Browsing by Author "Moya-Alvarado, Guillermo"
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- ItemAn 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.
- ItemBDNF/TrkB signaling endosomes in axons coordinate CREB/mTOR activation and protein synthesis in the cell body to induce dendritic growth in cortical neurons(2023) Moya-Alvarado, Guillermo; Tiburcio-Felix, Reynaldo; Ibanez, Maria Raquel; Aguirre-Soto, Alejandro A.; Guerra, Miguel, V; Wu, Chengbiao; Mobley, William C.; Perlson, Eran; Bronfman, Francisca C.Brain-derived neurotrophic factor (BDNF) and its receptors tropomyosin kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the CNS. After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation.
- ItemBrain-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
- ItemCoumarin-Chalcone Hybrids as Inhibitors of MAO-B: Biological Activity and In Silico Studies(2021) Moya-Alvarado, Guillermo; Yanez, Osvaldo; Morales, Nicole; Gonzalez-Gonzalez, Angelica; Areche, Carlos; Nunez, Marco Tulio; Fierro, Angelica; Garcia-Beltran, OlimpoFourteen coumarin-derived compounds modified at the C3 carbon of coumarin with an alpha,beta-unsaturated ketone were synthesized. These compounds may be designated as chalcocoumarins (3-cinnamoyl-2H-chromen-2-ones). Both chalcones and coumarins are recognized scaffolds in medicinal chemistry, showing diverse biological and pharmacological properties among which neuroprotective activities and multiple enzyme inhibition, including mitochondrial enzyme systems, stand out. The evaluation of monoamine oxidase B (MAO-B) inhibitors has aroused considerable interest as therapeutic agents for neurodegenerative diseases such as Parkinson's. Of the fourteen chalcocumarins evaluated here against MAO-B, ChC4 showed the strongest activity in vitro, with IC50 = 0.76 +/- 0.08 mu M. Computational docking, molecular dynamics and MM/GBSA studies, confirm that ChC4 binds very stably to the active rMAO-B site, explaining the experimental inhibition data.
- ItemDlg Is Required for Short-Term Memory and Interacts with NMDAR in the Drosophila Brain(2022) Bertin, Francisca; Moya-Alvarado, Guillermo; Quiroz-Manriquez, Eduardo; Ibacache, Andres; Kohler-Solis, Andres; Oliva, Carlos; Sierralta, JimenaThe vertebrates' scaffold proteins of the Dlg-MAGUK family are involved in the recruitment, clustering, and anchoring of glutamate receptors to the postsynaptic density, particularly the NMDA subtype glutamate-receptors (NRs), necessary for long-term memory and LTP. In Drosophila, the only gene of the subfamily generates two main products, dlgA, broadly expressed, and dlgS97, restricted to the nervous system. In the Drosophila brain, NRs are expressed in the adult brain and are involved in memory, however, the role of Dlg in these processes and its relationship with NRs has been scarcely explored. Here, we show that the dlg mutants display defects in short-term memory in the olfactory associative-learning paradigm. These defects are dependent on the presence of DlgS97 in the Mushroom Body (MB) synapses. Moreover, Dlg is immunoprecipitated with NRs in the adult brain. Dlg is also expressed in the larval neuromuscular junction (NMJ) pre and post-synaptically and is important for development and synaptic function, however, NR is absent in this synapse. Despite that, we found changes in the short-term plasticity paradigms in dlg mutant larval NMJ. Together our results show that larval NMJ and the adult brain relies on Dlg for short-term memory/plasticity, but the mechanisms differ in the two types of synapses.
- ItemMultiple Labeling of Compartmentalized Cortical Neurons in Microfluidic Chambers(2024) Moya-Alvarado, Guillermo; Aguirre-Soto, Alejandro; Bronfman, Francisca C.Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells.
- ItemThe Rab11-regulated endocytic pathway and BDNF/TrkB signaling: Roles in plasticity changes and neurodegenerative diseases(2022) Moya-Alvarado, Guillermo; Guerra, Miguel V.; Tiburcio, Reynaldo; Bravo, Evelyn; Bronfman, Francisca C.Neurons are highly polarized cells that rely on the intracellular transport of organelles. This process is regulated by molecular motors such as dynein and kinesins and the Rab family of monomeric GTPases that together help move cargo along microtubules in dendrites, somas, and axons. Rab5-Rab11 GTPases regulate receptor trafficking along early-recycling endosomes, which is a process that determines the intracellular signaling output of different signaling pathways, including those triggered by BDNF binding to its tyrosine kinase receptor TrkB. BDNF is a well-recognized neurotrophic factor that regulates experience-dependent plasticity in different circuits in the brain. The internalization of the BDNF/TrkB complex results in signaling endosomes that allow local signaling in dendrites and presynaptic terminals, nuclear signaling in somas and dynein-mediated long-distance signaling from axons to cell bodies. In this review, we briefly discuss the organization of the endocytic pathway and how Rab11-recycling endosomes interact with other endomembrane systems. We further expand upon the roles of the Rab11-recycling pathway in neuronal plasticity. Then, we discuss the BDNF/TrkB signaling pathways and their functional relationships with the postendocytic trafficking of BDNF, including axonal transport, emphasizing the role of BDNF signaling endosomes, particularly Rab5-Rab11 endosomes, in neuronal plasticity. Finally, we discuss the evidence indicating that the dysfunction of the early-recycling pathway impairs BDNF signaling, contributing to several neurodegenerative diseases.