Browsing by Subject "Autophagy"

Now showing 1 - 20 of 43
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    Advances in understanding mechanisms of long-term sperm storage-the soft-shelled turtle model
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Chen, Hong; Liu, Tengfei; Holt, William V.; Yang, Ping; Zhang, Linli; Zhang, Li; Han, Xiangkun; Bian, Xunguang; Chen, Qiusheng
    Long-term sperm storage is a special reproductive strategy, which can extend the time window between mating and fertilization in some animal species. Spermatozoa of the soft-shelled turtle, Pelodiscus sinensis, can be stored in the epididymis and oviduct for at least six months and one year, respectively. How spermatozoa can be stored in vivo for such a prolonged period is yet to be explained. We analyze the mechanisms that contribute to long-term sperm storage in P. sinensis, and compare them with other species from three different perspectives: the spermatozoon itself, the storage microenvironment and the interaction between the spermatozoon and microenvironment. Characteristics of soft-shelled turtle spermatozoa itself, such as the huge cytoplasmic droplet with its content of several large lipid droplets (LDs) and onion-like mitochondira, facilitate long-term sperm storage. The microenvironment of reproductive tract, involving in the secretions, structural barriers, exosomes, androgen receptors, Toll-like receptors and survival factor Bcl-2, are important for the maintenance of spermatozoa long-term storage. Sperm heads are always embedded among the oviductal cilia and even intercalate into the apical hollowness of the ciliated cells, indicating that the ciliated cells support the stored spermatozoa. RNA seq is firstly used to detect the molecular mechanism of sperm storage, which shows that autophagy, apoptosis and immune take part in the long-term sperm storage in this species
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    An insight into the role of autophagy in cell responses in the aging and neurodegenerative brain
    (F. Hernandez y JuanF. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología., 2012) Caballero, B; Coto-Montes, A.
    Oxidative stress, inflammation and the aggregation of oxidized, misfolded or aberrant proteins in the brain induces deregulations in programmed cell death: apoptosis and autophagy. Apoptosis is one of processes implicated in aging and neurodegenerative pathologies, and for the last decade, has been one of the most studied processes due to its essential role, not only in aging, but also in many neurodegenerative diseases, including Parkinson’s, Alzheimer’s and Huntington’s. However, autophagy being the major intracellular pathway for the degradation and recycling of long-live proteins and organelles is widely involved in the pathogenesis or prevention of many age-related diseases, including neurodegenerative conditions. Recently, autophagy activation has been considered as part of the cellular responses to elevated oxidative stress, eliminating unwanted, damaged and oxidative structures; thus favouring, in this way, the key anti-aging mechanism associated with the caloric restriction. Longevity factors, such as sirtuins, and redox-sensitive transcriptional factors, such as NF-κB and p53, can also regulate basal autophagy in cells, with a direct impact on longevity and the development of inflammation and neurodegeneration. Here, we reviewed the critical changes of autophagy in the aging and neuro-degenerative brain and the role of key regulators of autophagy, which are directly related to oxidative stress, inflammation and longevity pathways.
