal biopsies have shown that prostate after castration (Leger et al., 1987; Bettuzzi it is an invariable component of the terminal compleet al., 1989). This biological situation was chosen as a ment complex SC5b-9 whose vascular and tubular model of programmed cell death in order to search for basement membrane deposits are markedly increased novel RNA involved in this biological process. Actually, subsequent to renal injury. Similar observations situathe lack of androgens leads to a rapid involution of the tions can be made in interstitial nephritis and following prostate mediated by cell death. Furthermore, products allograft rejection (Murphy et al., 1989a). SC5b-9 is of genes expressed during the androgen-programmed known to contain seven proteins, including the termicell death can potentially be used as markers of urogen- nal components of the complement system (C5b, C6, ital injury (Bandyk et al., 1990). Induction of this gene C7, C8, C9), as well as S-protein (vitronectin) and was further generalized to other cases of tissue regres- SGP-2; and to be cytolytically inactive. As mentioned sion naturally occurring during embryonic develop- above, SGP-2 inhibits C5b-6 or C56f-initiated reactive ment or caused by experimental traumatisms. For ex- hemolysis (Jenne and Tschopp, 1989; Choi et al., 1989; ample, the regression of interdigital tissue during rat Murphy et al., 1989b; OBryan et al., 1990). This may limb bud formation, the renal atrophy which follows indicate that in the early stages of cell death, dying cells ureteral obstruction or the chemotherapeutic regres- can express SGP-2 as an inhibitor of complement comsion of a tumor are all associated with SGP-2 message plex formation, in order t o protect themselves against accumulation (Buttyan et al., 1989; Connor et al., autoimmune attack. In contrast, it has also been proposed that SGP-2, by acting as a cytolytic enhancer, can 1991). At present, SGP-2 is the only satisfactory probe for promote complement-mediated cytolysis. For example, programmed cell death in a variety of tissues and cell SGP-2 has been shown to stimulate hemolysis strongly types, including the CNS (Buttyan et al., 1989; Connor when in contact with cells before or at the same time as et al., 1991; Ucker, 1991). SGP-2 induction has been the C56f, C7, C8, and C9 components of complement reported in all cases of cell or tissue autolysis tested so (Choi et al., 1989). In that regard, it is of interest that far. Its involvement was recently extended to the well an activation of the classical complement pathway has defined glucocorticoid-induced thymocyte apoptosis been described in nephropathy (Murphy et al., 1989a; Cybulski et al., 1986) and in Alzheimer’s disease (Mc(Bettuzzi et al., 1991). Several phenotypic manifestations of programmed Geer et al., 1989a).An increased frequency of autoanticell death appear to be common to all types of tissues, bodies directed against cells of the nervous system has such as loss of adhesion to adherent cells and break- often been observed in Alzheimer’s brains, suggesting down of the nuclear envelope. Since these features are that associated neuronal death could be mediated by also characteristic of mitosis, it was proposed that cell the immunological activation of various components of suicide proceeds by an abortive mitosis mechanism the complement. The presence of numerous immuno(Ucker, 1991). This hypothesis is strikingly in keeping globulins in amyloid plaques (Ishii et al., 1988) supwith the fact that the SGP-2 avian gene transcription ports this hypothesis. SGP-2 is capable of physically interacting with the strictly requires the presence of classical mediators of the mitogenic stimuli, such as the junlfos Ap-1 complex cytolytic complement components but also shows struc(Herault et al., 1991). Consistently, brain lesions, heat tural relationships with this protein family. A consenstress and seizures were shown to produce induction of sus amino acid sequence typical of the terminal compoc-fos immunoreactivity (Morgan and Curran, 1991) in nents of the complement is conserved in the SGP-2 various brain areas enriched with SGP-2 mRNA proteins from mammalian as well as avian origin (Fig. (Chabot et al., 1991). Although SGP-2 has been pro- 1).When interacting with the complement system, the posed as a putative marker of cell death, it is still un- SGP-2 protein is regularly associated with the S protein clear whether SGP-2 overexpression is the cause or the (vitronectin),containing the tripeptide RGD that is spe-
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Fig. 1. Related cysteine-rich motifs in SP-40,40 and components of the complement terminal components incorporated in the membrane attack complexes. (Modified from firszbaum et al.. 1989.)
