Browsing by Author "Burgos, Patricia, V"

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    Clathrin adaptor AP-1-mediated Golgi export of amyloid precursor protein is crucial for the production of neurotoxic amyloid fragments
    (2022) Januario, Yunan C.; Eden, Jessica; de Oliveira, Luan S.; De Pace, Raffaella; Tavares, Lucas A.; da Silva-Januario, Mara E.; Apolloni, Vinicius B.; Wilby, Elise L.; Altmeyer, Randolf; Burgos, Patricia, V; Correa, Sonia A. L.; Gershlick, David C.; daSilva, Luis L. P.
    One of the hallmarks of Alzheimer's disease is the accumulation of toxic amyloid-beta (A beta) peptides in extracellular plaques. The direct precursor of A beta is the carboxyl-terminal fragment beta (or C99) of the amyloid precursor protein (APP). C99 is detected at elevated levels in Alzheimer's disease brains, and its intracellular accumulation has been linked to early neurotoxicity independently of A beta. Despite this, the causes of increased C99 levels are poorly understood. Here, we demonstrate that APP interacts with the clathrin vesicle adaptor AP-1 (adaptor protein 1), and we map the interaction sites on both proteins. Using quantitative kinetic trafficking assays, established cell lines and primary neurons, we also show that this interaction is required for the transport of APP from the trans-Golgi network to endosomes. In addition, disrupting AP-1-mediated transport of APP alters APP processing and degradation, ultimately leading to increased C99 production and A beta release. Our results indicate that AP-1 regulates the subcellular distribution of APP, altering its processing into neurotoxic fragments.
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    GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation
    (2020) Arriagada, Cecilia; Cavieres, Viviana A.; Luchsinger, Charlotte; Gonzalez, Alexis E.; Munoz, Vanessa C.; Cancino, Jorge; Burgos, Patricia, V; Mardones, Gonzalo A.
    Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.
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    Herpes Simplex Virus Type 1 Enhances Expression of the Synaptic Protein Arc for Its Own Benefit
    (2019) Acuna-Hinrichsen, Francisca; Munoz, Mariela; Hott, Melissa; Martin, Carolina; Mancilla, Evelyn; Salazar, Paula; Leyton, Luis; Zambrano, Angara; Concha, Margarita, I; Burgos, Patricia, V; Otth, Carole
    Herpes simplex virus type 1 (HSV-1) is a neurotropic virus able to reach the central nervous system (CNS) after primary infection in oronasal mucosa. HSV-1 establishes latency inside neurons due the repression of its gene expression process, which is related to periodic reactivations in response to cellular stress conditions, constituting a risk factor for neurodegenerative diseases such as Alzheimer's disease (AD). The immediate-early gene Arc plays an essential role in neuronal morphology, synaptic plasticity and memory formation. Arc acts as a hub protein, interacting with components of the endocytic machinery required for AMPA receptor (AMPAR) recycling as well as with proteins of the post-synaptic density and actin cytoskeleton. However, to date, no studies have evaluated whether persistent neurotropic HSV-1 infection modulates the expression or function of Arc protein in brain tissue. Here, we report that neuronal in vivo and in vitro infection of HSV-1 significantly increases Arc protein levels, showing a robust perinuclear distribution in neuronal cell lines, a process that is dependent on an active HSV-1 replication cycle. Finally, we found that silencing Arc protein caused a decrease in HSV-1 proteins and viral progeny, suggesting that Arc is involved in the lifecycle of HSV-1. Our studies strongly suggest that pathogenicity of HSV-1 neuronal reactivations in humans could be mediated in part by Arc neuronal upregulation and its potential role in endocytic trafficking and AMPA-neuronal function impairment. Further studies are necessary to define whether this phenomenon could have repercussions in cognition and learning processes in infected individuals.
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    Palmitic acid control of ciliogenesis modulates insulin signaling in hypothalamic neurons through an autophagy-dependent mechanism
    (SPRINGERNATURE, 2022) Avalos, Yenniffer; Paz Hernandez-Caceres, Maria; Lagos, Pablo; Pinto-Nunez, Daniela; Rivera, Patricia; Burgos, Paulina; Diaz-Castro, Francisco; Joy-Immediato, Michelle; Venegas-Zamora, Leslye; Lopez-Gallardo, Erik; Kretschmar, Catalina; Batista-Gonzalez, Ana; Cifuentes-Araneda, Flavia; Toledo-Valenzuela, Lilian; Rodriguez-Pena, Marcelo; Espinoza-Caicedo, Jasson; Perez-Leighton, Claudio; Bertocchi, Cristina; Cerda, Mauricio; Troncoso, Rodrigo; Parra, Valentina; Budini, Mauricio; Burgos, Patricia, V; Criollo, Alfredo; Morselli, Eugenia
    Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.