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    ATG7 immunohistochemical expression in malignant pleural mesothelioma. A preliminary report
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Rapisarda, Venerando; Broggi, Giuseppe; Caltabiano, Rosario; Lombardo, Claudia; Castorina, Sergio; Trovato, Angela; Ledda, Caterina; Filetti, Veronica; Loreto, Carla
    Literature evidence has demonstrated a high incidence of asbestos-related malignant pleural mesothelioma (MPM) in a Sicilian town (Biancavilla, Italy), where fluoro-edenite (FE) fibers were discovered some decades ago. As ATG7 immunohistochemical analysis has been ascribed as a prognostic tool of improved survival, we decided to investigate, in MPM patients, exposed and not exposed to FE fibers, the immunohistochemical expression of this autophagyrelated protein named ATG7. We analyzed the correlation between ATG7 immunohistochemical level and clinicopathological parameters. Twenty MPM tissue samples, from patients with available clinical and follow-up data, were included in paraffin and processed for immunohistochemistry. The immunohistochemical results confirmed activation of the autophagic process in MPM. Densitometric and morphometric expressions of ATG7 were significantly increased in MPMs when compared to the control tissues. A significant association of a high level of ATG7 with increased survival was demonstrated, with a mean overall survival (OS) of 12.5 months for patients with high expression vs. a mean OS of 4.5 months for patients with low ATG7 expression. In addition, a significant correlation between ATG7 expression and the survival time of MPM patients was observed. This study represents a starting point to hypothesize the prognostic role of ATG7 which could be a reliable prognostic indicator in MPM
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    Autofagia y canibalismo como mecanismos subversivos en dos cuentos de Silvina Ocampo y Virgilio Piñera
    (2019-01-18) Izaguirre Fernández, Belén
    Contemporáneos y narrado- res hispanoamericanos, Virgilio Piñera y Silvina Ocampo compartieron no solo nexos culturales, influencias artísticas y gustos literarios, sino también argumen- tos, rasgos técnicos y formales. Esto es lo que demuestra el presente artículo, los vínculos entre ellos a través de los cuentos “La carne” de Virgilio Piñera y “Malva” de Ocampo. Ambos ensayan aquí con el tema del canibalismo y la autofagia de forma similar y con igual fin: la utilización del cuerpo humano como mecanismo subversivo, como forma de ruptura con las imposiciones y restricciones del gobierno de Fulgencio Baptista en el caso del primero, y contra el dominio patriarcal y las exigencias sobre el género y el cuerpo femenino en el cuento de la argentina.
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    Autophagy activation promotes removal of damaged mitochondria and protects against renal tubular injury induced by albumin overload
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2018) Tan, Jin; Wang, Miaohong; Song, Shuling; Miao, Yuyang; Zhang, Qiang
    Proteinuria (albuminuria) is an important cause of aggravating tubulointerstitial injury. Previous studies have shown that autophagy activation can alleviate renal tubular epithelial cell injury caused by urinary protein, but the mechanism is not clear. Here, we investigated the role of clearance of damaged mitochondria in this protective effect. We found that albumin overload induces a significant increase in turnover of LC3-II and decrease in p62 protein level in renal proximal tubular (HK-2) cells in vitro. Albumin overload also induces an increase in mitochondrial damage. ALC, a mitochondrial torpent, alleviates mitochondrial damage induced by albumin overload and also decreases autophagy, while mitochondrial damage revulsant CCCP further increases autophagy. Furthermore, pretreatment of HK-2 cells with rapamycin reduced the amount of damaged mitochondria and the level of apoptosis induced by albumin overload. In contrast, blocking autophagy with chloroquine exerted an opposite effect. Taken together, our results indicated autophagy activation promotes removal of damaged mitochondria and protects against renal tubular injury caused by albumin overload. This further confirms previous research that autophagy activation is an adaptive response in renal tubular epithelial cells after urinary protein overload.
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    Autophagy and the regulation of the immune response
    (2012-12) Macián, Fernando; Valdor Alonso, Rut; Bioquímica y Biología Molecular B e Inmunología
    Autophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a crucial role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins and regulating protein levels in response to extracellular signals. In the last few years specific functions for different forms of autophagy have been identified in many tissues and organs. In the Immune System, autophagy functions range from the elimination infectious agents and the modulation of the inflammatory response, to the selection of antigens for presentation and the regulation of T cell homeostasis and activation. Here, we review the recent advances that have allowed us to better understand why autophagy is a crucial process in the regulation of the innate and adaptive immune responses.
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    Autophagy in neurons a review
    (Murcia : F. Hernández, 2002) Larsen, K.E.; Sulzer, D.
