Seizure activity causes a rapid increase in sulfated glycoprotein-2 messenger RNA in the adult but not the neonatal rat brain

Neuroscienee Letters, 153 (1993) 17~0 © 1993ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940193/$06.00

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NSL 09414

Seizure activity causes a rapid increase in sulfated glycoprotein-2 messenger R N A in the adult but not the neonatal rat brain Steven S. Schreiber a, G e o r g e s T o c c o b, I m a d N a j m b a n d M i c h e l B a u d r y b aDepartment of Neurology, University of Southern California, School of Medicine, Los Angeles, CA 90033 (USA) and bNeurosciencesProgram, University of Southern California, University Park Campus, Los Angeles, CA (USA)

(Received 14 August 1992;Revised version received 12 January 1993;Accepted 12 January 1993) Key words. SGP-2; Hippocampus; Seizure;Degeneration;Gene expression; In situ hybridization

The present study investigated the changes in sulfated glycoprotein-2(SGP-2) messenger RNA at various times following kainic acid-induced seizure onset in adult and neonatal rat brain. Double labellingusing immunostaining of the astrocyte-specificglial fibrillaryacidic protein indicated that SGP-2expressionwas rapidly and transientlyincreasedin granule cells of the dentate gyrus up to 8 hours after seizureonset. Thereafter,and up to 7 days followingseizure onset, the majority of cells exhibitingincreased SGP-2 expressionwere astrocytes located in the molecular layer of the dentate gyrus and in the alveus, as well as in regions adjacent to CA3 and CA1 pyramidal cells. No increase in SGP-2 mRNA was detected in pyramidal neurons selectivelydamaged by KA. In addition, increased expression of SGP-2 following KA administration was not observed in neonatal rat hippocampusprior to postnatal day 21. The results argue against a role for SGP-2 in KA-inducedneuronal death and demonstrate a surprisingly rapid increase in astroglial gene expressionfollowingseizure activity,thus supporting a role for SGP-2 in synaptic plasticity.

A growing body of evidence suggests that the mechanisms leading to programmed cell death involve gene transcription and protein synthesis [2, 3, 21]. In this regard, investigations have focused on sulfated glycoprotein-2 (SGP-2), a 70 kDa protein constitutively expressed and secreted by Sertoli cells in the rat testis [5]. In the rat, a close homologue of SGP-2, testosterone-repressed prostate message-2 (TRPM-2), is induced in the regressing prostate gland following orchiectomy [3, 10]. Furthermore, a human homologue of SGP-2 has been isolated from hippocampi of patients with Alzheimer's disease [13]. These results have led many to conclude that increased SGP-2 expression may be a marker of cell death. However, a comparison of the cells expressing SGP-2 and those exhibiting cell death during central nervous system (CNS) development [8] suggests that increased SGP-2 expression does not accompany all types of degenerative processes. In the adult rat brain, SGP-2 expression is modulated under conditions associated with damage and synaptic remodeling, such as after deafferentation, hormonal manipulation and excitotoxic

Correspondence: S.S. Schreiber, Department of Neurology, University of Southern California, School of Medicine, Los Angeles, CA 90033, USA. Fax: (1) 213-225-2369.

injury suggesting that SGP-2 plays a role in CNS plasticity [6, 11-13, 17]. To further delineate the role of SGP-2 expression in the CNS, we examined the changes in SGP-2 m R N A following systemic administration of the excitatory neurotoxin, kainic acid (KA), which results in recurrent seizures followed by loss of neurons in selectively vulnerable regions of the adult rat limbic system [1]. In contrast, animals less than 17 days old do not exhibit KA-induced neuronal loss despite prolonged seizure activity [15]. This age-related and site-specific neuronal vulnerability offered a unique opportunity to more precisely clarify the role of SGP-2 expression in neurodegeneration. In the present report, we used the techniques of in situ hybridization and immunocytochemistry to localize regional changes in SGP-2 m R N A in the neonatal and adult rat brain at various times following the onset of KA-induced seizures. Sprague-Dawley rats between 4 and 25 days old, and 2 month old adults, were given subcutaneous K A at a dose which resulted in maximal sub-lethal seizure activity (doses ranging from 1 mg/kg to 10 mg/kg). Control animals received either saline injection or were untreated. The animals were sacrificed by decapitation at various times after seizure onset. Frozen brain sections, prepared as previously de-

