REGULATION OF APOLIPOPROTEIN E SECRETION IN RAT PRIMARY HIPPOCAMPAL ASTROCYTE CULTURES A. CEDAZO-MI è NGUEZ, a ; 1 U. HAMKER, b; 1 V. MESKE, b R. W. VEH, b R. HELLWEG, c C. JACOBI, b F. ALBERT, b R. F. COWBURN a * and T. G. OHM b a Karolinska Institutet, NEUROTEC, Section for Experimental Geriatrics, NOVUM, KFC, pl. 4, 141 86 Huddinge, Sweden b Institut fu « r Anatomie, Universita «tsklinikum Charite ¨, D-10098 Berlin, Germany c Psychiatrische Klinik der Freien Universita «t Berlin, Eschenallee 3, 14050 Berlin, Germany AbstractöApolipoprotein E isoforms may have di¡erential e¡ects on a number of pathological processes underlying Alzheimer's disease. Recent studies suggest that the amount, rather than the type, of apolipoprotein E may also be an important determinant for Alzheimer's disease. Therefore, understanding the regulated synthesis of apolipoprotein E is important for determining its role in Alzheimer's disease. We show here that in rat primary hippocampal astrocyte cultures, dibutyryl-cAMP increased apolipoprotein E secre- tion with time in a dose-dependent manner (to 177% at 48 h) and that retinoic acid potentiated this e¡ect (to 298% at 48 h). Dibutyryl-cAMP also gave a rapid, albeit transient, increase of apolipoprotein E mRNA expression (to 200% at 1 h). In contrast, the protein kinase C activator phorbol 12-myristate 13-acetate decreased both apolipoprotein E secretion (to 59% at 48 h) and mRNA expression (to 22% at 1 h). Phorbol 12-myristate 13-acetate also reversed the e¡ects of dibutyryl-cAMP. Apolipoprotein E secretion was also modulated by receptor agonists for the adenylyl cyclase/ cAMP pathway. Isoproterenol (50 nM, a L-adrenoceptor agonist) enhanced, while clonidine (250 nM, an K2-adrenocep- tor agonist) decreased, secreted apolipoprotein E. We also analysed the e¡ects of agonists for the phospholipase C/protein kinase C pathway. Arterenol (1 WM, an K1-adrenoceptor agonist) and serotonin (2.5 WM) enhanced, whereas carbachol (10 WM, an acetylcholine muscarinic receptor agonist) decreased secreted apolipoprotein E. The e¡ects of these non- selective receptor agonists were modest, probably due to e¡ects on di¡erent signalling pathways. Arterenol also poten- tiated the isoproterenol-mediated increase. We also show that phorbol 12-myristate 13-acetate and dibutyryl-cAMP have opposite e¡ects on nerve growth factor, as compared to apolipoprotein E, secretion, suggesting that the results obtained were unlikely to be due to a general e¡ect on protein synthesis. We conclude that astrocyte apolipoprotein E production can be regulated by factors that a¡ect cAMP intracellular concentration or activate protein kinase C. Alterations in these signalling pathways in Alzheimer's disease brain may have consequences for apolipoprotein E secretion in this disorder. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: Alzheimer's disease, nerve growth factor, cAMP, protein kinase C. Apolipoprotein E (apoE) is an abundant component of plasma lipoproteins (Havel et al., 1980) that plays a key role in lipid transport and cholesterol homeostasis via the low density lipoprotein receptor (Mahley, 1988). In the CNS, apoE is synthesised and secreted by glial cells, especially astrocytes (Boyles et al., 1985; Pitas et al., 1987; Mouchel et al., 1995; Nakai et al., 1996). ApoE synthesis in the peripheral nervous system (PNS) takes place in macrophages and non-myelinating Schwann cells (Boyles et al., 1985). ApoE has been implicated in the maintenance of membranes and in nerve regeneration and growth. ApoE synthesis in both the CNS and PNS is increased dramatically after injury (Ignatius et al., 1986; Boyles et al., 1989). ApoE has also been implicated in several neurodegenerative disorders including Alzheim- er's disease (AD), Lewy body disease and frontal lobe dementia (Hardy et al., 1994; Weisgraber et al., 1994; Stevens et al., 1997). The apoE gene shows polymorphism, with three di¡er- ent alleles, termed O2, O3 and O4. The involvement of apoE in AD is suggested by studies showing that the frequency of the O4 allele is greatly increased in both familial and sporadic AD (Corder et al., 1998). This variation in the apoE genotype can account for up to two decades di¡erence in the mean age of onset of 651 1 These authors contributed equally to the work. *Corresponding author. Tel.: +46-8-585-83884; fax: +46-8-585- 83880. E-mail address : richard.cowburn@neurotec.ki.se (R. F. Cowburn). Abbreviations : a-apoE-AB, anti-apoE antibody ; AD, Alzheimer's disease; ANOVA, analysis of variance; apoE, apolipoprotein E; dibutyryl-cAMP or dBcAMP, N 6 ,2P-O-dibutyryladenosine 3P :5P-cyclic monophosphate ; DIG, digoxigenin; ECL, enhanced chemiluminescence ; ELISA, enzyme-linked immunosorbent assay; FBS, foetal bovine serum; 5-HT, 5-hydroxytryptamine or serotonin; NGF, nerve growth factor; NGS, normal goat serum ; PBS, phosphate-bu¡ered saline ; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate ; PNS, peripheral nervous system ; RA, retinoic acid; SSC, saline sodium citrate ; TBS, Tris-bu¡ered saline ; TBST, TBS with Triton X-100. NSC 5069 10-8-01 www.elsevier.com/locate/neuroscience Neuroscience Vol. 105, No. 3, pp. 651^661, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII:S0306-4522(01)00224-X 0306-4522 / 01 $20.00+0.00