AKT Signaling Mediates IGF-I Survival Actions on Otic Neural Progenitors Maria R. Aburto 1,2 , Marta Magarin ˜ os 1,2,3 , Yolanda Leon 1,3 , Isabel Varela-Nieto 1,2 * . , Hortensia Sanchez- Calderon 1,2. 1 Instituto de Investigaciones Biomedicas ‘‘Alberto Sols’’, CSIC-UAM, Madrid, Spain, 2 CIBERER, Unit 761, ISCIII, Madrid, Spain, 3 Departamento de Biologia, Universidad Autonoma de Madrid, Madrid, Spain Abstract Background: Otic neurons and sensory cells derive from common progenitors whose transition into mature cells requires the coordination of cell survival, proliferation and differentiation programmes. Neurotrophic support and survival of post- mitotic otic neurons have been intensively studied, but the bases underlying the regulation of programmed cell death in immature proliferative otic neuroblasts remains poorly understood. The protein kinase AKT acts as a node, playing a critical role in controlling cell survival and cell cycle progression. AKT is activated by trophic factors, including insulin-like growth factor I (IGF-I), through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by phosphatidylinositol 3-kinase (PI3K). Here we have investigated the role of IGF-dependent activation of the PI3K-AKT pathway in maintenance of otic neuroblasts. Methodology/Principal Findings: By using a combination of organotypic cultures of chicken (Gallus gallus) otic vesicles and acoustic-vestibular ganglia, Western blotting, immunohistochemistry and in situ hybridization, we show that IGF-I-activation of AKT protects neural progenitors from programmed cell death. IGF-I maintains otic neuroblasts in an undifferentiated and proliferative state, which is characterised by the upregulation of the forkhead box M1 (FoxM1) transcription factor. By contrast, our results indicate that post-mitotic p27 Kip -positive neurons become IGF-I independent as they extend their neuronal processes. Neurons gradually reduce their expression of the Igf1r, while they increase that of the neurotrophin receptor, TrkC. Conclusions/Significance: Proliferative otic neuroblasts are dependent on the activation of the PI3K-AKT pathway by IGF-I for survival during the otic neuronal progenitor phase of early inner ear development. Citation: Aburto MR, Magarin ˜ os M, Leon Y, Varela-Nieto I, Sanchez-Calderon H (2012) AKT Signaling Mediates IGF-I Survival Actions on Otic Neural Progenitors. PLoS ONE 7(1): e30790. doi:10.1371/journal.pone.0030790 Editor: Rafael Linden, Universidade Federal do Rio de Janeiro, Brazil Received July 1, 2011; Accepted December 29, 2011; Published January 23, 2012 Copyright: ß 2012 Aburto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the ‘‘Ministerio de Ciencia e Innovacion’’ (SAF2008-00470; http://www.micinn.es/) and the ‘‘Fundacion Mutua Madrilen ˜ a’’ (http://www.fundacionmutua.es/) to IVN. The funders had no role in the study design, data collection and analysis, decision to publish or the preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ivarela@iib.uam.es . These authors contributed equally to this work. Introduction The vertebrate inner ear is a complex sensory organ responsible for the senses of sound and balance. The inner ear derives from an ectodermal placode that invaginates to form the otic vesicle, or otocyst. This structure contains the information required to carry out an autonomous program of development resulting in generation of most of the cells that form the adult inner ear. Inner ear development requires the transition of otic progenitors through states of active cell proliferation, cell fate specification, synchronized cell cycle exit and differentiation to generate the distinctly fated cell populations within the highly ordered mosaic of the organ of Corti in mammals and the basilar papilla in birds [1], [2], [3]. Neurons of the acoustic and vestibular ganglia have a common origin in the otic placode. Neuronal progenitors are specified in the neurogenic otic epithelia, from where they migrate to form the acoustic-vestibular ganglion (AVG) [2]. The AVG later develops into the acoustic and vestibular ganglia that connect the sensory epithelia to the brain through the VIII cranial nerve [4]. Programmed cell death contributes to otic development and neurogenesis by controlling neuroblast cell number [5], [6], [7]. Neurodevelopmental programmed cell death occurs either in proliferating progenitors or upon loss of neurotrophins [8]. The postmitotic dependence of otic neurons on neurotrophins has been the subject of intense study [9], [10], [11], but the regulation of cell death during the expansion period of proliferative otic neuroblasts remains poorly understood. IGF-I deficiency is associated with severe cochlear defects and human sensorineural deafness (http://www.ncbi.nlm.nih.gov/ omim/608747), [12], [13], [14], [15]. In the chicken embryo, IGF-I is required for the early morphogenesis and survival of otic vesicle epithelial cells [16], [17], as well as for AVG neurogenesis [18]. Binding of IGF-I to high affinity receptors, IGF1R, activates two main downstream signalling pathways, namely the RAF- MEK-ERK phosphorylation cascade and the phosphatidylinositol 3 kinase-AKT (PI3K-AKT) pathway [7], [19], [20]. Igf1 2/2 null PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30790