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A

B

C

D

E

F

Fig. I. Regional distribution of SGP-2 mRNA at differenttimes followingseizure onset in adult rats. Film autoradiographs obtained after in situ hybridizationwith a 35S-labeledSGP-2 cRNA probe on coronal half-brain sections (in duplicate) from a control rat (A), KA-treated rat sacrificed 1 h (B), 4 h (C), 8 h (D), 4 days (E) and 7 days (F) after seizure onset. The large and small arrowheads denote the granule cell and molecularlayers of the dentate gyrus, respectively.Similar results were obtained in at least 5 animals per group, 6 sections per animal. scribed [18], were fixed in 4% paraformaldehyde, washed several times in phosphate-buffered saline (PBS), and treated with 3% hydrogen peroxide in methanol. Following a brief PBS rinse, the primary antibody, i.e. a mouse anti-human glial fibrillary acidic protein (GFAP, Boehringer Mannheim), was applied for 1 h. Following 3 brief PBS rinses the sections were incubated with goat anti-mouse IgG (Vectastain, Vector Laboratories) for 30 rain, washed in PBS, treated with an avidin-biotin complex (Vectastain) as per the manufacturer's protocol, washed in PBS and stained with diaminobenzidine (DAB, Sigma). All solutions were made with diethylpyrocarbonate (DEPC)-treated water. Control sections were subjected to identical conditions without the primary antibody. A 3SS-labeled antisense SGP-2 c R N A probe was trancribed from a rat c D N A template [5]. In situ hybridization, film and emulsion autoradiography were performed as previously described [18]. In some experiments, immunocytochemisty was performed prior to in situ hybridization for double labelling of G F A P protein and SGP-2 mRNA. Following staining with DAB, the sections were rinsed in DEPC-treated water, transferred to 0.1 M triethanolamine and further processed for in situ hybridization. Sections were dipped in Kodak NTB2 emulsion and were exposed at 4°C for 3-5 days; they were developed in D-19 and then counterstained with cresyl violet. In situ hybridization with a sense c R N A probe revealed a low level of non-specific background signal (data not shown). In control adult animals, low basal levels of SGP-2

m R N A were observed in most brain structures at the level of the dorsal hippocampus (Fig. 1A). In contrast, high constitutive levels of expression were observed in the choroid plexus (Fig. 1A,B). One hour following the onset of KA-induced seizures a rapid increase in SGP-2 m R N A was observed in the granule cell layer of the dentate gyrus (n = 6, Fig. 1B). Peak levels of SGP-2 m R N A in the granule cell layer were reached by 8 h following the onset of seizure activity (n = 5, Fig. 1D). In addition, 4 h following seizure onset an increase in SGP-2 m R N A was evident in the outer molecular layer of the dentate gyrus adjacent to the hippocampal fissure, in the stratum lacunosum moleculare of field CA1 (n = 8, Fig. 1C), and in the alveus. These changes gradually increased and were maintained for up to 1 week following seizure onset (n = 10, Fig. 1E,F). These results were corroborated by combined immunocytochemistry/in situ hybridization to identify the cellular localization of the changes in SGP-2 m R N A (Fig. 2). Under basal conditions relatively little SGP-2 m R N A was present in either GFAP-immunopositive or immunonegative cells. In the hippocampus, low amounts of SGP-2 m R N A were seen in both granule and pyramidal neurons, as well as astrocytes (Fig. 2A). Between 1 and 8 h after seizure onset SGP-2 m R N A progressively increased in the granule cells of the dentate gyrus (Fig. 2B,C). By 16 h, the expression of SGP-2 in dentate gyrus granule cells approached baseline levels (n = 9, Fig. 2D). In contrast, GFAP-immunopositive cells with increased amounts of SGP-2 m R N A were evident around the hippocampal fissure, both in the outer molecular layer and