cifically recognized by a vitronectin receptor belonging to the integrin family (Jenne and Stanley, 1985). Interestingly, the vitronectin receptor is implicated in the macrophage recognition of cells undergoing programmed cell death that leads t o their phagocytosis (Savill et al., 1990). This situation is likely to occur in Alzheimer brain tissue, in which some areas contain both extracellular vitronectin and microglial cells strongly positive for a vitronectin receptor: these two immunoreactivities being present in senile plaques (Akiyama et al., 1991). Recently, an additional activity of SGP-2 has been reported in relation with the immune system, namely its ability to bind immunoglobulins. This property makes possible to retain circulating SGP-2 from plasma by immunoglobulin affinity (Wilson et al., 1991) and may have several implications potentially important in the physiological context. First, this can account for the inhibiting activity of SGP-2 on complement-mediated hemolysis (Jenne and Tschopp, 1989; Murphy et al., 1989b) through a competitive interaction with a single domain of the constant fraction (Fc) of immunoglobulins, as suggested by the ability of SGP-2 to prevent the antagonizing effect of the complement on immunoprecipitation (Wilson et al., 1991). Second, because of its dual ability to bind cells and immunoglobulins, SGP-2 is a good candidate for the antibody-dependent cellmediated cytotoxicity (ADCC), including the natural killer cells and macrophages. The abundance of immunoglobulins (Ishii et al., 1988) and phagocytic cells (McGeer et al., 198913) in Alzheimer’s brains could permit such an activity of SGP-2. It remains to be established whether the complement system is required for SGP-2 activity. Indeed, the presence of complement molecules have been established in ovarian follicular fluid (D’Cruz et al., 1990) and can perhaps be functionally associated to sperm SGP-2. Thus, structural similarities between SGP-2 and complement proteins are certainly most intriguing and should be further investigated. The hypothesis that SGP-2 expressed at high levels in various experimentally induced injury and pathological conditions, is a cell death inducer, can also be reconciled with observations about transformed cells. The
overexpression of SGP-2 by transformed cells (Michel et al., 1989) and in gliomas (Danik et al., 1991) can constitute a suicide attempt aimed to preserve the organism integrity. In this respect, it is interesting to note that the RSV-transformed cells that express high levels of SGP-2 (Michel et al., 19891, are easily rejected in vivo, the maintenance of tumor being only the consequence of the viral recruitment of novel infected cells (Halpern et al., 1991). CORRELATION BETWEEN SGP-2 OVEREXPRESSION AND CERTAIN BRAIN DISORDERS If one assumes that the scrapie agent possesses a nucleotidic genome, the molecular cloning of such sequences would be achieved by adapting the differential screening procedure to the comparison of genes expressed between infected and non infected brains. Unexpectedly, such a strategy have so far failed to point to any viral sequence, but have shown that several cellular genes are clearly up-regulated upon scrapie infection. One of them is the glial fibrillary acidic protein, already known to be highly expressed in infected brain (Duguid et al., 1989). SGP-2 was also identified as a gene newly overexpressed after scrapie infection (Duguid et al., 1989). Two groups subsequently reported the overexpression of the same gene in Alzheimer disease by probing mRNA in postmortem hippocampi (Duguid et al., 1989; May et al., 1990).Moreover, there is a good correlation between the occurrence of SGP-2 like immunoreactive material and plaque densities in cortical and hippocampal areas in normal aged and Alzheimer brain tissues (Moyse et al., 1991). However, overexpression of SGP-2 mRNA and protein is not restricted to Alzheimer disease. We recently observed that brain gliomas (Fig. 2A) but not primitive neurectodermal tumor overexpressed SGP-2 (Danik et al., 1991). Similarly, overexpression of the message was observed in epileptic foci (Danik et al., 1991) (Fig. 2B). Moreover, expression of SGP-2 transcripts is also detected in normal aged brain tissues. For example, in the temporal cortex of a 81-year-old patient devoid of major neuropathological deficits, we observed hybridization signals throughout the gray matter (Danik et
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Fig. 2. Low-power dark-field photomicrographs and autoradiographs demonstrating the results of in situ hybridization with SGP-2 riboprobe. A Note the high labeling density in malignant glioma. B: Some clusters of silver grains were found in epileptic focis. C: Darkfield autoradiograph at the level of the hippocampal formation and choroid plexus of young adult rat. A very strong hybridization signal is detected over the choroid plexus. In the hippocampus, moderate labeling is observed over the pyramidal cell layer (py)and the granular cell layer of the dentate gyrus (GrDG). Note the prominent hybridization signal over the hippocampal fissure (HiF). D: Dark-field autoradiograph a t the level of the hippocampus in kainate-lesioned rat. Unilateral injection (1 PI) of kainic acid (2 kg/kl) was made into the dorsal hippocampus of adult rat. The contralateral hippocampus was used as
