TAM Receptors Support Neural Stem Cell Survival, Proliferation and Neuronal Differentiation

December TAM Receptors Support Neural Stem Cell Survival, Proliferation and Neuronal Differentiation Rui Ji 0 1 4 5 Lingbin Meng 1 4 5 Xin Jiang 2 4 5 Naresh Kumar CVM 0 4 5 Jixiang Ding 3 4 5 Qiutang 4 5 Li 0 4 5 Qingxian Lu 0 1 4 5 0 Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine , Louisville, Kentucky, 40202 , United States of America, 1 Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine , Louisville, Kentucky, 40202 , United States of America, 2 Department of Radiation Oncology, The First Hospital of Jilin University , Changchun, 130021, China, 3 Birth Defects Center, University of Louisville School of Dentistry , Louisville, Kentucky, 40202 , United States of America 4 Funding: This work was supported by the National Institutes of Health (R01-EY018830), National Eye Institute (http://www.nei.nih.gov/) to Q. Lu, and NIH (R01-EY019891), National Eye Institute (http:// www.nei.nih.gov/) to Q. Li. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manu- script 5 Editor: Alexander G. Obukhov, Indiana University School of Medicine , United States of America Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (btubulin III+) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF. Differentiation Neurogenesis takes place in adult central nervous system in many vertebrates including human [1]. The multipotent NSCs are located in the in the subgranular zone (SGZ) of hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricles [2]. The glial fibrillary acidic protein (GFAP)-positive radial glia-like cells in those regions are considered as primary stem cells normally remaining in the quiescent state, but have capacity for self-renewal and multipotential differentiation. Once activated, they develop into proliferating intermediate progenitor cells and the undifferentiated neuroblasts that will further maturate into dentate granule cells in hippocampus or interneurons in the olfactory bulb, accordingly [3, 4]. These newly generated neurons are capable to incorporate into the existing neural circuitry and contribute to brain functions [5]. Such adult neurogenesis event is a dynamic process and modulated by a variety of intrinsic and extrinsic factors including growth factors and cell surface receptors, signal transduction molecules, transcriptional factors and cytokine/ chemokines [6]. Interruption of adult neurogenesis leads to impairment in hippocampus-dependent learning and behavior [612]. Many physiological and pathological conditions affect neurogenesis in adult brains. Infection and the invoked inflammation inhibit NSC proliferation and neuronal differentiation [8, 13]. Inflammation has been recognized as a major negative impact on adult neurogenesis [8, 13]. We have recently shown TAM receptors are all expressed by astrocytes and microglia, and they play an important role in regulating brain inflammation. It was found that hyperreactive microglia in the Tyro32/2Axl2/ 2Mertk2/2 triple knockout (TAM TKO) mice produced increased level of proinflammatory cytokines that are detrimental to the neural stem cell proliferation and differentiation [14]. However, detailed comparison of b-tubulin III+ neurons showed a significantly decreased neuronal differentiation from the TKO NSCs than those from the WT NSCs that had been treated with LPS-treated microglia-conditioned medium. In addition, in vivo studies on the LPS-induced inhibition of NSC proliferation and differentiation demonstrated that the adult TKO brains manifested even severer reduction in neurogenesis than the WT brains that had undergone the LPS-induced inflammation [14]. These data imply that TAM receptors might play an intrinsic functional role in NSC proliferation and neuronal differentiation. Tyro3, Axl and Mertk belong to the structurally and functionally closely-related TAM family of receptor tyrosine kinases, expressed on the cell surface and playing divergent functional roles ranging from cell differentiation to cell death [15]. Both Gas6 and Protein S serve as ligands for this family of receptors [1618]. Although originally cloned from many fast growing or transformed cells, TAM receptors are now considered as intrinsic growth trophic factors. They sustain cell growth and survival, support PC12 cell neuronal differentiation upon neuronal growth factor stimulation [19]. Genome-wide analysis of the genes differentially expressed between neuronal progenitor and the differentiated neuronal cells revealed that all three members of the TAM family are expressed in the embryonic cortical neuronal progenitor cells [20]. Mice lacking both Axl and Mertk caused early differentiation and migration of SVZ NSCs [20], and knockout of their common ligand, Gas6, reduced the NSC numbers in the SVZ [21]. These evidences indicate that TAM receptors might play important roles in maintenance of the cortical neuronal progenitor cell identity, in regulation of the NSCs survival, proliferation, and differentiation. In the present study, we demonstrated that the primary NSCs express all three members of the TAM receptors to provide trophic support for themselves to ensure the survival, proliferation and differentiation into immature neurons in vitro. Under normal cultur (...truncated)