Browsing by Author "Orellana, Juan A."

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    A preclinical mice model of multiple sclerosis based on the toxin-induced double-site demyelination of callosal and cerebellar fibers
    (2024) Vejar, Sebastián; Pizarro, Ignacio S.; Pulgar-Sepúlveda, Raúl; Vicencio, Sinay C.; Polit, Andrés; Amador, Cristian A.; Rio, Rodrigo del; Varas, Rodrigo; Orellana, Juan A.; Ortiz, Fernando C.
    Background: Multiple sclerosis (MS) is an irreversible progressive CNS pathology characterized by the loss of myelin (i.e. demyelination). The lack of myelin is followed by a progressive neurodegeneration triggering symptoms as diverse as fatigue, motor, locomotor and sensory impairments and/or bladder, cardiac and respiratory dysfunction. Even though there are more than fourteen approved treatments for reducing MS progression, there are still no cure for the disease. Thus, MS research is a very active field and therefore we count with different experimental animal models for studying mechanisms of demyelination and myelin repair, however, we still lack a preclinical MS model assembling demyelination mechanisms with relevant clinical-like signs. Results: Here, by inducing the simultaneous demyelination of both callosal and cerebellar white matter fibers by the double-site injection of lysolecithin (LPC), we were able to reproduce CNS demyelination, astrocyte recruitment and increases levels of proinflammatory cytokines levels along with motor, locomotor and urinary impairment, as well as cardiac and respiratory dysfunction, in the same animal model. Single site LPC-injections either in corpus callosum or cerebellum only, fails in to reproduce such a complete range of MS-like signs. Conclusion: We here report that the double-site LPC injections treatment evoke a complex MS-like mice model. We hope that this experimental approach will help to deepen our knowledge about the mechanisms of demyelinated diseases such as MS.
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    Chronic stress decreases the expression of sympathetic markers in the pineal gland and increases plasma melatonin concentration in rats
    (WILEY, 2006) Dagnino Subiabre, Alexies; Orellana, Juan A.; Carmona Fontaine, Carlos; Montiel, Juan; Diaz Veliz, Gabriela; Seron Ferre, Maria; Wyneken, Ursula; Concha, Miguel L.; Aboitiz, Francisco
    Chronic stress affects brain areas involved in learning and emotional responses. Although most studies have concentrated on the effect of stress on limbic-related brain structures, in this study we investigated whether chronic stress might induce impairments in diencephalic structures associated with limbic components of the stress response. Specifically, we analyzed the effect of chronic immobilization stress on the expression of sympathetic markers in the rat epithalamic pineal gland by immunohistochemistry and western blot, whereas the plasma melatonin concentration was determined by radioimmunoassay. We found that chronic stress decreased the expression of three sympathetic markers in the pineal gland, tyrosine hydroxylase, the p75 neurotrophin receptor and alpha-tubulin, while the same treatment did not affect the expression of the non-specific sympathetic markers Erk1 and Erk2, and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, these results were correlated with a significant increase in plasma melatonin concentration in stressed rats when compared with control animals. Our findings indicate that stress may impair pineal sympathetic inputs, leading to an abnormal melatonin release that may contribute to environmental maladaptation. In addition, we propose that the pineal gland is a target of glucocorticoid damage during stress.
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    Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage
    (2024) Gómez, Gonzalo I.; Alvear, Tanhia F.; Roa, Daniela A.; Farias-Pasten, Arantza; Vergara, Sergio A.; Mellado, Luis A.; Martinez-Araya, Claudio J.; Prieto Villalobos, Juan; García-Rodríguez, Claudia; Sánchez, Natalia; Sáez, Juan C.; Ortíz, Fernando C.; Orellana, Juan A.