    Macroautophagy is a process of regulated turnover of cellular constituents that occurs during development and under conditions of stress such as starvation. Defects in autophagy have serious consequences, as they have been linked to neurodegenerative disease, cancer, and cardiomyopathy. This process, which exists in all eukaryotic cells, is tightly controlled, but in extreme cases results in the death of the cell. While major insights into the molecular and biochemical pathways involved have come from genetic studies in yeast, little is known about autophagic pathways in mammalian cells, particularly in neurons. Recently, research in neuronal culture models has begun to identify some characteristics of neuronal macroautophagy. The results suggest that macroautophagy in neurons may provide a neuroprotective mechanism. Here, we review the defining characteristics of autophagy with special attention to its role in neurodegenerative disorders, and recent efforts to delineate the pathway of autophagic protein degradation in neurons.
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    Autophagy in the immunosuppressive perivascular microenvironment of glioblastoma
    (MDPI, 2019-12-31) Molina Gallego, María Luisa; Martínez Pérez, Salvador; García Bernal, David; Valdor Alonso, Rut; Bioquímica y Biología Molecular B e Inmunología
    Glioblastoma (GB) has been shown to up-regulate autophagy with anti- or pro-oncogenic effects. Recently, our group has shown how GB cells aberrantly up-regulate chaperone-mediated autophagy (CMA) in pericytes of peritumoral areas to modulate their immune function through cell-cell interaction and in the tumor’s own benefit. Thus, to understand GB progression, the effect that GB cells could have on autophagy of immune cells that surround the tumor needs to be deeply explored. In this review, we summarize all the latest evidence of several molecular and cellular immunosuppressive mechanisms in the perivascular tumor microenvironment. This immunosuppression has been reported to facilitate GB progression and may be differently modulated by several types of autophagy as a critical point to be considered for therapeutic interventions.
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    Autophagy related markers (Beclin-1 and ATG4B) are strongly expressed in Wilms' tumor and correlate with favorable histology
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2019) Guimei, Maha; Ahmed Eladl, Mohamed; Vinod Ranade, Anu; Manzoor, Shaista
    Background. Wilms’ tumor treatment has achieved great success in the last decade. Nevertheless, some cases still fail to respond to the current multimodality therapy. These cases fall mainly in the unfavorable histology group with very few belonging to the favorable histology group. In recent years, autophagy manipulation whether inhibition or stimulation has been shown to affect cancer cell behavior and has emerged as a novel mechanism to improve cancer cell response to currently used therapeutic regimens. Objective. The current study aimed to investigate the expression of autophagy related markers (ATG4B and Beclin1) in WT, its association with the different clinicpathological parameters and its impact on patient survival. Methods. Twenty-one formalin fixed paraffin embedded (FFPE) WT specimens were immunohistochemically stained using autophagy related markers; Beclin-1 and ATG4B. All clinical, radiological and follow up data were retrieved from the patient records. Results. All specimens showed positive expression of both Beclin-1 and ATG4B. The staining score for Beclin1 varied between 50 and 300, and its expression was significantly associated with favorable histology (p=0.007). Similarly, ATG4B expression was significantly higher in favorable histology tumors compared to unfavorable histology (p=0.046). A statistically significant positive correlation between Beclin-1 and ATG4B expression was observed. The cumulative disease-free survival in patients with favorable histology was significantly higher compared to patients with unfavorable histology (p=0.0027). Conclusions. Beclin-1 and ATG4B expression were both found to be statistically significant discriminators of survival. Collectively these findings suggest that the expression of autophagy-related markers is associated with a favorable histology and could predict better survival in these patients.
