TRIP6透過YAP調控出生後小鼠側腦室-嗅球路逕上神經幹細胞的特性以及醫學上可能的應用

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(1)國立臺灣師範大學生命科學系博士論文. TRIP6 透過 YAP 調控出生後小鼠側腦室-嗅球路逕上神經幹細胞的特性以及醫學上可能的應用. YAP Mediates TRIP6-Promoted Neural Stem Cell Maintenance in the Postnatal Mammalian Subventricular Zone-Olfactory Pathway and Its Applications. 研究生：李明洋 Ming-Yang Li. 指導教授：王慈蔚博士 Tsu-Wei Wang 中華民國 108 年 2 月.

(2) Contents. Abstract in Chinese……………………………………………………..7. Abstract……………………………………………………….…………9. Chapter one: Introduction……………………………………………...10 1. Postnatal neurogenesis………………………………………………10 2. Methods to study postnatal NSCs…………………………………...10 3. The NSC niche and extrinsic factors………………………………...13 4. Intrinsic factors……………………………………………………....14 5. Potential functions of postnatal neurogenesis……………………….15 6. Aims of the thesis……………………………………………………17 7. References……………………………………………………….......18. Chapter two: TRIP6 Regulates Neural Stem Cell Maintenance in the Postnatal Mammalian Subventricular Zone…………….25 1. Abstract……………………………………………………………...26. 2. Introduction………………………………………………………….27 3. Materials and methods………………………………………………30 3.1 Animals 3.2 Plasmids 1.

(3) 3.3 Fixation and sectioning 3.4 RT-PCR 3.5 NIH3T3 cell culture 3.6 Immunoblotting 3.7 Immunofluorescence 3.8 Neurosphere culture and electroporation 3.9 Transfection and differentiation of NSCs 3.10 Luciferase assay 3.11 Statistical analysis. 4. Results……………………………………………………………….38 4.1 TRIP6 is expressed by NSCs in the embryonic and adult mouse brain 4.2 TRIP6 maintains self-renewal and proliferation of NSCs 4.3 TRIP6 inhibits differentiation of postnatal NSCs 4.4 TRIP6 activates the Notch signaling pathway. 5. Discussion…………………………………………………………...46. 6. References…………………………………………………………...50 7. Figures……………………………………………………………….59. Chapter three: YAP Mediates TRIP6-Promoted Neural Stem Cell Maintenance in the Postnatal Mammalian Subventricular 2.

(4) Zone-Olfactory Pathway………………………………72. 1. Abstract……………………………………………………………...73. 2. Introduction………………………………………………………….74 3. Materials and methods………………………………………………77 3.1 Animals 3.2 Fixation and sectioning 3.3 Plasmids 3.4 Primary NSC cultures 3.5 Immunofluorescence 3.6 P19 cell culture 3.7 HEK293T cell culture 3.8 Co-immunoprecipitation 3.9 Western blot analysis 3.10 Luciferase assay 3.11 Image acquisition and statistical analysis. 4. Results……………………………………………………………….86 4.1 YAP is necessary and sufficient for self-renewal in postnatal NSCs 4.2 YAP promotes cell proliferation in postnatal NSCs 4.3 YAP inhibits differentiation in postnatal NSCs 4.4 YAP is important for the maintenance and proliferation of NSCs 4.5 TRIP6 induces the transcription activity of YAP through inhibiting LATS 3.

(5) 4.6 TRIP6 recruits PP1A to inhibit the phosphorylation of LATS1 4.7 TRIP6 promotes stem cell maintenance in postnatal NSCs through YAP. 5. Discussion…………………………………………………………...98. 6. References………………………………………………………….103 7. Figures……………………………………………………………...111. Chapter four: Ginkgolide B Promotes Neuronal Differentiation Through the Wnt/β-catenin Pathway in Neural Stem Cells of the Postnatal Mammalian Subventricular Zone…………...131. 1. Abstract…………………………………………………………….132. 2. Introduction………………………………………………………...133 3. Materials and methods……………………………………………..136 3.1 Chinese herbal medicines 3.2 P19 cell culture 3.3 Primary NSC cultures 3.4 Plasmid for knockdown experiments 3.5 Immunofluorescence 3.6 Nuclear fractionation 4.

(6) 3.7 Western blot 3.8 Luciferase assay 3.9 Image and statistical analysis 4. Results……………………………………………………………...142 4.1 GBE promotes neuronal differentiation in P19 cells. 4.2 GBE promotes neuronal differentiation in postnatal NSCs. 4.3 GB promotes neuronal differentiation in postnatal NSCs. 4.4 GB increases the level of nuclear β-catenin and activates the Wnt pathway. 4.5 GB promotes neuronal differentiation through the Wnt pathway in postnatal NSCs.. 5. Discussion………………………………………………………….150. 6. References………………………………………………………….154 7. Figures……………………………………………………………...162. Chapter five: Discussion……………………………………………...171 1. Summary of the thesis work………………………………………..171 2. TRIP6 and NSC anchoring…………………………………………171 3. Mechanical force and YAP activity in postnatal NSCs…………….172 4. Clinical applications I: YAP and induced pluripotent stem (iPS) cells…………………………………………………………………173 5.

(7) 5. Clinical applications II: TRIP6, YAP and tumor progression……...174 6. Future direction I: TRIP6 and the Notch pathway ………………...175 7. Future direction II: YAP and the Shh pathway………………..........175 8. Future direction III: TRIP6, NSC anchoring and the Notch pathway…………………………………………………………….176 9. Conclusion………………………………………………………….178 10. References………………………………………………………...179. 6.

(8) 摘要出生後的哺乳動物腦中有持續的神經元新生，分別發生在海馬迴齒狀迴，以及側腦室下區到嗅球的路徑上。報導指出內生性的神經幹細胞有治療腦傷或神經退化性疾病的潛力，研究調控神經幹細胞的因子和機制因此有醫學上的應用價值。已知神經幹細胞的特性受到多種因子調控，然而 TRIP6、YAP 以及銀杏萃取物 ginkgolide B 等對於出生後哺乳動物神經幹細胞的影響仍是未知的。TRIP6 的蛋白質結構具有三個 LIM 區位，可以和多種蛋白質進行交互作用而調控細胞增生、存活及移動。我們發現 TRIP6 不表現在會移動的神經母細胞中，而表現在神經幹細胞中。TRIP6 促進神經幹細胞的維持、增生，並且抑制分化。且促進神經幹細胞自我更新的 Notch 訊息傳遞路徑能被 TRIP6 活化。Hippo 訊息傳遞路徑藉由抑制 YAP 來調控細胞增生，控制器官大小。我們發現 TRIP6 透過 PP1A 來抑制 Hippo 訊息傳遞路徑，活化 YAP。並且 TRIP6 透過 YAP 來促進神經幹細胞的維持、增生，以及抑制分化。在神經幹細胞的分化上，我們則發現 ginkgolide B 透過 Wnt 訊息傳遞路徑促進神經元新生。我們這一系列的研究指出 TRIP6 透過 YAP 維持神經幹細胞的特性，而給予 ginkgolide B 則可以促進神經元新生。. 7.

(9) 關鍵詞：神經幹細胞，出生後神經元新生，TRIP6，YAP， ginkgolide B. 8.

