Identification The Signaling Network Regulated By Rho-GDI-γ During Neural Stem Cell Differentiation&the Function And Interaction Of CDC50A And P4-ATPases

Neural stem cells (NSCs) are promising candidates for a variety of neurological diseasesdue to their ability to differentiate into neurons, astrocytes, and oligodentrocytes.Duringthis process, Rho GTPases are heavily involved in neuritogenesis, axon formation anddendritic development, due their effects on the cytoskeleton through downstream effectors.The activities of Rho GTPases are controlled by Rho-GDP-dissociation inhibitors (Rho-GDIs). As shown in our previous study, these are also involved inthe differentiation ofneural stem cells, however, little is known about the underlying regulatory mechanism.Here, we describe how the transcription factors hepatic nuclear factor4-1(HNF4-1) andmyc-associated zinc finger protein (MAZ-1) regulate the expression of Rho-GDIγ in thestimulation of NSC differentiation. Using a transcription factor decoy strategy, to blockthe activity of a specific transcription factor through the binding of synthetic double-stranded binding sequence oligodeoxynucleotides (ODNs),we examined the effects ofHNF4-1and MAZ-1on NSC differentiation in the neural stem cell line C17.2. Our resultsshow that HNF4-1and MAZ-1decoy ODNssignificantly knock down Rho-GDIγ genetranscription, leading to NSC differentiation towards neurons and an increase in cellmigration. We observed that HNF4-1and MAZ-1decoy ODNs are able enter the cellnucleolus and specifically bind to their target transcription factors. Furthermore, theexpression of Rho-GDIγ-mediated geneswas identified, suggesting that the regulatorymechanism for the differentiation and migration of neural stem cells is triggered by thetranscription factors MAZ-1and HNF4-1. These findings indicate that HNF4-1and MAZ-1regulate the expression of Rho-GDIγ and contribute to the differentiation of NSCs. Ourwork lays the foundation for the exploration of mechanisms fundamental to pluripotentNSCs differentiation.In addition, a novel systems biology approach was presented here to identify putativesignaling pathways regulated by Rho-GDI-γ during NSCs differentiation, and thesepathways can provide insights into the NSCs differentiation mechanisms. In particular, ourproposed approach combines the predictive power of computational biology and molecular experiments. With different biological experiments, the genes in thecomputationally identified signaling network were validated to be indeed regulated byRho-GDI-γ during the differentiation of NSCs. In particular, one randomly selectedpathway involving Vcp, Mapk8, Ywhae and Ywhah was experimentally verified to beregulated by Rho-GDI-γ. These promising results demonstrate the effectiveness of ourproposed systems biology approach, indicating the potential predictive power ofintegrating computational and experimental approaches. P4-ATPases are a relatively novel family of membrane proteins implicated in the active transportof phospholipids across cell membranes in order to maintain and change the membraneasymmetry. Deficiency or mutation of P4-ATPases in different tissues is associated with manysevere pathophysiological diseases. The majority of mammalian P4-ATPases consist of a largecatalytic subunit, and a smaller accessory subunit (CDC50A or CDC50B) that is crucial for thecorrect folding of P4-ATPases. In this study, a sensitive mass spectroscopic technique togetherwith immunoaffinity chromatography was used to identify P4-ATPases that bind to CDC50A indifferent tissues, as an initial step in defining their roles in structure, function, trafficking anddisease.In summary, we identified P4-ATPases from four of the five subfamilies of P4-ATPases. Thereare five P4-ATPases detected in the retina, seven in testis and only two in the brain. ATP8A1and ATP8A2have previously been shown to be expressed in the brain and retina, respectively.In addition, the ATP11subfamily was detected in the retina and testis, which has not beenpreviously reported. Moreover, ATP8A1, ATP8A2, ATP8B3and ATP8B4are all shown to bepresent in testis, indicating P4-ATPases are most likely very important in germ cell generation.Western blotting and immunofluorescence microscopy confirmed the presence of the novel P4-ATPases in the different tissues.In addition, three uncharacterized P4-ATPases, ATP11Aand ATP11C were found to bindCDC50A in the retina or testis. These genes were cloned, expressed in cultured cells,immunoaffinity purified and new α-ATP11C and ATP10A antibodies were generated.Immunochemistry studies showed co-localization of ATP11A and ATP11C with a Golgi markerwhen co-expressed with CDC50A and CDC50B. In contrast, the majority of ATP11A andATP11C co-localized with calnexin in the ER without CDC50proteins. ATP10A does not co-localize with the Golgi marker GM130, but instead co-localizes with CDC50A partially withinthe ER.ATP11A and ATP11C expressed in HEK293T cells assemble with CDC50A to generatea heteromeric complex that actively transports phosphatidylserine across membranes. These results, along with the proteomics data, suggest that P4-ATPases that bind to CDC50Aplay a role in transporting aminophospholipid across the membrane bilayer using ATP as anenergy source. The P4-ATPases identified in this study are a valuable resource for furtheranalysis and elucidation of the role of these transporters in cell structure, function, and disease.