Browsing by Author "Stuardo, Nicolas"

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    An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons
    (2020) Stuardo, Nicolas; Moya-Alvarado, Guillermo; Ramirez, Carolina; Schiavo, Giampietro; Bronfman, Francisca C.
    Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
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    Brain-Derived Neurotrophic Factor (BDNF) Regulates Rab5-Positive Early Endosomes in Hippocampal Neurons to Induce Dendritic Branching
    (2018) Moya-Alvarado, Guillermo; Gonzalez, Andres; Stuardo, Nicolas; Bronfman C., Francisca
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    c-Abl Tyrosine Kinase Is Required for BDNF-Induced Dendritic Branching and Growth
    (2023) Chandia-Cristi, America; Stuardo, Nicolas; Trejos, Cristian; Leal, Nancy; Urrutia, Daniela; Bronfman, Francisca C.; Rojas, Alejandra Alvarez
    Brain-derived neurotrophic factor (BDNF) induces activation of the TrkB receptor and several downstream pathways (MAPK, PI3K, PLC-gamma), leading to neuronal survival, growth, and plasticity. It has been well established that TrkB signaling regulation is required for neurite formation and dendritic arborization, but the specific mechanism is not fully understood. The non-receptor tyrosine kinase c-Abl is a possible candidate regulator of this process, as it has been implicated in tyrosine kinase receptors' signaling and trafficking, as well as regulation of neuronal morphogenesis. To assess the role of c-Abl in BDNF-induced dendritic arborization, wild-type and c-Abl-KO neurons were stimulated with BDNF, and diverse strategies were employed to probe the function of c-Abl, including the use of pharmacological inhibitors, an allosteric c-Abl activator, and shRNA to downregulates c-Abl expression. Surprisingly, BDNF promoted c-Abl activation and interaction with TrkB receptors. Furthermore, pharmacological c-Abl inhibition and genetic ablation abolished BDNF-induced dendritic arborization and increased the availability of TrkB in the cell membrane. Interestingly, inhibition or genetic ablation of c-Abl had no effect on the classic TrkB downstream pathways. Together, our results suggest that BDNF/TrkB-dependent c-Abl activation is a novel and essential mechanism in TrkB signaling.