(10) Abstract Postnatal neurogenesis in the dentate gyrus and subventricular zone (SVZ)-olfactory bulb pathway in mammals is regulated by extrinsic and intrinsic factors. Since endogenous neural stem cells (NSCs) in the adult brain have potential to treat neurodegenerative disorders, studying mechanisms regulating postnatal NSCs may provide clinical applications. However, the role of TRIP6, YAP and ginkgolide B in postnatal NSCs remain unclear. TRIP6 belongs to zyxin family of LIM proteins, which have been shown to interact with various proteins to regulate cell proliferation, survival and migration. We find that TRIP6 is expressed by adult NSCs in the SVZ but not migrating neuroblasts. TRIP6 is necessary and sufficient for self-renewal and proliferation of adult NSCs, but inhibits their differentiation. We also find that TRIP6 activates the Notch signaling, a pathway required for NSC self-renewal. Previous studies show that the Hippo pathway regulates cell proliferation and organ size through inhibiting YAP. We find that TRIP6 inhibits the Hippo pathway and activates YAP through PP1A. TRIP6 promotes NSC maintenance and proliferation and inhibits neuronal differentiation through YAP. During differentiation of NSCs, we also find that ginkgolide B promotes neurogenesis through the Wnt pathway. These findings show that YAP acts downstream of TRIP6 to promote adult NSC maintenance, whereas ginkgolide B promotes neurogenesis in the postnatal NSCs.. Key words: neural stem cells, postnatal neurogenesis, TRIP6, YAP, ginkgolide B 9.

(11) Chapter one: Introduction 1. Postnatal neurogenesis Postnatal neurogenesis is a process that neural stem cells (NSCs)/progenitor cells generate functional neurons continuously after birth (Ming and Song, 2011). NSCs are spatially restricted in two specific brain regions, the subventricular zone (SVZ) adjacent to the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus (Ming and Song, 2011). In the rodent brain, neuroblasts born in the SVZ migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) and differentiate into OB interneurons (Lledo et al., 2006). However, postnatal neurogenesis in humans is different. There are no newborn neurons detected in the human OB (Ernst et al., 2014). Instead of OB neurogenesis, NSCs in the SVZ provide striatal neurogenesis in the adult human brain (Ernst et al., 2014). NSCs in the SGZ generates dentate granule neurons (Lledo et al., 2006). Newborn neurons in the adult brain are reported to be incorporated into the neural circuits (Lledo et al., 2006). Importantly, postnatal neurogenesis is induced or increased in the injured cerebral cortex, hippocampus or striatum and neurodegenerative diseases such as Alzheimer’s disease (AD) and Huntington’s disease (HD) (Curtis et al., 2003; Darsalia et al., 2005; Jin et al., 2004; Jin et al., 2006; Magnusson et al., 2014). Therefore, studying the mechanisms regulating postnatal neurogenesis may shed light on clinical applications.. 2. Methods to study postnatal NSCs 10.

(12) NSCs have two characteristics, self-renewal to generate NSCs and multipotency to differentiate into neurons, astrocytes or oligodendrocytes (Ming and Song, 2011). These two properties can be studied by the neurosphere culture in vitro (Gil-Perotin et al., 2013). Neurosphere assay provides an in vitro condition to culture NSCs dissected from neurogenic niches (Gil-Perotin et al., 2013). In a serum-free medium and a nonadhesive culture environment, epidermal growth factors (EGF) and fibroblast growth factors (bFGF) promote self-renewal of NSCs to form sphere like structures named neurospheres (Doetsch et al., 1999; GilPerotin et al., 2013). NSCs derived from neurospheres can be cultured in the adherent culture condition to induce their differentiation into neurons, astrocytes and oligodendrocytes (Gritti et al., 2002). Hence, neurosphere culture provides a method to study self-renewal and multi-potency of NSCs. Organotypic brain slice culture provides a three-dimensional system in vitro with the preserved cell architecture close to in vivo to explore the cell-cell interaction of neural cells (Humpel, 2015). The SVZ-OB slice culture with the NSC niche, RMS and OB provides a model to study postnatal neurogenesis (Wang et al., 2005). NSCs/progenitors in the SVZ can be labeled with lipophilic carbocyanine dye DiI, the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) during S phase of the cell cycle or forced expression of green fluorescent protein (GFP) to study the SVZ-OB neurogensis (Wang et al., 2005). Labeled migrating neuroblasts are observed in the RMS and the OB (Wang et al., 2005). Therefore, SVZ-OB slice culture provides a platform to study SVZ-OB neurogenesis 11.

(13) in vitro. To trace the NSC progenies in vivo, diverse approaches have been used. For example, viruses or transgenic mice were designed to label NSCs and migrating neuroblasts with reporter genes (Coskun et al., 2008; Jessberger et al., 2008). Retrovirus infects dividing progenitors and causes long-term expression of GFP in NSC progenies with viral promoters (Jessberger et al., 2008). In transgenic mice, Cre recombinase under the control of NSC-specific promoters such as Nestin or GFAP is used to remove the stop codon in front of a GFP gene under the control of Rosa26 promoter (Coskun et al., 2008). After genetic manipulations, GFP-positive NSCs of the SVZ or SGZ generate GFP-positive neuroblasts to differentiate into OB interneurons or DG granule neurons, respectively (Coskun et al., 2008). Since NSCs/progenitors are proliferating cells, BrdU is incorporated into NSCs/progenitors during S phase of the cell cycle (Lledo et al., 2006). BrdU-positive newborn cells are added in the SVZ-OB pathway or DG (Lledo et al., 2006). Live images of tdTomato-labeled NSCs and their progenies induced by CreloxP system were recorded and categorized based on cell morphologies (Pilz et al., 2018). By using live images, it has been shown that NSCs generate progenitors, which divide several times before differentiating into neurons (Pilz et al., 2018). Live images also show that NSCs generate NSCs, whereas NSCs progenies do not generate NSCs (Pilz et al., 2018). In summary, in vivo and in vitro methods provide strategies to study NSC properties.. 12.

(14) 3. The NSC niche and extrinsic factors The NSC niche is a microenvironment to house NSCs and regulates the maintenance and differentiation of NSCs (Ming and Song, 2011). NSC progenies, mature neurons, astrocytes and blood vessels are major components of the NSC niche (Ming and Song, 2011). Extracellular factors including secreted proteins, growth factors and neurotransmitters contribute to the niche signaling to regulate NSC properties through specific receptors (Ming and Song, 2011; Zhao et al., 2008b). Notch, Shh, Wnt and EGF signaling regulate NSCs maintenance (Ming and Song, 2011). Jagged and Delta are Notch ligand to activate Notch receptors on NSCs (Kovall et al., 2017). Activation of the Notch pathway leads to cleavage of Notch intracellular domain (NICD) by γ-secretase (Kopan and Ilagan, 2009). NICD forms a complex with RBPjκ to promote the expression of Hes1 and Sox2 to inhibit differentiation and promote NSC maintenance (Ehm et al., 2010; Kovall et al., 2017; Ross et al., 2003). EGF transduces its signaling through EGF receptors (EGFR) and promotes the expression of Sox2 (Hu et al., 2010). The secreted protein Shh binds to PATCH1 receptors to activate transcription factor Gli1/2 through Smoothen and promote NSC maintenance (Balordi and Fishell, 2007; Rimkus et al., 2016). Wnt disinhibits β-catenin from GSK3β to promote target gene expression to promote cell proliferation and neurogenesis (Hur and Zhou, 2010; Kuwabara et al., 2009; Qu et al., 2013). Therefore, extrinsic factors regulate NSC properties. During the processes of differentiation, extrinsic factors play important roles in cell fate determination (Lledo et al., 2006). BMP promotes 13.