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Fig. 2. Double-labellingof GFAP protein and SGP-2mRNA at differenttimes followingseizure onset in adult rats. Bright-fieldphotomicrographs demonstrating the localizationof GFAP protein and SGP-2mRNA within the granule cell layer of the dentate gyrus of a control rat (A), 1 h (B), 8 h (C) and 16 h (D) followingseizure onset. Arrowheads denote the upper and lower borders of the granule cell layer. Note the increasein grains over granule cellsin B and C. In D, increased SGP-2expressionis evidentin cellsalong the lowerborder of the granule celllayer as well as the hilus. Black arrow points to GFAP-immunoreactivecell abundant in SGP-2mRNA. Bar = 50/lm. in the stratum lacunosum moleculare of CA1 by 4 h, and in the hilus by 12-16 h (Fig. 2D). By 16 h the majority of astrocytes within the hilus and molecular layer exhibited increased SGP-2 expression (Fig. 2D). This astrocytic response was maintained for a prolonged period as, at 4 and 7 days following seizure onset, an abundance of hypertrophic-appearing astrocytes containing elevated levels of SGP-2 m R N A were observed particularly in the hilar and CA3 regions. No increase in SGP-2 m R N A was detected in damaged GFAP-immunonegative cells, located in the CA1 and CA3 pyramidal cell layers. In rats less than 3 weeks old there was no increase in SGP-2 m R N A expression following KA-induced seizures (n = 28, data not shown). However, at postnatal day 21, increased SGP-2 m R N A was observed in the granule cell layer of the dentate gyrus 1 hour after seizure onset (n = 4, not shown). By 16 h, double-labelling experiments revealed that, as in adult animals, the majority of cells exhibiting increased levels of SGP-2 m R N A were present in regions surrounding the hippocampal fissure and were GFAP-immunoreactive (data not shown). The present results indicate that KA-induced seizure

activity produces a very rapid increase in SGP-2 m R N A within heterogeneous cell populations of the hippocampus. In granule cells, SGP-2 message increases between 1 and 8 h after seizure onset and returns toward basal levels by 16 h. At around 4 h, astrocytes located within the outer molecular layer and the stratum lacunosum moleculare also begin to exhibit increased amounts of SGP-2 mRNA. In contrast to granule cells, the increased expression of SGP-2 in astrocytes persists for up to 7 days after seizures. Previous reports have demonstrated biochemical and structural changes in astrocytes following seizure activity [4, 15, 20]. Elevated levels of G F A P m R N A have also been observed in hippocampal astrocytes 1 day after a single seizure [19]. In comparison with these studies, our results demonstrate that modulation of astroglial gene expression can occur within a few hours of seizure onset. The relatively rapid induction of SGP-2 in astrocytes may possibly be associated with an increase in the expression of the cytoskeletal protein GFAP, since it precedes the astrocytic hypertrophy and proliferation that occur at later times as a result of KA toxicity. Our results also indicate that there is no direct correla-

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tion between the expression of SGP-2, a gene putatively involved in some forms of programmed cell death, and KA-induced neurodegeneration. Specifically, hippocampal neurons that are susceptible to KA toxicity and in many cases obviously damaged, i.e. CA1 and CA3 pyramidal cells, did not exhibit any increase in SGP-2 expression up to 1 week after seizure onset. In contrast, granule cells, i.e. neurons that survive KA treatment, showed increased expression of SGP-2 m R N A relatively soon after seizure onset. These results are at variance with those of May et al. who reported increased SGP-2 immunoreactivity within pyramidal neurons 14 days after the intraventricular administration of KA [13]. In that study, however, the identity of the cells expressing SGP-2 was not firmly established. Nonetheless, our results do not rule out the possibility that SGP-2 protein is secreted by reactive astrocytes and taken up by damaged hippocampal neurons, as has previously been suggested

[12, 17]. The increase in astrocytic SGP-2 expression following KA-induced seizure activity is consistent with similar findings following hormonal manipulation and deafferentation [6, 11-13, 17]. Our results, therefore, lend further support for the importance of SGP-2 in the glial response to CNS injury. Although a specific role for SGP-2 remains elusive, current evidence suggests that it is a multifunctional protein [8, 14]. In particular, sequence similarity between SGP-2 and apolipoprotein J [7], complement cytolysis inhibitor [9] and adrenal glycoprotein III [16] has suggested roles in lipid transport, immune function and extracellular secretion, respectively [8]. These functions could conceivably be important in the CNS response to injury, and, as such, may be one way that vital glial-neuronal interactions occur under adverse conditions. This work was supported by Grants NS 01337 to S.S.S. and NS 18427 from NINDS to M.B. The authors thank Pierre Risold for assisting in the analysis of the double-labeled results. 1 Ben-Ari, Y., Limbic seizure and brain damage produced by kainic

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