3.06 Tesis doctorado

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Browsing 3.06 Tesis doctorado by browse.metadata.categoriaods "03 Salud y bienestar"

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    Control of motivation for palatable food by kappa opioid receptor signaling and the neuropeptide Orexin-A
    (2023) Sandoval Caballero, Carolina Loreto; Pérez Leighton, Claudio; Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas
    Eating is a motivated behavior that provides nutrients to allow life. Animals evolved to perceive food as a positive reinforcer, resulting in increased motivation for food when the animal is hungry and decreased motivation when it is satiated. Motivation evolved to seek and eat food in an environment of food scarcity; however, it can become maladaptive in an environment with plenty food access, favoring excessive eating, weight gain, and obesity. Understanding the neuronal mechanisms regulating food motivation could improve therapies against obesity.Opioids and the hypothalamic Orexin/Dynorphin neurons are part of the brain mechanisms that regulate food intake and motivation for food. Opioids bind mainly to three G-protein coupled receptors (kappa, mu, and delta), which are expressed in brain regions that regulate food intake and motivation. Still, the contribution of each opioid receptor to food intake and motivation for food is unclear. The Orexin/Dynorphin neurons co-release the excitatory neuropeptides Orexins (e.g., Orexin-A and Orexin-B) and the inhibitory opioids Dynorphin (e.g., Dynorphin-A 1-13) into different areas, including the Oxytocin neurons from the paraventricular hypothalamic nucleus. The effects of Orexin and Dynorphin peptides in eating behavior are brain-site specific. For example, in the paraventricular hypothalamic nucleus, Dynorphin-A1-13 increases and Orexin-A decreases palatable food intake (i.e., tasty food, generally high in sugars and fats). Still, the opposite effect is observed in the ventral tegmental area. Dynorphin-A1-13 binds mainly to the kappa opioid receptor, which is expressed in the anorexigenic oxytocin neurons of the paraventricular hypothalamic nucleus; however, whether inhibition of oxytocin neurons is involved in the orexigenic effects of Dynorphin-A1-13 in the paraventricular hypothalamic nucleus is unknown. Also, in this brain region, Orexin-A blocks the orexigenic effects of Dynorphin-A1-13. Still, it is unclear whether these effects are mediated by changes in motivation for palatable food and whether the endogenous kappa opioid receptor signaling in the paraventricular hypothalamic nucleus regulates motivation for food. We hypothesize that in the paraventricular hypothalamic nucleus, Dynorphin-A1-13 signaling through the kappa opioid receptor promotes motivation for sucrose, and Orexin-A inhibits this behavior. We evaluated this hypothesis through four specific aims. [1] To determine through a meta-analysis the effects of kappa, mu, and delta opioid receptor ligands on food intake and motivation. We found that: (1) agonists increased food intake after central injections while antagonists decreased food intake after central and peripheral injections. (2) We did not find differences between the effects of opioid receptor ligands on standard rodent food intake after injections in the paraventricular hypothalamic nucleus compared to other brain sites. (3) Mu agonists had the strongest orexigenic effect compared to kappa and delta agonists, with the most significant effect on fat intake. (4) Whereas only centrally administered kappa antagonists reduce feeding, peripherally administered antagonists for all opioid receptor subtypes reduce feeding; (5) peripheral administration of antagonists decreases motivation for food regardless of food type. [2] To determine whether in the paraventricular hypothalamic nucleus signaling through kappa opioid receptor mediates the ability of Dynorphin-A1-13 to increase motivation for sucrose. We assessed motivation for sucrose using a progressive ratio schedule in which mice must perform work licks to obtain sucrose. After each sucrose reward obtention, there was a 20 sec time-out period in which licks were recorded, but sucrose could not be obtained. We found that in the paraventricular hypothalamic nucleus and relative to the vehicle: (1) bilateral administration of the kappa opioid antagonist nor-binaltorphimine increased the time to obtain sucrose rewards and decreased sucrose work and time-out licks in the early and middle stages of the sucrose progressive ratio. These effects were also found after subcutaneous administration of nor-binaltorphimine. (2) Unilateral Dynorphin-A1-13 administration increased the time to obtain sucrose rewards and decreased sucrose work licks in the middle and late stages of the sucrose progressive ratio without changing time-out licks. (3) Bilateral Dynorphin-A1-13 administration did not change the time to obtain sucrose rewards. However, it strongly decreased sucrose work licks in the early and middle stages of the sucrose progressive ratio. Also, nor-binaltorphimine did not block Dynorphin-A1-13 effects. Finally, we could not downregulate the expression of the kappa opioid receptor in the paraventricular hypothalamic nucleus and thus, these effects in motivation for sucrose were not performed. [3] To determine whether oxytocin neurons in the paraventricular hypothalamic nucleus express the kappa opioid receptor and whether blocking the kappa opioid receptor activates Oxytocin neurons in this brain region. Published