(15) gliogenesis, whereas Noggin antagonizes gliogenesis and promotes neurogenesis through binding to BMP (Groppe et al., 2002; Lledo et al., 2006). Neuroblast migration is repelled by SLIT1/2 expressed in the septum and SVZ (Lledo et al., 2006). Ephrins and the receptor tyrosine kinase ErbB4 direct neuroblast migration (Lledo et al., 2006). Neurotransmitter GABA decreases the migration speed through GABAA receptor on neuroblasts (Zhao et al., 2008b). Neurotransmitters such as glutamate and GABA from mature neurons in the OB and DG promote survival and maturation of newborn neurons (Lledo et al., 2006; Zhao et al., 2008b). In summary, extrinsic factors regulate NSC maintenance, neuroblast migration, differentiation and survival.. 4. Intrinsic factors Cell-cycle inhibitors and transcription factors are major intrinsic factors to regulate NSC properties (Ming and Song, 2011). NSCs are quiescent cells maintained by cell cycle inhibitors p16, p21, p53 and Notch target gene Hes1, a basic helix-loop-helix transcription factor (Ming and Song, 2011; Sang et al., 2008). The Notch pathway and EGF signaling upregulate the expression of transcription factor SOX2 through RBPjκ to maintain NSCs (Ehm et al., 2010; Hu et al., 2010). SOX2 promotes EGFR expression to form EGF-SOX2-EGFR positive feedback loop (Hu et al., 2010). SOX2 promotes expression of Nestin, an intermediatefilament required for self-renewal (Park et al., 2010; Tanaka et al., 2004). The expression of secreted protein Shh and Wnt are also upregulated by SOX2 and contributes to NSC niche signalings (Favaro et al., 2009). 14.

(16) Downregulation of Sox2 expression by transcription factor E2f3a result in neuronal differentiation, whereas E2f3b promotes Sox2 expression and NSC maintenance (Julian et al., 2013). Cell cycle inhibitor p21 down regulates the expression of Sox2 to avoid senescence and DNA damage in NSCs (Marques-Torrejon et al., 2013). Taken together, intrinsic factors form regulatory network to regulate gene expression and maintain NSCs. The Wnt pathway target genes including Cyclin D1, Id2, NeuroD1, Neurogenin1 and Neurogenin2 regulate cell proliferation and neuronal differentiation (Hirabayashi et al., 2004; Kuwabara et al., 2009; Qu et al., 2013; Rockman et al., 2001; Ross et al., 2003). Cyclin D1 and Id2 promote proliferation of neural progenitor cells (Paolella et al., 2011; Qu et al., 2013; Ross et al., 2003). Id2 responds to mitogens to regulate proliferation and neuronal differentiation (Wang et al., 2001). With mitogens, Id2 disinhibits the transcription factor E2f from Rb proteins (Hindley and Philpott, 2012; Ross et al., 2003; Wang et al., 2001). Active E2f then promotes cell proliferation via the expression of cell cycle related genes (Chong et al., 2009). When mitogens are absent, Id2 is kept in the cytosol, which results in the activation of Rb proteins to inhibit E2f-mediated cell proliferation (Wang et al., 2001). Rb proteins are also reported to bind to and activate NeuroD1, which promotes cell cycle exit and terminal neuronal differentiation (Batsche et al., 2005; Hindley and Philpott, 2012; Ross et al., 2003). In summary, extrinsic and intrinsic factors tightly regulate NSC properties.. 5. Potential functions of postnatal neurogenesis 15.

(17) There are main olfactory bulb (MOB) system and accessory olfactory bulb (AOB) system in the rodent brain (Firestein, 2001). MOB receives signals from the main olfactory epithelium (MOE) to mediate odor discrimination (Firestein, 2001). AOB receives signals from the vomeronsal organ (VNO) to respond to pheromones (Firestein, 2001). The cellular architecture and the process of postnatal neurogenesis in the MOB and AOB are similar (Doetsch and Hen, 2005; Firestein, 2001). Migrating neuroblasts derived from the SVZ differentiate into two kinds of interneurons, periglomerular cells and granule cells (Lledo et al., 2006). Sensory neurons in the MOE or VNO and mitral cells in the OB are not regenerated (Lledo et al., 2006). Dopaminergic periglomerular cells regulate signal transmission between sensory neurons and mitral cells at the glomeruli (Lledo et al., 2006). GABAergic granule cells regulate the activity of mitral cells (Lledo et al., 2006). Newborn granule cells and periglomerular cells are activated to provide lateral inhibition on the mitral cell and the glomeruli, respectively (Magavi et al., 2005; Ming and Song, 2011). Newborn granule cells and periglomerular cells in the OB participate in odor discrimination, perceptual learning and memory of mating partners (Doetsch and Hen, 2005; Moreno et al., 2009; Zhao et al., 2008b). Therefore, postnatal neurogenesis in the OB is required for survival and reproductive behaviors in rodents. The hippocampus contributes its functions in learning and memory (Zhao et al., 2008b). Granule neurons in the DG receives inputs from the entorhinal cortex and send axonal projections to the hippocampal CA3 region via the mossy fiber (Zhao et al., 2008b). CA1 receives signals 16.

(18) from the CA3 via the Schaffer collateral inputs and sends projections back to the neocortex (Zhao et al., 2008b). Newborn granule neurons from the SGZ participate in hippocampal-dependent learning and memory (Zhao et al., 2008b). DG neurogenesis is also involved in appropriate response to stress (Tsai et al., 2015). Together, postnatal neurogenesis in the DG participates not only in hippocampal-dependent learning and memory, but also anti-depression.. 6. Aims of the thesis Based on previous studies, we have learned that intrinsic and extrinsic factors regulate NSC properties. We are interested in finding new factors and studying their regulatory mechanisms in postnatal NSCs. Since adaptor protein TRIP6 and the Hippo pathway component YAP are expressed in NSCs derived from adult mouse SVZ without knowing their functions (Ramalho-Santos et al., 2002), we studied the role of TRIP6 (Chapter two), YAP and the interaction between TRIP6 and the Hippo pathway (Chapter three) in postnatal NSCs. Chinese herbal medicines such baicalin and curcumin show their clinical potential via inducing neurogenesis (Kim et al., 2008; Li et al., 2012). We studied Ginkgolide A (GA) and B (GB), the effective components of Ginkgo biloba extract (GBE) in postnatal NSCs (Chapter four). Postnatal neurogenesis contributes to the production of functional newborn neurons. Studying the mechanisms regulating postnatal neurogenesis may provide clinical applications.. 17.

