Developmental Brain Research, 67 (1992) 205-210 Elsevier
205
BRESD 51453
Developmental expression of the proenkephalin and prosomatostatin genes in cultured cortical and cerebellar astrocytes Hisaharu Shinoda, Ann M. Marini and Joan P. Schwartz Clinical Neuroscience Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, (U.S.A.) (Accepted 29 January 1992) Key words: Astrocyte; Enkephalin; Somatostatin; Cortex; Cerebellum; Trophic
Astrocytes were prepared from rats of 4 ages, embryonic day 20, postnatal days 3 and 8, and adult, in order to study the developmental time course of expression of enkephalin and somatostatin (SS). Glial fibrillary acidic protein (GFAP) content was constant in both cortical and cerebellar astrocytes prepared from all ages. SS mRNA and peptide decreased over this developmental time course in cerebellar astrocytes; the time course of changes in SS mRNA paralleled that for rat cerebellum. Proenkephalin (PE) mRNA increased about 3-fold in eerebeUar astrocytes from embryonic day 20 to adult but remained constant in cortical astrocytes; in contrast, PE mRNA showed a 10- to 12-fold increase in rat cerebellum and cortex developmentally. For both cerebella' and cortical astrocytes, free met-enkephalin decreased from embryonic day 20 to adult, whereas total met-enkephalin (measured following trypsin-earboxypeptidase B digestion of the extracts) increased. These results suggest (1) that there is a developmental regulation of the expression of both enkephalin and SS peptides in astrocytes, and (2) that the regulation occurs at the level of transcription for SS but at the level of precursor processing for PE. Possible trophic functions for astrocyte-derived peptides early in CNS development are discussed. INTRODUCTION
The discovery that astrocytes could express some of the neuropeptide genes, including proenkephalin (PE) and prosomatostatin (SS), has occurred very recently. An initial report 37 that met-enkephalin-like immunoreactivity could be detected by immunohistochemistry in cerebellar gila was directly confirmed using in situ hybridization to localize PE mRNA to cerebellar astrocytes, as well as neurons, in vivo 26 and in cerebellar explants7. PE mRNA has now been identified in cultured astrocytes from many brain regions, including striatum, cerebellum, cortex, hippocampus, and hypothalamus 7'~4' 21,25,26,31, suggesting that virtually all type 1 astrocytes can express the PE gene. Not only PE but also sraaller enkephalin-containing peptides and free enkephalins have been identified in astrocyte extracts ~4'25, suggesting that astrocytes are capable of processing neuropeptide precursors. In vivo results have shown that with increasing age, processing of PE declines 26, raising the question of whether that is also true in astrocytes. In contrast, somatostatin gene expression occurs in cerebellar astrocytes but not those from cortex or striatum25; little is known about developmental expression or processing in astroeytes. Both the enkephalin and somatostatin peptides are
thought to function as neuropeptide transmitters when released by specific groups of neurons, but their functions when synthesized by astrocytes are essentially unknown. In this p a p e r , we have analyzed the developmental time course of expression of the P E and SS genes in astrocytes: the results show that the peptides are expressed early in development, suggesting the possibility that they m a y play trophic roles in C N S development. MATERIALS AND METHODS Materials Sources were as follows: tissue culture reagents, Gibco; fetal bovine serum, Whittaker-MA Bioproducts hybridoma serum; TPCKtrypsin and carboxypeptidase B, Sigma Chemical Co.; [Iz~Ityr~]met-enkephalin (2200 Ci/mmol ), [125I-tyrl]somatostatin-14 (2200 Ci/mmol), New England Nuclear; [1251]donkey anti-rabbit IgG (10 #Ci/~tg), Amersham. Cell culture Cells were prepared from a litter of embryonic day 20 fetuses or postnatal day 3 or 8 rat pups, or from 3-4 adult (10- to 15-weekold) Sprague-Dawley rats, and cultured in 75 cm 2 flasks as described 22, in Dulbecco's Modified Eagle Medium, containing 10% fetal bovine serum. When cultures became confluent, the flasks were shaken overnight and the medium changed the next day. This was repeated twice, after which the cells were trypsinized, replated, and treated with 10 ~¢M cytosine arabinoside for 24 h. When the flasks became confluent again (7-14 days), the c:lis were subcuitured: all experiments were carried out following :his subculturing, at which point 99% of the cells were GFAP-positive type 1 astro-
Correspondence: J.P. Schwartz, CNB, NINDS, NIH, Building 9, Room 1Wl15, Bethesda, MD 20892, U.S.A. Fax: (1) (301) 402-0117.