    Background: Alcohol, a widely abused drug, significantly diminishes life quality, causing chronic diseases and psychiatric issues, with severe health, societal, and economic repercussions. Previously, we demonstrated that non-voluntary alcohol consumption increases the opening of Cx43 hemichannels and Panx1 channels in astrocytes from adolescent rats. However, whether ethanol directly affects astroglial hemichannels and, if so, how this impacts the function and survival of astrocytes remains to be elucidated. Results: Clinically relevant concentrations of ethanol boost the opening of Cx43 hemichannels and Panx1 channels in mouse cortical astrocytes, resulting in the release of ATP and glutamate. The activation of these large-pore channels is dependent on Toll-like receptor 4, P2X7 receptors, IL-1β and TNF-α signaling, p38 mitogen-activated protein kinase, and inducible nitric oxide (NO) synthase. Notably, the ethanol-induced opening of Cx43 hemichannels and Panx1 channels leads to alterations in cytokine secretion, NO production, gliotransmitter release, and astrocyte reactivity, ultimately impacting survival. Conclusion: Our study reveals a new mechanism by which ethanol impairs astrocyte function, involving the sequential stimulation of inflammatory pathways that further increase the opening of Cx43 hemichannels and Panx1 channels. We hypothesize that targeting astroglial hemichannels could be a promising pharmacological approach to preserve astrocyte function and synaptic plasticity during the progression of various alcohol use disorders.
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    Evidence for TGF-β1/Nrf2 Signaling Crosstalk in a Cuprizone Model of Multiple Sclerosis
    (2024) Guevara, Coram; Vicencio, Sinay C.; Pizarro, Ignacio S.; Villavicencio-Tejo, Francisca; Quintanilla, Rodrigo A.; Astudillo, Pablo; Ampuero, Estibaliz; Varas, Rodrigo; Orellana, Juan A.; Ortiz, Fernando C.
    Multiple sclerosis (MS) is a chronic and degenerative disease that impacts central nervous system (CNS) function. One of the major characteristics of the disease is the presence of regions lacking myelin and an oxidative and inflammatory environment. TGF-beta 1 and Nrf2 proteins play a fundamental role in different oxidative/inflammatory processes linked to neurodegenerative diseases such as MS. The evidence from different experimental settings has demonstrated a TGF-beta 1-Nrf2 signaling crosstalk under pathological conditions. However, this possibility has not been explored in experimental models of MS. Here, by using the cuprizone-induced demyelination model of MS, we report that the in vivo pharmacological blockage of the TGF-beta 1 receptor reduced Nrf2, catalase, and TGF beta-1 protein levels in the demyelination phase of cuprizone administration. In addition, ATP production, locomotor function and cognitive performance were diminished by the treatment. Altogether, our results provide evidence for a crosstalk between TGF-beta 1 and Nrf2 signaling pathways under CNS demyelination, highlighting the importance of the antioxidant cellular response of neurodegenerative diseases such as MS.
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    Hemichannels contribute to mitochondrial Ca2+ and morphology alterations evoked by ethanol in astrocytes
    (2024) Alvear, Tanhia F.; Farias-Pasten, Arantza; Vergara, Sergio A.; Prieto-Villalobos, Juan; Silva-Contreras, Antonia; Fuenzalida, Fernando A.; Quintanilla, Rodrigo A.; Orellana, Juan A.
    Alcohol, a toxic and psychoactive substance with addictive properties, severely impacts life quality, leading to significant health, societal, and economic consequences. Its rapid passage across the blood-brain barrier directly affects different brain cells, including astrocytes. Our recent findings revealed the involvement of pannexin-1 (Panx1) and connexin-43 (Cx43) hemichannels in ethanol-induced astrocyte dysfunction and death. However, whether ethanol influences mitochondrial function and morphology in astrocytes, and the potential role of hemichannels in this process remains poorly understood. Here, we found that ethanol reduced basal mitochondrial Ca2+ but exacerbated thapsigargin-induced mitochondrial Ca2+ dynamics in a concentration-dependent manner, as evidenced by Rhod-2 time-lapse recordings. Similarly, ethanol-treated astrocytes displayed increased mitochondrial superoxide production, as indicated by MitoSox labeling. These effects coincided with reduced mitochondrial membrane potential and increased mitochondrial fragmentation, as determined by MitoRed CMXRos and MitoGreen quantification, respectively. Crucially, inhibiting both Cx43 and Panx1 hemichannels effectively prevented all ethanol-induced mitochondrial abnormalities in astrocytes. We speculate that exacerbated hemichannel activity evoked by ethanol may impair intracellular Ca2+ homeostasis, stressing mitochondrial Ca2+ with potentially damaging consequences for mitochondrial fusion and fission dynamics and astroglial bioenergetics.
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    SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics
    (2023) Prieto Villalobos, Juan Carlos; Lucero, Claudia M.; Rovegno, Maximiliano; Gómez, Gonzalo I.; Retamal, Mauricio A.; Orellana, Juan A.
    Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels. Results: We found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP. Conclusions: We hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.