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(26) Chapter two: TRIP6 Regulates Neural Stem Cell Maintenance in the Postnatal Mammalian Subventricular Zone This work has been published in Developmental Dynamics (2014) 243:1130–1142. DOI: 10.1002/DVDY.24161. Author Contributions Jui-Chen Tsai performed experiments and analyzed data of Figure 1. Chen-Yao Yang performed experiments and analyzed data of Figure 2, 3 and 4. Kao-Hua Huang performed experiments and analyzed data of Figure 3 and 4. Yun-Ju Lai wrote the manuscript. Ming-Yang Li designed and performed experiments and analyzed data of cell cultures and biochemical experiments in Figure 5, Figure 6, Figure 7, Figure 8 and wrote the manuscript. Tsu-Wei Wang designed all experiments and wrote the manuscript.. 25.

(27) 1. Abstract Postnatal neurogenesis persists throughout life in the subventricular zone (SVZ)-olfactory bulb pathway in mammals. Extrinsic or intrinsic factors have been revealed to regulate neural stem cell (NSC) properties and neurogenesis. Thyroid hormone receptor interacting protein 6 (TRIP6) belongs to zyxin family of LIM proteins, which have been shown to interact with various proteins to mediate cellular functions. However, the role of TRIP6 in NSCs is still unknown. By performing double immunofluorescence staining, we found that TRIP6 was expressed by SOX2-positive NSCs in embryonic and postnatal mouse forebrains. To study the function of TRIP6 in NSCs, we performed overexpression and knockdown experiments with neurospheres derived from postnatal day 7 SVZ. We found that TRIP6 was necessary and sufficient for self-renewal and proliferation of NSCs, but inhibited their differentiation. To further investigate the mechanism of TRIP6 in NSCs, we performed Luciferase reporter assay and found that TRIP6 activated the Notch pathway, a pathway required for NSC self-renewal. Our data suggest that TRIP6 regulates NSC maintenance and it may be a new marker for NSCs.. 26.

(28) 2. Introduction Neurogenesis is a tightly regulated process from the embryonic stage through adulthood (Gotz and Huttner, 2005). During the developmental stage of the mammalian nervous system, neural stem cells (NSCs) present at the ventricular zone (VZ) of the neural tube generate neurons and glia through symmetric and asymmetric cell division (Gotz and Huttner, 2005). Postnatally, NSCs are restricted at the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) (Corotto et al., 1993; Luskin, 1993; Seki and Arai, 1993). During embryonic and postnatal neurogenesis, newly generated cells exit cell cycle and undergo guided migration to reach their final destination and differentiate into neurons (Gotz and Huttner, 2005; Kriegstein and Alvarez-Buylla, 2009). In the postnatal forebrains, glial fibrillary acidic protein (GFAP)- and Sox2-expressing neural stem cells in the SVZ generate transit-amplifying cells, which give rise to doublecortin (DCX)positive neuroblasts. Neuroblasts migrate tangentially along the rostral migratory stream (RMS) to the olfactory bulb (OB) and differentiate into interneurons, whereas neuroblasts generated in the SGZ migrate radially into the nearby DG and differentiate into granule cells (Ming and Song, 2011). In these neural stem cell niches, morphogens and transcription factors play important roles in regulating self-renewal and multipotency, two properties of NSCs (Gong et al., 2007; Lin et al., 2012; Wang et al., 2006; Wang et al., 2011b; Wang et al., 2005). The major morphogens, including wingless-type MMTV integration site family (Wnt), bone morphogenetic 27.

(29) proteins (BMPs), fibroblast growth factor (FGF), sonic hedgehog (Shh), and Notch, mediate the activation of different transcription factors to regulate self-renewal and differentiation of NSCs (Liu and Zhang, 2011; Yao et al., 2012). Many transcription factors have also been shown to regulate embryonic neural development and postnatal neurogenesis (Liu and Zhang, 2011). For example, SOX2 promotes stem cell proliferation and maintenance (Ferri et al., 2004), whereas Pax6 and Tbr1 induce neuronal differentiation (Englund et al., 2005; Hevner et al., 2001). Whether there are other factors controlling NSC properties remains to be investigated. Thyroid hormone receptor interacting protein-6 (TRIP6) is first identified as a thyroid hormone receptor b1 interacting protein using a yeast two-hybrid screening system (Lee et al., 1995). It contains a proline-rich region in its amino-terminus and three LIM domains (named for homeodomain proteins Lin-11, Isl-1, and Mec-3) in the carboxyterminus (Murthy et al., 1999; Yi and Beckerle, 1998). With these features, TRIP6 and other focal adhesion proteins, such as zyxin (Beckerle, 1997), lipoma preferred partner (LPP) (Petit et al., 1996), Ajuba (Kanungo et al., 2000), and LIMD1 (Kiss et al., 1999) all belong to the zyxin family proteins. Most zyxin family members localize at sites of focal adhesions, but may also shuttle between the plasma membrane and nucleus to mediate different signaling events (Wang and Gilmore, 2001). Through the three LIM domains, the PDZ-binding motif, the Crk SH2binding motif, and/or other protein-interacting domains, TRIP6 serves as a platform for the recruitment of a number of molecules involved in actin 28.

(30) assembly, cell motility, survival, and transcriptional control (Bai et al., 2007; Chastre et al., 2009; Hadjipanayis and Van Meir, 2009; Lai et al., 2005; Lai et al., 2010; Lai et al., 2007; Li et al., 2005; Solaz-Fuster et al., 2006; Xu et al., 2004). Besides, TRIP6 is also involved in telomere protection (Sheppard and Loayza, 2010) and host-pathogen interactions (Williams et al., 1998; Worley et al., 2006). Interestingly, the expression of TRIP6 mRNA is reported to be elevated in different lineages of stem cells, including neural stem cells (Ramalho-Santos et al., 2002). This study implicates that TRIP6 may play roles in regulating properties of NSCs. To understand the function of TRIP6 in NSCs, we examined the expression pattern of TRIP6 in stem cell niches of embryonic and adult mouse forebrains. We found that TRIP6 was majorly expressed by SOX2-positive NSCs. Besides, TRIP6 promoted the self-renewal and proliferation of postnatal NSCs and inhibited their differentiation. Furthermore, TRIP6 may regulate stem cell maintenance through the Notch signaling pathway.. 29.

(31) 3. Materials and methods 3.1 Animals Handling of mice was according to university guidelines and the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Normal University (Approval Number 101026). Adult and pregnant CD1 mice at gestation day 15.5 and 16.5 (E15.5, E16.5) were exposed to photoperiod (12L: 12D) in IVC system with unlimited food and water. The sample size of each group of experiments was at least three.. 3.2 Plasmids For the TRIP6-expressing construct, cDNA sequence of human TRIP6 was inserted into the pEGFP-C1 expressing vector with CMV promoter (Clontech, Palo Alto). Short hairpin TRIP6 RNA (shTRIP6) and scrambled shRNA were inserted in pSUPER and pLVTHM vector (Lai et al., 2005; Lai et al., 2007; Lin et al., 2013). Enhanced green fluorescence protein (GFP), renilla Luciferase, and the human Notch intracellular domain (NICD) were inserted into the US2 vector with human Ubiquitin C promoter. Luciferase reporter constructs CBF1-WT and CBF1-MT (4xwtCBFLuc and 4xmtCBFLuc), Gli-WT and Gli-MT (8x3’Gli-BS and 8xm3’Gli-BS), TCF/LEF binding site and mutant sites (8xTOP and 8xFOP), and the human NICD (US2-NICD), Gli2 expression construct (pcDNA3.1-HisB-Gli2), and β-catenin expression construct (CAGEYFPCAG-ctnnb1) were described previously (Lin et al., 2012).. 30.