206 cytes"". In some experiments, cells were incubated in serum-free medium for 24 h prior to analysis: this did not affect the content of peptides or GFAP.
~-, . c_
o t,..-
RNA analysis
RNA was isolated and analyzed by slot blot hybridization as previously described TM- The RNA was prepared from individual 100-ram Petri dishes, with 4 dishes per treatment group, and each RNA sample was spotted onto the filter in 3 dilutions. PE mRNA ~'* and SS mRNA ~ were measured using specific rat eDNA probes and quantitated relative to a constitutively expressed nonchanging RNA by dehybridizing the blots and hybridizing with a cDNA probe for tlae class I clone IBI5 (cyclophilin) ts. which corrects for mRNA recovery and/or degradation. The area of the peak for the PE or SS mRNA band. obtained by densitometric scan of the autoradiogram. was divided by the area of the peak for IBI5. and defined as 1 unit of mRNA.
Met.enkephalin radioimmunoassay Radioimmunoassay (RIA) of ME (met-enkephalin-like immunoreactivity) was carried out in a total volume of 0.3 ml in RIA buffer (100 mM Tris-HCI pH 8, 0.1% bovine serum albumin, 50 mM NaCI, 0.1% fl.mercaptoethanol) containing [t:'~l]met-enkephalin (10,000 cpm per tube) and antiserum ~ at a final dilution of 1:30, 000. The assay was sensitive to 1.0 pg ME. Relative to ME (100%), the cross-reactivities were as follows: LeuS-enkephalin (11.6%): Mer%enkephalin-Arg6 (2.8%); MetS-enkephalin-Arg~-Phe ~ (1.3%) ; MerLenkephalin.Arg~'.GlyLLeus (
205
BRESD 51453
Developmental expression of the proenkephalin and prosomatostatin genes in cultured cortical and cerebellar astrocytes Hisaharu Shinoda, Ann M. Marini and Joan P. Schwartz Clinical Neuroscience Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, (U.S.A.) (Accepted 29 January 1992) Key words: Astrocyte; Enkephalin; Somatostatin; Cortex; Cerebellum; Trophic
Astrocytes were prepared from rats of 4 ages, embryonic day 20, postnatal days 3 and 8, and adult, in order to study the developmental time course of expression of enkephalin and somatostatin (SS). Glial fibrillary acidic protein (GFAP) content was constant in both cortical and cerebellar astrocytes prepared from all ages. SS mRNA and peptide decreased over this developmental time course in cerebellar astrocytes; the time course of changes in SS mRNA paralleled that for rat cerebellum. Proenkephalin (PE) mRNA increased about 3-fold in eerebeUar astrocytes from embryonic day 20 to adult but remained constant in cortical astrocytes; in contrast, PE mRNA showed a 10- to 12-fold increase in rat cerebellum and cortex developmentally. For both cerebella' and cortical astrocytes, free met-enkephalin decreased from embryonic day 20 to adult, whereas total met-enkephalin (measured following trypsin-earboxypeptidase B digestion of the extracts) increased. These results suggest (1) that there is a developmental regulation of the expression of both enkephalin and SS peptides in astrocytes, and (2) that the regulation occurs at the level of transcription for SS but at the level of precursor processing for PE. Possible trophic functions for astrocyte-derived peptides early in CNS development are discussed. INTRODUCTION
The discovery that astrocytes could express some of the neuropeptide genes, including proenkephalin (PE) and prosomatostatin (SS), has occurred very recently. An initial report 37 that met-enkephalin-like immunoreactivity could be detected by immunohistochemistry in cerebellar gila was directly confirmed using in situ hybridization to localize PE mRNA to cerebellar astrocytes, as well as neurons, in vivo 26 and in cerebellar explants7. PE mRNA has now been identified in cultured astrocytes from many brain regions, including striatum, cerebellum, cortex, hippocampus, and hypothalamus 7'~4' 21,25,26,31, suggesting