Involvement of Fractin in TGF-b-Induced Apoptosis in Oligodendroglial Progenitor Cells RAMONA SCHULZ, 1 TANJA VOGEL, 1 TETSUO MASHIMA, 2 TAKASHI TSURUO, 2,3 AND KERSTIN KRIEGLSTEIN 1,4 * 1 Center of Anatomy, Department of Neuroanatomy, Georg-August-University, Goettingen, Germany 2 Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan 3 Department of Structural Biology, Institute of Molecular and Cellular Biosciences, Tokyo, Japan 4 Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg, Germany KEY WORDS cytoskeleton; Bcl-xl; caspases ABSTRACT Transforming growth factor-b (TGF-b) induces apoptotic cell death during the development of the nervous system. We recently identified that TGF-b induced apoptosis in oli- godendroglial progenitor cells (primary cells as well as oli- godendroglial cell line OLI-neu) is characterized by down- regulation of Bcl-xl. In this report, we now focused on mechanisms that mediate TGF-b dependent Bcl-xl down- regulation in oligodendroglial cells. We showed that the cas- pase-specific cleavage product Fractin is produced in oligo- dendroglial cells during TGF-b-mediated apoptosis, which represents an early event of the cascade. Cleavage of actin into Fractin was dependent on functional actin and cas- pases, and occurred simultaneously with a Fractin-Bcl-xl- interaction. This Fractin-Bcl-xl interaction indicated a connection between Bcl-xl down-regulation and Fractin appearance, since Bcl-xl regulation was also dependent on caspases and functional actin, and an overexpression of Fractin induced a Bcl-xl protein down-regulation. Further analysis of Fractin-Bcl-xl interaction in other culture sys- tems confirmed these data. In conclusion, we show that Fractin is not only an apoptotic marker, but has indeed a functional role in apoptotic signaling in oligodendro- cytes. V V C 2009 Wiley-Liss, Inc. INTRODUCTION Apoptosis plays a major role during organogenesis and morphogenesis during development as well as during tissue homeostasis and in degenerative diseases. During brain development cells are generated in excess and in specific coordinated processes the dispensible and super- fluous cells are eliminated through programmed cell death (Oppenheim, 1991). Ontogenetic cell death has first been shown for many populations of neurons in the peripheral as well as central nervous system (Oppen- heim, 1991), but has also been documented for oligoden- droglial development in vivo (Barres et al., 1992). Newly generated oligodendrocytes (20–50%) are eliminated dur- ing development in vivo (Butts et al., 2008; Casaccia- Bonnefil, 2000), e.g., in the optic nerve (Barres et al., 1992; Raff, 1996) and in the neocortex (Trapp et al., 1997). Specifically, it has been shown that oligodendro- cyte progenitors and newly formed oligodendrocytes are very susceptible to apoptosis (Barres et al., 1992; Butts et al., 2008; Casaccia-Bonnefil, 2000; Schuster and Krieglstein, 2002; Scurlock and Dawson, 1999). Transforming growth factor (TGF)-b-induced apoptosis leads to selective elimination of cells during the develop- ment of the nervous system (Krieglstein et al., 2000). In chick retina, TGF-b mediated apoptosis (Duenker et al., 2001) involves a down-regulation of Bcl-xl protein (Schuster et al., 2002b). Experiments with mouse retina confirm apoptotic functions of TGF-b by regulation of the Bcl-2 protein and the caspase cascade (Duenker et al., 2005). In the murine, oligodendroglial progenitor cell line OLI-neu, an established TGF-b responsive model system (Jung et al., 1995), apoptosis and G1- arrest are accompanied by caspase activation and down- regulation of Bcl-xl following TGF-b1 treatment (Schuster et al., 2002a, 2003). Primary oligodroglial cell cultures, as well as selected cell lines have been shown to serve as suitable models to study molecular signaling of TGF-b in oligodendroglial apoptotic cell death (Marushige and Marushige, 1994; Schuster et al., 2002a; Schuster and Krieglstein, 2002; Yu et al., 2000) as well as other cytokines, growth and trophic factors in oligodendroglial determination by apo- ptosis/survival (Butts et al., 2008; Casaccia-Bonnefil, 2000; D’Souza et al., 1996; Pang et al., 2005; Schuster et al., 2002a, 2003). To affirm apoptotic processes and to distinguish apo- ptotic from necrotic cells, the usage of a specific apopto- sis marker is indispensable. The actin cleavage product Fractin is an established marker for apoptosis in patho- logical cell models, especially in neuronal systems of neurodegeneration (Adamec et al., 2001; Ariano et al., 2005; Rossiter et al., 2000). Cleavage of actin at a highly conserved caspase-cleavage site into 15-kDa tActin and 32-kDa Fractin was identified during development of ap- optosis in tumor cell lines (Chen et al., 1996; Mashima Additional Supporting Information may be found in the online version of this article. Grant sponsor: Deutsche Forschungsgemeinschaft. *Correspondence to: Kerstin Krieglstein, Institute of Anatomy and Cell Biology, University of Freiburg, Albertstrasse 17, 79104 Freiburg, Germany. E-mail: kerstin.krieglstein@anat.uni-freiburg.de Received 9 October 2008; Accepted 20 February 2009 DOI 10.1002/glia.20875 Published online 27 March 2009 in Wiley InterScience (www.interscience. wiley.com). GLIA 57:1619–1629 (2009) V V C 2009 Wiley-Liss, Inc.