(32) 3.3 Fixation and sectioning Brain fixation and sectioning was described previously (Wu et al., 2013). In brief, adult mice were deeply anesthetized with Avertin and perfused with saline and 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO). Brains were then postfixed with 4% PFA, cryoprotected, and frozenly cut into 40-mm coronal sections with a microtome (Leica). For embryonic brains, mother mice were anesthetized and perfused as described above. Embryonic brains were frozen and cut into 30-mm coronal sections with a cryostat (Leica, Exton, PA). Three embryonic forebrain sections (120 mm apart/section) from each animal were selected for staining. Six equivalent SVZ sections (160 mm apart/section) from each adult animal throughout the anterior to posterior part were selected after the corpus callosum and before the anterior commissure were connected from both sides of the brain.. 3.4 RT-PCR Brain tissues of female pregnant mice and their fetuses at E16.5 were subjected to RNA extraction using Trizol® (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Two micrograms of total RNA were reverse transcribed by GoScriptTM kit (Promega, Madison, WI) and then 2 ml of cDNA was used for PCR reaction by GoTaq® Green Master Mix (Promega). Mouse TRIP6 forward primer: 50GAAGCCCAGTGGAGGTGCTG-30. Reverse primer: 50ACTCCAGAAGGTCCCTCCGG-30.. 31.

(33) 3.5 NIH3T3 cell culture NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) with 10% calf serum (Invitrogen). Two micrograms of pLVTHM-scamble shRNA or pLVTHM-shTRIP6 were transfected into 60 to 80% confluent 3T3 cells in 6-wells using Lipofectamine® 2000 (Invitrogen) according to the manufacturer’s manual. Sixteen hours after transfection, cells were starved by 0.1% BSA containing medium overnight and focal adhesions were induced by 10% FBS DMEM for 15 min (Xu et al., 2004). Cells were then fixed by 3% formaldehyde (Bionovas, Bremerton, WA) for immunofluorescence.. 3.6 Immunoblotting Brain tissues of female pregnant mice and their fetuses at E15.5 were lysed with 1xSDS lysis buffer (10% SDS, 60 mM Tris-HCl pH 6.8) and sonicated for 20 sec. Sixty micrograms of lysates were then applied to SDS-PAGE analysis and TRIP6 (1:2,000, Bethyl Laboratories) and GAPDH (1:5,000, GeneTex) were detected by their specific antibodies. Immunoblotting for 3T3 cells, 4 µg plasmids were used for transfection. Two days after transfection, cells were lysed by 1xSDS lysis buffer and 40 µg of cell lysates were then used for SDS-PAGE analysis. TRIP6 (1:2,000, Clone 16, BD Bioscience, Franklin Lakes, NJ) and β-actin (1:2,000, Sigma-Aldrich) were detected by their specific antibodies.. 3.7 Immunofluorescence Immunofluorescence for 3T3 cells was described previously (Xu et al., 32.

(34) 2004). In brief, fixed cells were permeablized with 0.2% Triton X-100 and blocked with phosphate-buffered saline containing 2% BSA. TRIP6 was detected by its specific antibody (1:250, Clone 16, BD Bioscience) and DyLightTM-549 goat anti-mouse antibody (1:500, Jackson ImmunoResearch, West Grove, PA). Cell nuclei were stained by DAPI (Invitrogen). Cells were photographed by the inverted fluorescence microscope (Leica DMI3000, Exton, PA). The immunostaining procedure on brain slices was described in Wu et al. (2013). Brain sections were incubated in blocking buffer (10% goat serum in TBS buffer) for 1 hr followed by incubation with primary antibodies overnight at 4°C. After washing, sections were incubated with fluorescence-conjugated secondary antibodies for 2 hr and with DAPI (Invitrogen) for 30 min. Stained sections were then mounted with antifade solution (Invitrogen) and photographed by a confocal microscope (TCS SP2, Leica) in the Image Core at NTNU. Antibodies used: mouse anti-TRIP6 (1:250; BD Bioscience), rabbit anti-SOX2 (1:1,000; Millipore), rabbit anti-S100β (1:1,000; Millipore), rabbit anti-Ki67 (1:250; Leica), guinea pig anti-DCX (1:5,000; Millipore), rabbit antiGFAP (1:350; Sigma), rabbit anti-MAP2 (1:1,000; Millipore), rabbit antiIba1 (1:1,000; Wako), DyLightTM 488 goat-anti-mouse, DyLightTM549 goat-anti-rabbit (1:500; Thermo Scientific), and DyLightTM549 donkeyanti-guinea pig (Jackson ImmunoResearch). Cells in the SVZ area were counted in 2-mm confocal sections. Differentiated NSCs were fixed in 4% PFA for 15 min. After wash and blocking, cells were incubated overnight at 4°C with the following 33.

(35) primary antibodies: mouse anti-neuronal class III β-tubulin (Tuj1, 1:1,000, Covance, Princeton, NJ), mouse anti-GFAP (1:1,000, Millipore, Billerica, MA), and rabbit anti-GFP (1:1,000, Invitrogen). Labeling was visualized with DyLightTM-550 goat anti-mouse and DyLightTM-488 goat anti-rabbit secondary antibody (1:1,000, Abcam, Cambridge, MA). Cell nuclei were stained by DAPI (Invitrogen). Cells were photographed by an inverted fluorescence microscope in the Image Core at NTNU.. 3.8 Neurosphere culture and electroporation Neurosphere (NS) cultures were prepared as previously described (Wang et al., 2005) with modifications. P7 CD1 mice were sacrificed by cervical dislocation. SVZ tissue dissected from 2-mm-thick coronal brain sections was minced and dissociated with trypsin (Sigma), hyaluronidase (Sigma), and Kynurenic acid (Sigma). SVZ cells were cultured in a 24-well dish (1.5 mice per well) with Dulbecco’s modified Eagle’s medium (DMEM/F12) (Invitrogen) with 1% N2 (Invitrogen), 10 ng/ml bFGF (Sigma), 20 ng/ml EGF (Sigma), 2 mg/ml Heparin (Sigma), and 1 % Penicillin-Streptomycin-Gluatmax (Invitrogen) at 37°C, 5 % CO2 incubator for 5 days. Half of the medium were replaced every 2 days. For each experiment, SVZ explants from two litters of P7 mice were combined and cultured to form primary (1’) NS first. These 1’ NS were then dissociated and transfected with different constructs for either secondary (2’) NS formation, proliferation or differentiation experiments. Electroporation was performed with 1x106 NSCs dissociated from 1’ NS using Nucleofector (LONZA Amaxa) with Program A-033. In TRIP6 34.