control. Ten days later, rats were sacrificed, and brains were prepared for in situ hybridization histochemistry. Note the difference of labeling between the lesioned and intact hippocampus. E: Dark-field montage photomicrograph of emulsion-coated sections from control human temporal cortex. Numerous clusters of silver grains representing SGP-2 mRNA-positive cells are detected throughout gray matter areas (a).Note the absence of signal on a consecutive section incubated with the sense probe (b). F Dark-field autoradiograph at the level of the cerebellum and brainstem. Note the important labeling seen over certain brainstem nuclei. G: Low-power dark-field photomicrograph in the gigantocellular reticular nucleus. Intense clusters of silver grains are seen over motoneurons. For more details on SGP-2 in situ hybridization histochemistry, see Danik et al. (1991).
al., 1991)(Fig. 2E). In normal rat brain tissues, we have also observed a widespread distribution of SGP-2 transcripts (Figs. BC,F,G) (Chabot et al., 1991).The highest level of expression was found in the choroid plexus and ependyma. Prominent labeling was detected over the pyramidal cell layer of the hippocampus, the granular
cell layer of the dentate gyrus, and the hippocampal fissure. In a few hypothalamic nuclei, some neurons were SGP-2 mRNA positive. In the brainstem, certain populations of motoneurons also displayed intense labelling (Fig. 2F,G). In all cases, the sense probe gave little or no hybridization signal demonstrating the spec-
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ificity of the observed labelling (data not shown). Similar results are recently been reported in adult rat brain (Garden et al., 1991). The expression of SGP-2 mRNA in gliomas and the hippocampal fissure suggests that astrocytes are likely associated to the basal expression of SGP-2 in normal brain tissues. On the other hand, neurons are also likely to express this mRNA, since some were labeled in epileptic foci, human temporal cortex and certain regions of the rat brain. Although the precise role of this protein in the CNS remains to be established, we recently suggested that SGP-2 overexpression is a general characteristic of neural cell deathlor plasticity on the basis of data obtained in brain tumors, epileptic foci and Alzheimer hippocampi (May et al., 1990; Danik et al., 1991). However, its overexpression in healthy brain tissues, such as ependymocytes and motoneurons (Chabot et al., 1991; Garden et al., 1991), suggests that SGP-2 may play additional roles, such as an involvement in lipid transport, fluid production, and neurotransmitter secretion process. In addition, it was recently suggested that SGP-2 may be a marker of neuronal differentiation (Garden et al., 1991),a hypothesis that certainly deserves further consideration. Concerning its possible implication in cell death (or brain plasticity), it has recently been shown that manipulations that are accelerating brain aging and cell death such as experimental neuronal injuries (Day et al., 1990;May et al., 1990; Pasinetti and Finch, 1991) or the withdrawal of testicular and adrenal hormones (Day et al., 1990; Laping et al., 1991), enhanced the expression of SGP-2 mRNA. We also recently observed a marked increase of the SGP-2 hybridization signals in kainate-lesioned rat striatum and hippocampus (Chabot et al., 1991)(Fig. 2D). A great number of astrocytes in the vicinity of the lesion sites as well as degenerative neurons were SGP-2 mRNA positive (data not shown). Such a response of neuronal cells to major physiological disturbances had already been established from a differential screening experiment performed with avian neuroretinal cells (Michel et al., 1989).Actually, the most abundant mRNA synthesized in these cells after in vitro infection by the Rous sarcoma retrovirus (RSV) carrying the v-src oncogene is the avian counterpart of SGP-2, termed T64 (Michel et al., 1989).This gene expression is the consequence of an increase of its transcription rate and experiments are now in progress to determine whether the induction of this gene is directly linked to the intense cell death associated with RSV infection. Finally, a fourth differential screening experiment, aimed a t the search for genes involved in programmed cell death has identified a SGP-2 homologue as a gene induced in rat ventral prostate regressing after castration (Bandyk et al., 1990).Taken together, these data strongly suggest that SGP-2 is a key gene associated with cell response to various biological stressors.
