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Xie X.,University of Western Ontario | Hu J.,University of Western Ontario | Liu X.,University of Western Ontario | Qin H.,University of Western Ontario | And 3 more authors.
International Journal of Biological Sciences | Year: 2010

NIP/DuoxA, originally cloned as a protein capable of binding to the cell fate determinant Numb in Drosophila, was recently identified as a modulator of reactive oxygen species (ROS) production in mammalian systems. Despite biochemical and cellular studies that link NIP/DuoxA to the generation of ROS through the dual oxidase (Duox) enzyme, the in vivo function of NIP/DuoxA has not been characterized to date. Here we report a genetic and functional characterization of nip in Drosophila melanogaster. We show that nip is essential for Drosophila development as nip null mutants die at the 1st larval instar. Expression of UAS-nip, but not UAS-Duox, rescued the lethality. To understand the function of nip beyond the early larval stage, we generated GAL4 inducible UAS-RNAi transgenes. daG32-GAL4 driven, ubiquitous RNAi-mediated silencing of nip led to profound abnormality in pre-adult development, crinkled wing and markedly reduced lifespan at 29°C. Compared to wild type flies, da-GAL4 induced nip-RNAi transgenic flies exhibited significantly reduced ability to survive under oxidative stress and displayed impaired mitochondrial aconitase function. Our work provides in vivo evidence for a critical role for nip in the development and oxidative stress response in Drosophila. © Ivyspring International Publisher. Source

Jones E.V.,Center for Research in Neuroscience | Cook D.,Center for Research in Neuroscience | Murai K.K.,Center for Research in Neuroscience
Methods in Molecular Biology | Year: 2012

Astrocytes secrete factors that promote neuron survival, synapse formation, and plasticity. Understanding how these factors perform these roles requires a robust in vitro system that can effectively assess the impact of individual glial factors on neuronal properties. A classical approach to studying neuron-glial interactions in vitro uses a system where dissociated embryonic rat neurons are suspended over a feeder layer of rat astrocytes. Here, we describe a useful "sandwich" co-culture system where postnatal mouse hippocampal neurons are grown suspended above a feeder layer of mouse hippocampal astrocytes. We demonstrate that neurons in these cultures remain healthy beyond 3 weeks in vitro and develop more synapses compared to neurons grown without astrocytes. An advantage of this method is that astrocytes and neurons can be prepared separately from postnatal transgenic or knock-out mouse lines allowing one to study, for example, how wild-type neurons develop in the presence of astrocytes from a knock-out mouse line that lacks the expression of a specific astrocyte-secreted factor. We find this culture system to be a convenient and powerful approach to study the contribution of astrocyte-secreted molecules to neuron development. © 2012 Springer Science+Business Media, LLC. Source

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