(36) loss-of-function experiments, 4 µg of GFP plasmid was co-electroporated with 6 µg of shTRIP6 or scrambled shRNA plasmid. In TRIP6 gain-offunction experiments, 4 µg of GFP plasmid was co-electroporated with 6 µg of pEGFP-TRIP6 or pEGFP-C1 control plasmid. Post-electroporated NSCs were cultured at 2.5x105 cells per 6-well to form 2’ NS.. 3.9 Transfection and Differentiation of NSCs 1x105 NSCs dissociated from 1’ NS were plated in 24-well dishes and cultured in DMEM/F12 with 1% N2 and 1% FBS without antibiotics for 1–2 hr before transfection. In TRIP6 loss-of-function experiments, cells were co-transfected with 0.25 µg of US2-GFP and 0.35 µg of shTRIP6 or scrambled shRNA-harboring plasmid. In TRIP6 gain-of-function experiments, cells were co-transfected with 0.25 µg of US2-GFP and 0.35 µg of GFP-TRIP6 or GFP-expressing vector. Lipofectamine® 2000 (Invitrogen) was used for transfection according to the manufacturer’s instruction. The medium was replaced by DMEM/F12 with 1% N2, 1% FBS, and 1% antibiotics 6 hr after transfection. For neuronal differentiation, NSCs were cultured for 3 days on cover slips coated with poly-L-lysine (Sigma) and laminin (Invitrogen).. 3.10 Luciferase assay P19 cells were maintained in MEMα medium with 7.5% calf serum, 2.5% FBS, and 1% antibiotics (Lin et al., 2012). For transfection, cells were plated in 12-well dishes at 80–90% confluence in MEMα with 10% serum without antibiotics. Lipofectamine® 2000 (Invitrogen) was used for 35.

(37) transfection according to the manufacturer’s instruction. P19 cells were co-transfected with 0.05 µg of US2-renilla Luciferase, 0.5 µg of pEGFPC1, pGFP-TRIP6, US2-NICD, Gli2, or ctnnb1, and 0.7 µg of Firefly Luciferase reporter constructs with either wild type (WT) or mutated (MT) transcription factor binding sites. Six hours after transfection, medium was replaced with Opti-MEM with 1% FBS and antibiotics. SHSY5Y cells were maintained in DMEM/F12 medium with 10% FBS, and 1% antibiotics. For transfection, 5x105 SH-SY5Y cells were plated in 12well dishes and cultured in DMEM/F12 with 10% FBS without antibiotics for one hour before transfection. The transfection protocol was the same as P19 cells. Six hours after transfection, medium was replaced with DMEM/F12 with 1% FBS and antibiotics. Reporter activity was measured 24 hr after transfection using Dual-Luciferase Assay System (Promega). For normalization, firefly Luciferase activity was first normalized to renilla Luciferase activity of each group. The obtained value from the wild-type binding sites (WT CBF1) was then normalized to that of the MT CBF1. Finally, the normalized value from the control vector group was set as 100% and the value from the TRIP6 group was shown as the percentage of the control group. Samples of each experiment were plated in duplicates and each experiment was repeated for three times (n=3).. 3.11 Statistical Analysis Two-group comparisons were analyzed by two-tailed Student’s t-test. Difference of two distributions was analyzed by Wilcoxons rank-sum 36.

(38) test. All data were presented as mean ± standard error of the mean (SEM). Significant level is p<0.05.. 37.

(39) 4. Results 4.1 TRIP6 is expressed by NSCs in the embryonic and adult mouse brain To study the role of TRIP6 in the nervous system, we asked whether it was present in the brains of embryonic and adult mice. For this, we first examined the specificity of the TRIP6 antibody, which has been reported to be able to detect endogenous TRIP6 by immunobloting (Xu et al., 2004) and immunostaining (Takizawa et al., 2006). In 3T3 cells, this antibody detected endogenous TRIP6 at focal adhesions in the scrambled shRNA-expressing cell, but not in the cell expressing shTRIP6 (Fig. 1A). Our TRIP6 antibody could not detect TRIP6 signal in shTRIP6transfected 3T3 cells (Fig. 1B). These results confirm the specificity of our TRIP6 antibody. From the whole brain extract, we found that TRIP6 mRNA was expressed in embryonic day 15.5–16.5 (E15.5–16.5) mouse brains, but the level was low or undetectable in adult ones (Fig. 1C). Consistent with the mRNA result, TRIP6 protein was only detected in embryonic, but not in adult brains (Fig. 1D). These results implicate that the role of TRIP6 in the nervous system may be mainly in neural development. After the nervous system is developed, the expression of TRIP6 is down-regulated. Since we found that TRIP6 was abundant in the embryonic brain (Fig. 1C, D) and TRIP6 has been reported to be enriched in NSCs (RamalhoSantos et al., 2002), we carried out immunofluorescence of TRIP6 with E16.5 forebrain sections to investigate whether embryonic NSCs express TRIP6. We found that there was a ventricle-to-mantle gradient of TRIP6 38.

(40) expression in the dorsal and ventral forebrain, where the primordium of cerebral cortex and striatum reside, respectively (Fig. 2A). Since embryonic NSCs are located at the VZ, we double labeled forebrain sections with antibodies against TRIP6 and the neural stem cell marker SOX2. We found that most of the TRIP6-expressing cells were also SOX2-positive in the dorsal and ventral VZ (Fig. 2B, C). The percentage of TRIP6-positive cells that also express SOX2 was 90.2 ± 2.7% (Fig. 2E). During the embryonic stage, NSCs are actively dividing. To examine whether TRIP6-positive cells are proliferative, E16.5 forebrain sections were double-labeled with antibodies against TRIP6 and the cell cycle marker Ki67 (Fig. 2D). We found that 34.1 ± 2.1% of TRIP6-positive cells were labeled with Ki67 (Fig. 2E). These results suggest that TRIP6 is mainly expressed by NSCs in the embryonic forebrain. The central nervous system is mostly developed during the embryonic stage, but neurogenesis persists in the SVZ-olfactory bulb pathway and the DG of adult brains. Since we found that embryonic NSCs expressed TRIP6, it is possible that TRIP6 may also be expressed by postnatal NSCs. Although we did not find TRIP6 expression in the adult brain by Western blot analysis, its mRNA was detected in some of the analyzed adult brains (Fig. 1C, D). Due to relatively fewer NSCs present in the adult nervous system, it is difficult to detect TRIP6 signal from whole brain extracts. Therefore, immunofluorescence can provide a better resolution of TRIP6-expressing cells in the adult brain. Indeed, we found that TRIP6 was expressed in the SVZ (Fig. 3A, C–H), but not in the SGZ (Fig. 3B). Consistent with the finding from embryonic brains, most 39.

