Enteric neuroblasts require the phosphatidylinositol 3-kinase/Akt/Forkhead pathway for GDNF-stimulated survival Shanthi Srinivasan, a, T Mallappa Anitha, a Simon Mwangi, a and Robert O. Heuckeroth b,c a Department of Medicine, Division of Digestive Diseases, Emory University, 615 Michael Street, Whitehead Research Building, Suite 246, Atlanta, GA 30322, USA b Department of Pediatrics, Division of Gastroenterology and Nutrition, Washington University School of Medicine, 660 Euclid Avenue, St. Louis, MO 63110, USA c Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 Euclid Avenue, St. Louis, MO 63110, USA Received 25 November 2004; revised 19 January 2005; accepted 7 February 2005 Glial cell line-derived neurotrophic factor (GDNF)/Ret signaling is required for enteric neural crest survival, proliferation, migration and process extension, but signaling pathways that mediate enteric nervous system (ENS) precursor development are poorly understood. We therefore examined GDNF effects on immunoselected ENS precursor survival and neuronal process extension in the presence of phospha- tidylinositol 3-kinase and mitogen-activated protein kinase pathway inhibitors. These studies demonstrated that GDNF promotes ENS precursor survival through phosphatidylinositol-3-kinase. Specifically, GDNF induces phosphorylation of Akt and loss of the Akt substrates FOXO1 and FOXO3a from the nucleus of ENS precursors. Further- more, dominant negative Akt or active FOXO1 constructs promote ENS precursor cell death while a dominant negative FOXO1 construct prevents cell death. In contrast, the MAPK kinase inhibitor PD98059 did not influence ENS precursor survival or neurite extension. These data demonstrate a critical role for PI-3 kinase/Akt/FOXO signaling, but not for MAPK in ENS precursor survival and neurite extension. D 2005 Elsevier Inc. All rights reserved. Introduction The enteric nervous system (ENS) controls intestinal motility, regulates intestinal secretion and blood flow and responds to sensory stimuli from the gut (Costa and Brookes, 1994; Furness, 2000; Kunze and Furness, 1999). This network of neurons and glia is derived from vagal, sacral and upper thoracic neural crest cells (Gariepy, 2001; Gershon, 1997; Newgreen and Young, 2002a,b) that migrate into the gut where they proliferate and then differentiate into a variety of distinct cell types. The gut micro- environment provides signals that critically direct cell migration, proliferation and differentiation by activating cell surface receptors and stimulating intracellular signal transduction pathways within ENS precursors. Defective signaling results in a variety of defects in ENS structure and function (Camilleri, 2001). The most severe problem of ENS development is complete distal intestinal aganglionosis (Hirschsprung’s disease, HSCR), which affects 1:5000 human infants (Parisi and Kapur, 2000; Skinner, 1996). While eight genes are known to cause HSCR in humans (Amiel and Lyonnet, 2001), inactivating mutations of Ret tyrosine kinase (Carrasquillo et al., 2002; Edery et al., 1994; Romeo et al., 1994) account for 50% of familial and 15 – 20% of sporadic HSCR. Ret is a cell surface receptor that is essential for the survival, proliferation, migration and maturation of many neuronal precur- sors. (Airaksinen and Saarma, 2002; Baloh et al., 2000; Schuchardt et al., 1994). Ret À/À mice have total intestinal aganglionosis and defects in the sympathetic, parasympathetic and sensory nervous system. Ret activation requires one of four GFRa co-receptors (GFRa1 – 4) that directly bind to the GDNF family of ligands (GFL; glial cell line-derived neurotrophic factor (GDNF), neu- rturin, artemin and persephin, respectively). Each GFL/GFRa pair has a unique expression pattern that determines its biological function. GDNF/GFRa1/Ret signaling, for example, is essential for the proliferation, survival, migration and maturation of ENS precursors during embryonic development (Barlow et al., 2003; Cacalano et al., 1998; Enomoto et al., 1998; Gianino et al., 2003; Kruger et al., 2003; Moore et al., 1996; Natarajan et al., 2002; Pichel et al., 1996; Sanchez et al., 1996; Taraviras et al., 1999; Young et al., 2001). In contrast, NRTN/GFRa2/Ret signaling is essential for the maintenance of mature enteric neurons and projection of acetylcholinesterase/Substance P-expressing neuronal fibers within the myenteric plexus (Heuckeroth et al., 1999; Rossi et al., 1999). Ret activation stimulates a variety of intracellular signaling pathways including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 (PI-3) kinase pathways (Air- aksinen and Saarma, 2002; Takahashi, 2001). While the PI-3- kinase pathway is critical for avian neural crest precursor 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.02.005 T Corresponding author. E-mail address: ssrini2@emory.edu (S. Srinivasan). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 29 (2005) 107 – 119