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    The Cervical and Meningeal Lymphatic Network as a Pathway for Retrograde Nanoparticle Transport to the Brain
    (2024) Ramos-Zaldivar, Hector; Polakovicova, Iva; Salas-Huenuleo, Edison; Yefi, Claudia P.; Silva-Ancahuail, David; Jara-Guajardo, Pedro; Oyarzun, Juan Esteban; Neira-Troncoso, Alvaro; Burgos, Patricia, V; Cavieres, Viviana A.; Arias-Munoz, Eloisa; Martinez, Carlos; Riveros, Ana L.; Corvalan, Alejandro H.; Kogan, Marcelo J.; Andia, Marcelo E.
    Introduction: The meningeal lymphatic vessels have been described as a pathway that transports cerebrospinal fluid and interstitial fluid in a unidirectional manner towards the deep cervical lymph nodes. However, these vessels exhibit anatomical and molecular characteristics typical of initial lymphatic vessels, with the absence of surrounding smooth muscle and few or absent valves. Given its structure, this network could theoretically allow for bidirectional motion. Nevertheless, it has not been assessed as a potential route for nanoparticles to travel from peripheral tissues to the brain. Methods: We employed superparamagnetic iron oxide nanoparticles (SPIONs), exosomes loaded with SPIONs, gold nanorods, and Chinese ink nanoparticles. SPIONs were prepared via chemical coprecipitation, while exosomes were isolated from the B16F10 melanoma cell line through the Exo-Spin column protocol and loaded with SPIONs through electroporation. Gold nanorods were functionalized with polyethylene glycol. We utilized C57BL/6 mice for post-mortem and in vivo procedures. To evaluate the retrograde directional flow, we injected each nanoparticle solution in the deep cervical lymph node. The head and neck were fixed for magnetic resonance imaging and histological analysis. Results: Here we show that extracellular vesicles derived from the B16F10 melanoma cell line, along with superparamagnetic iron oxide nanoparticles, gold nanorods, and Chinese ink nanoparticles can reach the meningeal lymphatic vessels and the brain of C57BL/6 mice after administration within the deep cervical lymph nodes post-mortem and in vivo, exclusively through lymphatic structures. Discussion: The functional anatomy of dural lymphatics has been found to be conserved between mice and humans, suggesting that our findings may have significant implications for advancing targeted drug delivery systems using nanoparticles. Understanding the retrograde transport of nanoparticles through the meningeal lymphatic network could lead to novel therapeutic approaches in nanomedicine, offering new insights into fluid dynamics in both physiological and neuropathological contexts. Further research into this pathway may unlock new strategies for treating neurological diseases or enhancing drug delivery to the brain.
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    The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-to-ER Retrograde Transport
    (2020) Bustamante, Hianara A.; Cereceda, Karina; Gonzalez, Alexis E.; Valenzuela, Guillermo E.; Cheuquemilla, Yorka; Hernandez, Sergio; Arias-Munoz, Eloisa; Cerda-Troncoso, Cristobal; Bandau, Susanne; Soza, Andrea; Kausel, Gudrun; Kerr, Bredford; Mardones, Gonzalo A.; Cancino, Jorge; Hay, Ronald T.; Rojas-Fernandez, Alejandro; Burgos, Patricia, V
    Ubiquitination regulates several biological processes, however the role of specific members of the ubiquitinome on intracellular membrane trafficking is not yet fully understood. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1187 genes of the human "ubiquitinome" using amyloid precursor protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 with Capzimin (CZM) caused a robust increase in APP levels at the Golgi apparatus and the swelling of this organelle. We showed that this phenotype is the result of rapid inhibition of Golgi-to-ER retrograde transport, a pathway implicated in the early steps of the autophagosomal formation. Indeed, we observed that inhibition of PSMD14 with CZM acts as a potent blocker of macroautophagy by a mechanism related to the retention of Atg9A and Rab1A at the Golgi apparatus. As pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and the K63-Ub chains, act as a crucial regulatory factor for macroautophagy by controlling Golgi-to-ER retrograde transport.