(41) TRIP6-expressing cells in the adult SVZ were also SOX2-positive (62.7 ± 1.6%; Fig. 3C, D, D’, I). However, only 8.0 ± 0.3% of TRIP6-positive cells were labeled with Ki67 (Fig. 3E, F, I). Since adult NSCs are mostly quiescent, it is reasonable that there are fewer TRIP6 and Ki67 doublepositive cells in the adult SVZ. It is also proposed that adult NSCs are ependymal cells lining the lateral ventricle (Spassky et al., 2005). We also double labeled adult SVZ sections with antibodies against TRIP6 and the ependymal cell marker S100β, and found that 44.4 ± 1.5% of TRIP6expressing cells were also S100β-positive (Fig. 3G, H, I). These results suggest that TRIP6 is mainly expressed by adult NSCs in the SVZ. To examine whether the progeny of adult NSCs also expresses TRIP6, we double-labeled adult SVZ sections with antibodies against TRIP6 and the neuroblast marker, DCX, the neuronal maker, microtubule-associated protein 2 (MAP2), or the astrocyte marker, GFAP. We found that only 6.7 ± 1.8% of TRIP6-expressing cells were DCX-positive (Fig. 4A). However, MAP2-positive neurons do not express TRIP6 (Fig. 4B). Interestingly, TRIP6 was not expressed by GFAP-positive astrocytes in the striatum and the corpus callosum (Fig. 4C and data not shown). To further examine whether TRIP6 is expressed by microglia, we doublelabeled SVZ sections with antibodies against TRIP6 and the microglia marker, ionized calcium-binding adapter molecule 1 (Iba1, Fig. 4D). We found that TRIP6 was not expressed by Iba1-positive microglia, either. Taken together, TRIP6 is mainly expressed by NSCs, ependymal cells, and some of the neuroblasts in the adult SVZ, but not in more differentiated cells including neurons, astrocytes, and microglia. These 40.

(42) results implicate that TRIP6 may regulate NSC properties.. 4.2 TRIP6 maintains self-renewal and proliferation of NSCs Postnatal NSCs from the SVZ can be cultured in vitro in suspension condition supplied with mitogenic growth factors to form primary neurospheres (1’ NS). 1’ NS can be dissociated and cultured in the same condition to form secondary NS (2’ NS). After growth factor removal, NS can differentiate into neurons, astrocytes and oligodendrocytes. Therefore, the rate of 2’ NS formation and neural differentiation are indices of self-renewal and multipotency of NSCs, respectively. To investigate whether TRIP6 is required for the self-renewal ability of NSCs, 1’ NS derived from postnatal day 7 (P7) SVZ were dissociated, transfected with scrambled shRNA (Ctrl) or shTRIP6 together with GFP plasmids, and cultured to form 2’ NS for 5 days. Scrambled control transfected cells formed 2’ NS (Fig. 5A). However, shTRIP6 ones did not (Ctrl: 55 ± 3.6 spheres/106 cells, TRIP6: 0.33 ± 0.3 spheres/106 cells, p<0.01; Fig. 5B, C). To further confirm this result, we also performed gain-of-function experiment by overexpression of TRIP6. 1’ NS were dissociated, transfected with control (Ctrl) or TRIP6 together with GFP vectors, and cultured to form 2’ NS. Transfected cells formed 2’ NS in both the control and TRIP6 groups (Fig. 5D, E). The number of 2’ NS was not increased by TRIP6 overexpression (TRIP6: 41 ± 4.93 spheres/106 cells). This could be due to the abundance of endogenous TRIP6 in neural stem cells. Therefore, we measured the sphere diameter as another index of self-renewal. For the control of the knockdown 41.

(43) experiment, the size of the 2’ NS tended to be slightly larger than that in the control of the overexpression experiment, which could be due to different vectors transfected. Still, TRIP6 shifted the distribution toward larger spheres where TRIP6 significantly decreased the number of smallsized spheres (100–150 mm; p<0.05) and increased the number of largesized ones (250–300 mm; p<0.05; Fig. 5F). Together, these results suggest that TRIP6 is necessary and sufficient for self-renewal of NSCs. Since TRIP6 increases the neurosphere size, it is possible that it positively regulates cell proliferation of NSCs. To test this hypothesis, 1’ NS were dissociated, transfected with scrambled shRNA (Ctrl) or shTRIP6, and cultured in differentiation condition for 1 day when cells were still actively dividing. BrdU, the thymidine analog, was added 2 hr before fixation. We found that BrdU-positive cells more significantly decreased in the shTRIP6 group than those in the control group (Ctrl: 100 ± 0%, shTRIP6: 77.3 ± 5.8%, p<0.05; Fig. 6A–C). We also overexpressed TRIP6 in dissociated 1’ NS and cultured them in differentiation condition for 2 days when cells started to exit cell cycle and underwent differentiation. BrdU was again added 2 hr before fixation. We found that BrdU-positive cells significantly increased in the TRIP6 group (Ctrl: 100 ± 0%, TRIP6: 132.7 ± 5.4%, p<0.05; Fig. 6D–F). These results together suggest that TRIP6 positively regulates NSC/neural progenitor cell proliferation.. 4.3 TRIP6 inhibits differentiation of postnatal NSCs Since TRIP6 positively regulates self-renewal of postnatal NSCs (Fig. 5), 42.

(44) TRIP6 may also be involved in NSC differentiation. To test whether TRIP6 modulates NSC differentiation, we started with the loss-offunction approach by knockdown of TRIP6. In our culture condition, NSCs mostly differentiate into astrocytes and neurons labeled by GFAP and class III b-tubulin (Tuj1) antibodies, respectively (Fig. 7), and a few differentiate into oligodendrocytes (data not shown). Therefore, we examined neuronal and astroglial differentiation after manipulation of TRIP6 expression. 1’ NS derived from P7 SVZ were dissociated, cotransfected with GFP and shTRIP6 constructs, and cultured in differentiation condition for 3 days. We found that knockdown of TRIP6 significantly enhanced neuronal differentiation of NSCs (Ctrl: 100 ± 0%, shTRIP6: 208 ± 20.8%, p<0.05; Fig. 7A, B, and E) at the expense of glial differentiation (Ctrl: 100 ± 0%, shTRIP6: 66.5 ± 3.3%, p<0.01; Fig. 7C, D, and E), suggesting that TRIP6 negatively regulates neuronal differentiation. To further confirm the role of TRIP6 in NSC differentiation, we transfected 1’ NS with control (Ctrl) or TRIP6 constructs and cultured them in differentiation condition for 3 days. Overexpression of TRIP6 decreased neuronal differentiation (Ctrl: 100 ± 0%, TRIP6: 50.8 ± 3.7%; p<0.01; Fig. 7F, G, and J) and reduced glial differentiation (Ctrl: 100 ± 0%, TRIP6: 71.2 ± 6.9%, p<0.05; Fig. 7H–J), suggesting that TRIP6 inhibits differentiation. Taken together, our results demonstrate that TRIP6 mainly maintains NSC identity and negatively regulates NSC differentiation.. 4.4 TRIP6 activates the Notch signaling pathway 43.

