PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARs) AND RELATED TRANSCRIPTION FACTORS IN DIFFERENTIATING ASTROCYTE CULTURES L. CRISTIANO, a A. CIMINI, a S. MORENO, b A. M. RAGNELLI a AND M. PAOLA CERÙ a * a Department of Basic and Applied Biology, University of L’Aquila, Via Vetoio 10, Coppito, L’Aquila, 67010 Italy b Department of Biology-LIME, University “Roma Tre,” Viale Marconi 446, Rome, 00146 Italy Abstract—Peroxisome proliferator-activated receptors (PPARs), retinoid X receptors (RXRs), CCAAT/enhancer binding pro- teins (C/EBPs) and -catenin are transcription factors in- volved in cell differentiation. The aim of this work was to investigate the occurrence and variations of these proteins during astrocyte differentiation. Primary cultures of mouse cortical astrocytes were characterized using nestin, A2B5 and glial fibrillary acidic protein (GFAP) as differentiation markers, during a period of 21 days in vitro (DIV). Glycogen and triglyceride accumulation were also studied. At 3 DIV the cultures were mainly constituted by neural progenitor cells, as assessed by their immunofluorescent pattern. At this time PPARs and -catenin were localized to the cytoplasm. Interestingly, some cells contained Oil Red O-positive lipid droplets. Between 7 and 21 DIV, nestin de- creased, while GFAP increased, indicating ongoing astroglial differentiation. -catenin, predominantly nuclear at 7 DIV, later localized to membranes. Redistribution of all three PPAR isotypes from the cytoplasm to the nucleus was ob- served starting from 7 DIV. Between 7 and 14 DIV, C/EBP, PPAR, RXR and glycogen content increased. Between 14 and 21 DIV, PPAR/ decreased, while PPAR, C/EBP and  and lipid droplet-containing cells increased. At 21 DIV both A2B5/GFAP and A2B5/GFAP cells were predominantly observed, indicating differentiation toward type-1 and type-2 astrocytes, although the presence of GFAP cells demon- strates the persistence of neural precursors in the culture even at this time point. In conclusion, our results, reporting modifications of PPARs, RXRs, C/EBPs and -catenin during culture time, strongly suggest the involvement of these transcription fac- tors in astrocyte differentiation. Specifically, -catenin trans- location from the nucleus to plasma membrane, together with PPAR/ decrease and C/EBP increase, could be related to decreased proliferation at confluence, while PPAR and  and all C/EBPs could participate in differentiation processes, such as glycogenesis and lipidogenesis. © 2005 IBRO. Pub- lished by Elsevier Ltd. All rights reserved. Key words: transcription factors, glycogen, lipid, brain, stem cells, differentiation. Astrocytes play fundamental roles in the physiology of the developing and adult CNS (for reviews see Deitmer, 2001; Hansson and Rönnbäck, 2003). Moreover, they have been suggested to include a population of neural stem cells, thus participating in neural cell replacement (Laywell et al., 2000; Doetsch, 2003; Marshall et al., 2003). Astrocyte differentiation has been thoroughly studied, especially in vitro, and cells along astroglial lineage are well character- ized (for reviews, see Lee et al., 2000; Holland, 2001). Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, that heterodimer- ize with retinoid X receptors (RXRs) to modulate the ex- pression of genes involved in lipid and glucose metabo- lism, cell proliferation and differentiation (for reviews, see Mangelsdorf and Evans, 1995; Escher and Wahli, 2000). The three PPAR isotypes (, / and ) have related but distinct activities. PPAR is predominantly expressed in tissues, such as liver and kidney, characterized by a high rate of lipid catabolism, while PPAR is abundant in adi- pose tissue, being implicated in lipidogenesis and adipo- cyte differentiation (for review, see Lee et al., 2003). As to the function of PPAR/, recent works suggest its involve- ment in several processes, including adipocyte, keratino- cyte, and oligodendrocyte differentiation (Bastie et al., 1999; Saluja et al., 2001; Michalik et al., 2001), and neu- ronal functions and maturation (Kremarik-Bouillaud et al., 2000; Woods et al., 2003; Moreno et al., 2004; Cimini et al., 2005). Interestingly, a role for PPAR/ in the regula- tion of the transcriptional activity of PPAR and  has also been suggested (Shi et al., 2002). We previously demonstrated that rat cortical and cer- ebellar astrocytes in highly purified cultures at confluence express both PPARs and RXRs (Cristiano et al., 2001). We here extend the study to primary cultures of mouse cortical astrocytes at different time points (3–21 days in vitro, DIV), in order to investigate possible variations in the expression and/or localization of the receptors during dif- ferentiation of these cells. To characterize the cultures, we examined known glial differentiation markers (for review, see Lee et al., 2000) and glycogen accumulation, known to reflect a specific function of differentiated astrocytes (Hevor, 1994). The presence of PPAR in our cultures prompted us to also investigate the occurrence of triglyc- eride droplets, as a marker for lipidogenesis (Bastie et al., 1999). *Corresponding author. Tel: +39-0862-433288; fax: +39-0862-433273. E-mail address: mariapaola.ceru@univaq.it (M. Paola Cerù). Abbreviations: BSA, bovine serum albumin; C/EBP, CCAAT/enhancer binding protein; DIV, days in vitro; EDTA, ethylenediaminetetraacetic acid; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; PBS, phosphate buffered saline; PCNA, proliferating cell nuclear antigen; PLL, poly-L-lysine; PPAR, peroxi- some proliferator-activated receptor; RT, room temperature; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TTBS, Tris-buffered saline containing 0.25% Tween-20. Neuroscience 131 (2005) 577–587 0306-4522/05$30.00+0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.11.008 577