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    Calnexin is essential for survival under nitrogen starvation and stationary phase in Schizosaccharomyces pombe
    (Public Library of Science, 2015-03-24) Núñez, Andrés; Dulude, Dominic; Jbel, Mehdi; Rokeach, Luis A.; Genética y Microbiología
    Cell fate is determined by the balance of conserved molecular mechanisms regulating death (apoptosis) and survival (autophagy). Autophagy is a process by which cells recycle their organelles and macromolecules through degradation within the vacuole in yeast and plants, and lysosome in metazoa. In the yeast Schizosaccharomyces pombe, autophagy is strongly induced under nitrogen starvation and in aging cells. Previously, we demonstrated that calnexin (Cnx1p), a highly conserved transmembrane chaperone of the endoplasmic reticulum (ER), regulates apoptosis under ER stress or inositol starvation. Moreover, we showedthat in stationary phase, Cnx1p is cleaved into two moieties, L_Cnx1p and S_Cnx1p. Here, weshowthattheprocessing of Cnx1p is regulated by autophagy, induced by nitrogen starvation or cell aging. The cleavage of Cnx1p involves two vacuolar proteases: Isp6, which is essential for autophagy, and its paralogue Psp3. Blocking autophagy through the knockout of autophagy-related genes (atg) results in inhibition of both, the cleavage and the trafficking of Cnx1p from the ER to the vacuole. We demonstrate that Cnx1pis required for cell survival under nitrogen-starvation and in chronological aging cultures. The death of the mini_cnx1 mutant (overlapping S_cnx1p) cells is accompanied by accumulation of high levels of reactive-oxygen species (ROS), a slowdown in endocytosis and severe cell-wall defects. Moreover, mutant cells expressing only S_Cnx1p showed cell wall defects. Co-expressing mutant overlapping the L_Cnx1p and S_Cnx1p cleavage products reverses the death, ROS phenotype and cell wall defect to wild-type levels. As it is involved in both apoptosis and autophagy, Cnx1p could be a nexus for the crosstalk between these pro-death and pro-survival mechanisms. Ours, and observations in mammalian systems, suggest that the multiple roles of calnexin depend on its sub-cellular localization and onits cleavage. The use of S. pombeshouldassist in further shedding light on the multiple roles of calnexin.
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    cAMP-Protein Kinase A and Stress-Activated MAP Kinase 1 signaling mediate transcriptional control of autophagy in fission yeast during glucose limitation or starvation.
    (Taylor and Francis Group, 2022-09-26) Pérez Díaz, Armando Jesús; Vázquez Marín, Beatriz; Vicente Soler, Jero; Prieto Ruiz, Francisco; Soto, Teresa; Franco, Alejandro; Cansado Vizoso, José; Madrid, Marisa; Genética y Microbiología
    Macroautophagy/autophagy is an essential adaptive physiological response in eukaryotes induced during nutrient starvation, including glucose, the primary immediate carbon and energy source for most cells. Although the molecular mechanisms that induce autophagy during glucose starvation have been extensively explored in the budding yeast Saccharomyces cerevisiae, little is known about how this coping response is regulated in the evolutionary distant fission yeast Schizosaccharomyces pombe. Here, we show that S. pombe autophagy in response to glucose limitation relies on mitochondrial respiration and the electron transport chain (ETC), but, in contrast to S. cerevisiae, the AMP-activated protein kinase (AMPK) and DNA damage response pathway components do not modulate fission yeast autophagic flux under these conditions. In the presence of glucose, the cAMP-protein kinase A (PKA) signaling pathway constitutively represses S. pombe autophagy by downregulating the transcription factor Rst2, which promotes the expression of respiratory genes required for autophagy induction under limited glucose availability. Furthermore, the stress-activated protein kinase (SAPK) signaling pathway, and its central mitogen-activated protein kinase (MAPK) Sty1, positively modulate autophagy upon glucose limitation at the transcriptional level through its downstream effector Atf1 and by direct in vivo phosphorylation of Rst2 at S292. Thus, our data indicate that the signaling pathways that govern autophagy during glucose shortage or starvation have evolved differently in S. pombe and uncover the existence of sophisticated and multifaceted mechanisms that control this self-preservation and survival response.