(45) Notch, Shh, and Wnt pathways have been reported to be involved in NSC maintenance (Ming and Song, 2011). Since we found that TRIP6 is necessary and sufficient to regulate self-renewal and negatively regulates differentiation of postnatal NSCs (Fig. 5, 7), it is plausible that these signaling pathways mediate the function of TRIP6. To examine these possibilities, we tested whether TRIP6 regulates Notch, Shh, or Wnt activity by Luciferase reporter assays in P19 mouse embryonic carcinoma cell line. P19 cells are pluripotent cells, which can be induced to differentiate into neurons by retinoic acid (RA) treatment or expression of neural basic helix-loop-helix (bHLH) transcription factors (Farah et al., 2000; Jones-Villeneuve et al., 1982; Jones-Villeneuve et al., 1983). First, we tested whether TRIP6 interacted with the Notch pathway. Upon ligand binding, Notch intracellular domain (NICD) is cleaved from the cell membrane and translocated into the nucleus to increase the transcription of genes containing CBF binding sites (Hsieh et al., 1996). We transfected P19 cells with Luciferase reporter constructs containing wildtype (WT) or mutant (MT) CBF binding sites and found that TRIP6 significantly promoted the Luciferase activity with WT CBF binding sites up to 2-fold (Ctrl: 100 ± 0%, TRIP6: 192.5 ± 26.2%, p<0.05; Fig. 8A). However, TRIP6 did not promote Luciferase activities with Gli or TCF binding sites, which are consensus binding sites for Shh and Wnt pathways, respectively (data not shown). To rule out the possibility that this is a P19 cell-specific effect, we repeated the same experiment with the SH-SY5Y neuroblastoma cell line, which can be induced to differentiate into neurons with RA (Biedler et al., 1978). Consistent with 44.

(46) the result from P19 cells, TRIP6 also significantly increased Notch activity in SH-SY5Y cells (Ctrl: 100 ± 0%, TRIP6: 260.4 ± 32.6%, P<0.05; Fig. 8B). Taken together, our results suggest that TRIP6 may activate the Notch pathway to maintain NSC identity.. 45.

(47) 5. Discussion As a focal adhesion molecule, studies of TRIP6 have been focused on its role in regulating cell migration, including cancer metastasis and pathogen invasiveness in the body. Its functions in progression and survival of cancer cells have also been reported (Lai et al., 2010; Lin et al., 2013). Since a microarray analysis of different lineages of stem cells reveals that TRIP6 may serve as a stemness gene (Ramalho-Santos et al., 2002), here we investigate its role in NSCs. We found that TRIP6 was expressed by NSCs in the embryonic and adult forebrain. It was required for their self-renewal and proliferation and inhibited differentiation. In addition, TRIP6 activated the Notch signaling pathway. Sox1, 2, and 3 belonging to SoxB1 transcription factors are expressed by NSCs (Wang et al., 2006; Zappone et al., 2000). SOXB1 transcription factors and the Notch pathway are well known for their roles in maintaining NSCs in an undifferentiated state (Bylund et al., 2003; Ferri et al., 2004; Ross et al., 2003). Moreover, the Notch pathway has been shown to activate Sox2 expression and inhibit neural differentiation (Ehm et al., 2010). It has also been reported that the Notch signaling is downregulated in RA-induced differentiation of glioblastoma stem cells, further suggesting that the Notch pathway is required to maintain stem cells in an undifferentiated state (Ying et al., 2011). Here, we found that TRIP6 was expressed by SOX2-positive NSCs in the embryonic and adult forebrain (Figs. 2, 3). TRIP6 maintained self-renewal ability and inhibited differentiation of postnatal NSCs (Figs. 5, 7). More importantly, TRIP6 increased the Notch activity (Fig. 8). In addition, the Notch signaling is 46.

(48) reported to inhibit the neuronal fate while inducing astrocyte differentiation in embryonic and postnatal neural stem cells (Grandbarbe et al., 2003; Tanigaki et al., 2001). Moreover, the Notch pathway has been shown to maintain adherens junctions of neural stem cells (Hatakeyama et al., 2014). Loss of adherens junctions promotes detachments of neural progenitor cells and induces precocious neurogenesis (Hatakeyama et al., 2014; Rousso et al., 2012). Here, we found that knockdown of TRIP6 increased neuronal differentiation at the expense of glial differentiation (Fig. 7). This result further supports that TRIP6 may act upstream of the Notch-SOX2 signaling pathway to maintain NSC identity and regulate their differentiation. During neurogenesis, adhesion status of NSCs regulates the balance of self-renewal and differentiation. NSCs form a self-supporting niche by adhering to the luminal surface of ventricle and neighboring progenitor cells (Meng and Takeichi, 2009; Zhang et al., 2010a). Loss of adhesion status correlates with differentiation, while secured adhesion promotes the self-renewal of NSCs (Malaguti et al., 2013; Rousso et al., 2012). These reports mainly concentrate on adherens junctions and cadherin proteins. However, studies of focal adhesion molecules in neurogenesis are limited and most of them focus on cell migration. Interestingly, the dynamics of focal adhesions has been shown to correlate with differentiation of NSCs (Lyu et al., 2013) and deficiency of a focal adhesion molecule, vinculin, disrupts neural tube formation (Xu et al., 1998). Here, we provide evidence that TRIP6 is involved in regulating of NSC self-renewal and differentiation. Taken together, this 47.

(49) finding suggests that focal adhesion molecules also play a role in neurogenesis. Previously, we found that TRIP6 regulates not only cell motility, but also cell proliferation and survival in cancer cells (Lai et al., 2005; Lai et al., 2010; Lin et al., 2013). It activates ERK, Akt, and NFkB pathways to promote cell motility, proliferation, and survival in ovarian cancer and glioblastoma cells (Lai et al., 2005; Lai et al., 2010; Lin et al., 2013). During neurogenesis, ERK, Akt, and NF-kB signaling pathways are also important regulators for proliferation and survival of NSCs (Shioda et al., 2009; Widera et al., 2006; Zhou and Miller, 2006). Therefore, TRIP6 may also regulate properties of NSCs through these pathways. In the adult SVZ, TRIP6 is also expressed by ependymal cells (Fig. 3). Ependymal cells are one kind of glial cells that remain in the VZ during neural development (Spassky et al., 2005). Johansson et al. have reported that ependymal cells serve as NSCs that give rise to new neurons in the adult olfactory bulb (Johansson et al., 1999). Ependymal cells can also respond to spinal cord injury and generate migratory astrocytes to the injured sites (Johansson et al., 1999). Our results show that TRIP6 is expressed in these cells and SOX2-positive cells, suggesting that TRIP6 is majorly expressed in cells with stem cell properties and may maintain the stemness features of these cells. Ependymoma is a glioma transformed from ependymal cells. According to the microarray data from the Neale Multi-cancer data set, TRIP6 mRNA expression is also higher in ependymoma and anaplastic ependymoma. In addition, we have studied the role of TRIP6 in glioma 48.

(50) tumorigenesis and found that TRIP6 is highly expressed in glioblastoma compared to control tissues (Lai et al., 2010). Moreover, TRIP6 enhances the anti-apoptotic ability of glioblastoma cells and promotes tumor growth in vivo (Lai et al., 2010; Lin et al., 2013). Consistent with this, the expression level of TRIP6 is reversely correlated with the overall survival of patients; poor prognosis of patients usually has a higher level of TRIP6 (Lin et al., 2013). These studies suggest that TRIP6 may be a key factor in various gliomas. Since our results showed that mature astrocytes do not express TRIP6 (Fig. 4C) but NSCs do (Figs. 2, 3), it is possible that the ectopic expression of TRIP6 in differentiated glial cells may transform them into cancer cells and make them tumorigenic. Therefore, TRIP6 may not only serve as a biomarker, but also be a therapeutic target for malignant brain diseases.. 49.

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