Browsing by Author "Arias-Munoz, Eloisa"

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    Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival
    (2022) Vargas, Gabriela; Cortes, Omar; Arias-Munoz, Eloisa; Hernandez, Sergio; Cerda-Troncoso, Cristobal; Hernandez, Laura; Gonzalez, Alexis E.; Tatham, Michael H.; Bustamante, Hianara A.; Retamal, Claudio; Cancino, Jorge; Varas-Godoy, Manuel; Hay, Ronald T.; Rojas-Fernandez, Alejandro; Cavieres, Viviana A.; Burgos, Patricia V.
    Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.
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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.