CONCLUDING REMARKS Cell death belongs to the critical biological processes necessary to both the development and the survival of pluricellular organisms. The cell death genetic program remains active in most tissues of adult organisms but constitutes an important aspect of morphogenesis. Autolysis of embryonic temporary structures, often reminiscent of ancestral schemes, permits edification of the definitive organization. It is also postulated that the development of the nervous system by “epigenesis” is based upon the spontaneous regression of synapses (Changeux et al., 1973). The massive neuronal death occurring during development may represent selective losses of neurons having erroneous or in adapted projections (Oppenheim, 1991). Among the complex and aleatory connections of the brain at birth, a few networks should be stabilized by their activation, while others are definitively lost, for lack of adequate stimuli occurring during a critical period of apprenticeship. Neuronal cell death should thus allow conservation of only those neuronal circuits adapted to environment. It is conceivable that an abnormal reactivation of genes involved in the cell death program among the selected neurons leads to degenerative processes and diseases. Such a reactivation can result either from an exogenous stimulus like a viral infection, or from an endogenous, yet undetermined defect. The elucidation of mechanisms which regulate the SGP-2 gene expression could allow for the identification of genetic deregulations occurring in neural tissues under pathophysiological conditions. REFERENCES Akiyama, H., Kawamata, T., Dedhar, S., and McGeer, P.L. (1991) Immunohistochemical localization of vitronectin, its receptor and beta-3 integrin in Alzheimer brain tissue. J. Neuroimmunol., 32:1928. Bandyk, M.G., Sawczuck, I S . , Olsson, C.A., Katz, A.E., and Buttyan, R. (1990) Characterization of the products of a gene expressed during androgen-programmed cell death and their potential use as a marker of urogenital injury. J . Urol., 143:407413. Bettuzzi, S., Hiipakka, R.A., Gilna, P., and Liao, S. (1989) Identification of an androgen-repressed mRNA in rat ventral prostate as coding for sulphated glycoprotein 2 by cDNA cloning and sequence analysis. Biochem. J., 257:293-296. Bettuzzi, S., Troiano, L., Davalli, P., Tropea, F., Ingletti, M.C., Grassilli, E., Monti, D., Corti, A,, and Franceschi, C. (1991) In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death. Biochem. Biophys. Res. Commun., 175:810-815. Boyle, J.K., Zoellner, C.D., Anderson, L.J., Kosick, L.M., Pitas, R.E., Weismaber. K.H., Hui. D.Y.. Mahlev. R.W.. Gebicke-Haeter. P.J.. Ignagus, M.J., and Shooter, E.M. (1989)A role for apolipoprotein E; apolipoprotein A-1 and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve. J . Clin. Invest., 83:1015-1031. Buttyan, R., Olsson, C.A., Pintar, J., Chang, C., Bandyk, M., Ng, P.-U., and Sawczuk, I.S. (1989) Induction of the TRPM-2 gene in cells undergoing programmed death. Mol. Cell. Biol., 9:3473-3481. Chabot, J.-G., Danik, M., Mercier, C., Benabid, A.-L., Quirion, R., and Suh, M. (1991) Expression of sulfated glycoprotein-2 (SGP-2) in human gliomas, epileptic foci and rat brain tissues. Presented a t the Twenty-first Annual Meeting of the Society for Neuroscience, 271.11,692. Changeux, J.P., Courrege, P., and Danchin, A. (1973) A theory of the epigenesis of neuronal networks by selective stabilization of synapses. Proc. Natl. Acad. Sci. USA, 70:2974-2978.
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