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    Chaperone-Mediated Autophagy in Pericytes: A Key Target for the Development of New Treatments against Glioblastoma Progression
    (MDPI, 2022-08-10) Salinas Hidalgo, María Dolores; Valdor Alonso, Rut; Bioquímica y Biología Molecular B e Inmunología
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    Clomiphene and dexamethasone inhibit apoptosis and autophagy via the ROS-JNK/MAPK-P21 signaling pathway in PCOS
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2025) Liu, Ruxia; Tang, Yuxiang; Chen, Xiangjun; Shang, Xintong
    Background. Polycystic ovarian syndrome (PCOS) is a complicated endocrine and metabolic disease, which seriously affects women’s health. However, the etiology and genetic basis of PCOS are complex, and the pathogenesis remains unclear. In this study, we aimed to explore the effects of clomiphene and dexamethasone on PCOS and their potential mechanisms. Methods. Sprague-Dawley (SD) rats were injected with dehydroepiandrosterone (DHEA) to establish a PCOS model. After treatment with clomiphene, dexamethasone, and their combination, ovarian tissue of rats was collected. The morphological changes in the ovary were observed by hematoxylin and eosin (HE) staining and Electron microscopy. The levels of oxidative stress and hormones were determined by ELISA. Apoptosis was assessed by TUNEL assay. The mechanism of clomiphene and dexamethasone effects on PCOS was explored by Immunohistochemical staining, real-time PCR, and western blotting. Results. Clomiphene and dexamethasone could improve the morphology of the ovary in PCOS. TUNEL assay and ELISA showed that clomiphene, dexamethasone, and their combination could inhibit apoptosis and significantly reverse the levels of ROS, T-SOD, CAT, T, and E2 in the ovary. Immunohisto-chemical staining revealed that clomiphene and dexamethasone could remarkably reduce the protein levels of Bax, Caspase-3, LC3II, p-JNK, p-P38 MAPK, and P21, and increase P62 and Bcl-2 protein expression. The mRNA levels of Bax, Bcl-2, and Caspase-3 were also modulated in the PCOS model with clomiphene and dexamethasone treatment. Additionally, western blotting indicated that clomiphene and dexamethasone significantly regulated the levels of Bax, Bcl-2, Caspase-3, LC3I, LC3II, P62, p-JNK, JNK, p-P38 MAPK, P38 MAPK, and P21 in PCOS rats. Conclusions. Clomiphene and dexamethasone can not only reduce oxidative damage, and inhibit apoptosis and autophagy, but they can also regulate the ROS-JNK/MAPK-P21 signaling pathway in PCOS rats. It provides an experimental basis for the clinical application of clomiphene and dexamethasone in PCOS
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    Datos de investigación Proyecto Estudio de la Biología del Pericito Dependiente de Autofagia Mediada por Chaperonas Como Diana Clave para el Desarrrollo de Terapias en Glioblastoma (PID2023-149111OB-I00)
    (2026-05-19) Salinas Hidalgo, María Dolores; Naranjo Sánchez, Elena; Valdor Alonso, Rut; Martínez González, Isabel María; Bioquímica y Biología Molecular B e Inmunología; Facultades de la UMU::Facultad de Medicina
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    Dian sanguis draconis improves pulmonary fibrosis by activating autophagy to regulate the PA/PAI-1 balance
    (Universidad de Murcia, Departamento de Histología e Histopatología, 2025) Song Jiayi; Li Qian; Yang Chunyan; Liu Qing; Li Jianmei; Fu Yi; Biología Celular e Histología
    Background. Pulmonary fibrosis (PF) is a severe lung disease that manifests as lung tissue destruction and collagen deposition and easily leads to respiratory failure. It is difficult to reverse these conditions using current treatment methods. This study focused on the exploration and development of novel drugs for the treatment of PF. Methods. This study simulated the pathological process of PF by using a bleomycin (BLM)-induced rat model and a TGF-β1-induced in vitro cell model. Dian sanguis draconis (DSD) was used for intervention, and the effects on the lung tissue structure, collagen fiber deposition, autophagy level and PA/PAI-1 balance were evaluated via pathological tissue staining, western blotting, and ELISA. Results. In untreated PF rats, severely disordered lung tissue, thickened alveolar septa, and excessive deposition of collagen fibers were observed. In addition, the level of autophagy was inhibited, and the balance of PA/PAI-1 in the lung tissue was disrupted. After treatment with DSD, these pathological injuries improved, as demonstrated by the restoration of lung tissue structure, reduction in collagen fiber deposition, recovery of autophagy levels, and remodeling of the PA/PAI-1 balance. In addition, mechanistically, DSD improves PF by increasing the level of autophagy-related proteins and regulating the PA/PAI-1 balance. Conclusion. This study confirmed the significant effect of DSD in alleviating PF. These findings provide new drug candidates for the treatment of PF