that virtually all type 1 astrocytes can express the PE gene. Not only PE but also sraaller enkephalin-containing peptides and free enkephalins have been identified in astrocyte extracts ~4'25, suggesting that astrocytes are capable of processing neuropeptide precursors. In vivo results have shown that with increasing age, processing of PE declines 26, raising the question of whether that is also true in astrocytes. In contrast, somatostatin gene expression occurs in cerebellar astrocytes but not those from cortex or striatum25; little is known about developmental expression or processing in astroeytes. Both the enkephalin and somatostatin peptides are
thought to function as neuropeptide transmitters when released by specific groups of neurons, but their functions when synthesized by astrocytes are essentially unknown. In this p a p e r , we have analyzed the developmental time course of expression of the P E and SS genes in astrocytes: the results show that the peptides are expressed early in development, suggesting the possibility that they m a y play trophic roles in C N S development. MATERIALS AND METHODS Materials Sources were as follows: tissue culture reagents, Gibco; fetal bovine serum, Whittaker-MA Bioproducts hybridoma serum; TPCKtrypsin and carboxypeptidase B, Sigma Chemical Co.; [Iz~Ityr~]met-enkephalin (2200 Ci/mmol ), [125I-tyrl]somatostatin-14 (2200 Ci/mmol), New England Nuclear; [1251]donkey anti-rabbit IgG (10 #Ci/~tg), Amersham. Cell culture Cells were prepared from a litter of embryonic day 20 fetuses or postnatal day 3 or 8 rat pups, or from 3-4 adult (10- to 15-weekold) Sprague-Dawley rats, and cultured in 75 cm 2 flasks as described 22, in Dulbecco's Modified Eagle Medium, containing 10% fetal bovine serum. When cultures became confluent, the flasks were shaken overnight and the medium changed the next day. This was repeated twice, after which the cells were trypsinized, replated, and treated with 10 ~¢M cytosine arabinoside for 24 h. When the flasks became confluent again (7-14 days), the c:lis were subcuitured: all experiments were carried out following :his subculturing, at which point 99% of the cells were GFAP-positive type 1 astro-
Correspondence: J.P. Schwartz, CNB, NINDS, NIH, Building 9, Room 1Wl15, Bethesda, MD 20892, U.S.A. Fax: (1) (301) 402-0117.
206 cytes"". In some experiments, cells were incubated in serum-free medium for 24 h prior to analysis: this did not affect the content of peptides or GFAP.
~-, . c_
o t,..-
RNA analysis
RNA was isolated and analyzed by slot blot hybridization as previously described TM- The RNA was prepared from individual 100-ram Petri dishes, with 4 dishes per treatment group, and each RNA sample was spotted onto the filter in 3 dilutions. PE mRNA ~'* and SS mRNA ~ were measured using specific rat eDNA probes and quantitated relative to a constitutively expressed nonchanging RNA by dehybridizing the blots and hybridizing with a cDNA probe for tlae class I clone IBI5 (cyclophilin) ts. which corrects for mRNA recovery and/or degradation. The area of the peak for the PE or SS mRNA band. obtained by densitometric scan of the autoradiogram. was divided by the area of the peak for IBI5. and defined as 1 unit of mRNA.
Met.enkephalin radioimmunoassay Radioimmunoassay (RIA) of ME (met-enkephalin-like immunoreactivity) was carried out in a total volume of 0.3 ml in RIA buffer (100 mM Tris-HCI pH 8, 0.1% bovine serum albumin, 50 mM NaCI, 0.1% fl.mercaptoethanol) containing [t:'~l]met-enkephalin (10,000 cpm per tube) and antiserum ~ at a final dilution of 1:30, 000. The assay was sensitive to 1.0 pg ME. Relative to ME (100%), the cross-reactivities were as follows: LeuS-enkephalin (11.6%): Mer%enkephalin-Arg6 (2.8%); MetS-enkephalin-Arg~-Phe ~ (1.3%) ; MerLenkephalin.Arg~'.GlyLLeus (