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    Enhanced autophagy and phagocytosis of apoptotic lymphocytes in splenic macrophages of acute ethanol-treated rats: Light and electron microscopic studies
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2024) Betsuyaku, Tsubasa; Ito, Yuko; Peake, Nicholas; Al Bari, Abdul Alim; El Akabawy, Gehan; Eid, Nabil
    Autophagy is a prosurvival mechanism for the clearance of damaged cellular components, specifically upon exposure to various stressors. In lymphoid organs, excessive ethanol consumption increases lymphocyte apoptosis, resulting in immunosuppression. However, ethanol-induced autophagy and related phagocytosis of apoptotic lymphocytes in the spleen have not been studied yet. Adult male Wistar rats were injected intraperitoneally either with 5 g/kg ethanol or phosphate-buffered saline (as a control group) and then sacrificed 0, 3, 6, and 24 hours after injection. Light and transmission electron microscopy (TEM) findings indicated enhanced T cell apoptosis in the white pulps of ethanol-treated rats (ETRs) compared with the control group, which peaked at 6h and was associated with the accumulation of tingible body macrophages (TBMs). These macrophages exhibited an upregulated autophagic response, as evidenced by enhanced LC3-II (a specific marker of autophagosomes) expression, which peaked at 24h. In addition, double labeling immunofluorescence of LC3-II with lysosomal markers revealed the enhanced formation of autolysosomes in TBMs of ETRs, which was associated with suppression of p62 immunostaining, indicating the enhanced autophagic flux. Interestingly, this elevated autophagic response in ETR TBMs was accompanied by evidence of LC3-associated phagocytosis (LAP) of apoptotic splenocytes. This is based on TUNEL/LC3-II double labeling and TEM observations of phagosomes containing apoptotic bodies, enclosed within phagosomal membranes adjacent to the autophagic vacuoles. It can be concluded that enhanced prosurvival autophagy in splenic TBMs of ETRs and clearing of apoptotic lymphocytes via LAP may contribute to preventing secondary necrosis and autoimmune diseases.
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    Ethanol enhances thymocyte apoptosis and autophagy in macrophages of rat thymi
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2017) Betsuyaku, Tsubasa; Eid, Nabil; Ito, Yuko; Tanaka, Yoshihisa; Otsuki, Yoshinori; Kondo, Yoichi
    Tingible body macrophages (TBMs) play essential roles in the phagocytosis of apoptotic lymphocytes, specifically under exposure to various stressors. Although excessive ethanol consumption may enhance thymocyte apoptosis, reports investigating the autophagic response of the thymus to ethanol toxicity are still lacking. We investigated apoptosis and autophagy in thymi of an animal model of binge ethanol exposure. Adult male Wistar rats were injected intraperitoneally either with 5 g/kg ethanol or phosphate buffer saline (for the control group) and sacrificed 0, 3, 6 and 24 hours after injection. Light and transmission electron microscopy (TEM) studies revealed enhanced formation of TBMs phagocytosing many apoptotic thymocytes in the thymic cortex of the ethanol-treated rats (ETRs), and this formation was particularly marked at 24 h. The macrophages showed signs of activation under TEM and immunofluorescence double labeling with RM4 (a macrophage marker) and iNOS. Additionally, in comparison to the control group, autophagy was enhanced in ETR thymic TBMs as evidenced ultrastructurally by accumulation of autophagic vacuoles, immunohistochemical increases in LC3 puncta, Western blot analysis of the latter protein, and colocalization of LC3 and RM4 in immunofluorescence double labeling. Immunoelectron microscopy also revealed LC3-labeled autophagic vacuoles and apoptotic cell phagosomes in ETR TBMs, suggesting the possibility of LC3-related phagocytosis. This was confirmed by enhanced colocalization of LC3 with lysosomal cathepsins in double labeling. These results indicate that enhanced autophagy in ETR thymic TBMs is not only a cytoprotective mechanism but could also be involved in the clearance of apoptotic thymocytes, thus preventing autoimmune reactions and suppressing inflammatory response.
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    Ex vivo 3D human corneal stroma model for Schnyder corneal dystrophy - role of autophagy in its pathogenesis and resolution
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2018) Szabó, Dóra Júlia; Nagymihály, Richárd; Veréb, Zoltán; Josifovska, Natasha; Noer, Agate; Liskova, Petra; Facskó, Andrea; Moe, Morten C.; Petrovski, Goran
    Introduction. Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. Methods. Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stromaderived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50nM), and inhibited by the autophagy inhibitor 3- methyladenine (3-MA, 10 mM) for 24 hours. Results. CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. Conclusions. MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.
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    Expression of long-chain noncoding RNA GAS5 in osteoarthritis and its effect on apoptosis and autophagy of osteoarthritis chondrocytes
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Ji, Qinghui; Qiao, Xiaofeng; Liu, Yongxiang; Wang, Dawei
    Objective. To investigate the expression of long-chain noncoding RNA GAS5 in osteoarthritis(OA) and the effect of silencing GAS5 on autophagy of osteoarthritis chondrocytes (OACs). Methods. OA rat models were constructed by cutting the anterior cruciate ligament, and the expressions of GAS5 in rat cartilage tissues at 4 weeks (early OA) and 12 weeks (late OA) after modeling were detected. The rat chondrocytes were isolated, cultured and transfected with si-GAS5 to silencing GAS5. Then, the changes of apoptosis and autophagy levels of OA chondrocytes were detected by transfection of GFP-LC3 and flow cytometry. Bioinformatic tools were used to analyze the miRNA binding to GAS5 and the downstream target genes, then luciferase reporter assay and GDC-0349 (inhibitor of mTOR) were used to verify their relationships. Results. The expression of GAS5 in cartilage tissue of OA rats was higher than control, which was higher in late OA than that in early OA. After silencing the GAS5, the autophagy ability of OACs was increased and the apoptosis rate was decreased. GAS5 was able to bind to miR-144 and regulate the expressin of mTOR. mTOR inhibitor GDC-0349 could reverse the inhibition of GAS5 on autophagy but could not reverse its effect on apoptosis. Conclusion. GAS5 expresses highly in OA cartilage tissues and increases with the progression of OA. GAS5 inhibits autophagy and promotes the apoptosis of OACs, and the inhibition of autophagy may be related to its regulation of mTOR
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    Focus on the Small GTPase Rab1: a key player in the pathogenesis of parkinson’s disease
    (MDPI, 2021-11-08) Martínez Menárguez, José Ángel; Martínez Alonso, Emma; Cara-Esteban, Mireia; Tomás, Mónica; Biología Celular e Histología; Facultad de Medicina
    Parkinson’s disease (PD) is the second most frequent neurodegenerative disease. It is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of large aggregates in the survival neurons called Lewy bodies, which mainly contain α-synuclein (α-syn). The cause of cell death is not known but could be due to mitochondrial dysfunction, protein homeostasis failure, and alterations in the secretory/endolysosomal/autophagic pathways. Survival nigral neurons overexpress the small GTPase Rab1. This protein is considered a housekeeping Rab that is necessary to support the secretory pathway, the maintenance of the Golgi complex structure, and the regulation of macroautophagy from yeast to humans. It is also involved in signaling, carcinogenesis, and infection for some pathogens. It has been shown that it is directly linked to the pathogenesis of PD and other neurodegenerative diseases. It has a protective effect against α–σψν toxicity and has recently been shown to be a substrate of LRRK2, which is the most common cause of familial PD and the risk of sporadic disease. In this review, we analyze the key aspects of Rab1 function in dopamine neurons and its implications in PD neurodegeneration/restauration. The results of the current and former research support the notion that this GTPase is a good candidate for therapeutic strategies. View Full-Text