data demonstrated that the kappa opioid receptor is expressed in oxytocin neurons in the paraventricular hypothalamic nucleus. Thus, we assessed whether nor-binaltorphimine activates oxytocin neurons and decreases food intake in mice. We found that nor-binaltorphimine administration in the paraventricular hypothalamic nucleus decreased cafeteria diet intake and abolished the negative correlation between the percentage of active caudal oxytocin neurons and cafeteria diet intake established after vehicle injection. [4] To determine whether activation of the kappa opioid receptor by Dynorphin-A1-13 and orexin receptors by Orexin-A in the paraventricular hypothalamic nucleus have opposing effects on motivation for sucrose. We found that in the paraventricular hypothalamic nucleus and relative to vehicle (1) Unilateral Orexin-A administration increased sucrose work and time-out licks in the middle and late stages of the sucrose progressive ratio without changing the time to obtain sucrose rewards and time-out licks. (2) Orexin-A decreases the demotivational effects of Dynorphin-A1-13.We conclude that signaling through orexin and opioid receptors modulates feeding behaviors and motivation for food. Those effects depend on variables including the dose and administration route of the drug, food tested, and circadian period in which the outcomes were measured. Dynorphin-A1-13 and nor-binaltorphimine in the paraventricular hypothalamic nucleus decreased sucrose work licks, and Orexin-A blocked the demotivational effects of Dynorphin-A1-13 over time. We reasoned that the demotivational effects of nor-binaltorphimine could involve the activation of anorexigenic oxytocin neurons, and the demotivational effects of Dynorphin-A1-13 could be explained by their signaling through other receptors besides the kappa opioid receptor, such as Mu opioid receptor. We conclude that during the active phase of the mice, Orexin-A potentiates the orexigenic mechanisms that are naturally active in mice and increases locomotion and motivation. Consequently, the orexigenic effects of Dynorphin-A1-13 could not be detected because orexigenic mechanisms reached a ceiling effect. Thus, instead of promoting motivation for sucrose, Dynorphin-A1-13 prompts allodynia that decreases feeding and motivated behaviors. 
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    OCRL1 Regulates the endocytic trafficking of APOER2 and reelin signaling
    (2024) Fuentealba Perez, Luz Maria; Marzolo Canales, Maria Paz; Pontificia Universidad Católica de Chile. Facultad de Ciencias Bológicas
    Lowe Syndrome is a severe disorder characterized by renal, ocular, and neurological signs. Mutations in the gene encoding the OCRL1 phosphatase are recognized as responsible for this syndrome. OCRL1 targets membrane phospholipids called phosphoinositides, with PI(4,5)P2 being the most common. The functions of OCRL1 are therefore associated with the regulation of intracellular trafficking and protein sorting. Mutations in OCRL1 affect different domains of the protein, both in enzymatic activity and in protein-binding domains, making it very difficult to describe a specific mechanism to explain this syndrome. Little is known about the nervous system, but neurological problems such as intellectual disability, behavioral disorders, and in some cases repetitive and stereotyped behaviors have been described. Interestingly, Reelin has functions in the development of the central nervous system, memory, and learning, and it signals by binding to membrane receptors of the LRP family, such as ApoER2/LRP8 and VLDLR, with ApoER2 being the most relevant. ApoER2 is internalized and recycled, and its intracellular trafficking is important for its function, as inhibiting its recycling significantly reduces Reelin signaling. Therefore, our study aims to elucidate whether the effects of OCRL1 on ApoER2 trafficking are reflected in a disturbance in Reelin signaling and, with this, to explain part of the neurological damage that occurs in Lowe Syndrome. Here, we show that ApoER2 passes through OCRL1-positive compartments and, furthermore, that the absence of OCRL1 disrupts the normal trafficking of ApoER2, affecting its availability at the cell surface and decreasing its half-life. Moreover, we detected a dysfunction in the signaling triggered by Reelin in our LS neuronal model. Thus, our study suggests that the disruption of the ApoER2/Reelin signaling pathway due to OCRL1 dysfunction may be a contributing factor to the neurological manifestations observed in Lowe Syndrome patients.
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    Rol de la E3 ligasa NEDD4-1 en la regulación de Mitofagia en células miogénicas
    Salas Venegas, Jeremy Antonio; Olguín Marín, Hugo César; Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas
    El músculo esquelético de los vertebrados cuenta con la capacidad de regenerarse tras sufrir un daño, gracias a una pequeña población de células troncales musculares conocidas como células satélite (CS), las cuales se mantienen en un estado quiescente o de reposo proliferativo. El daño muscular desencadena distintos eventos, incluyendo la liberación de señales que activan a las CS, induciendo la entrada al ciclo celular y proliferación, generando células hijas, las cuales irán a reparar una fibra dañada o formar una fibra muscular de novo. Parte de las CS activadas escapan del proceso de diferenciación celular, recuperando el estado troncal, permitiendo la renovación de la población de CS quiescentes. En este contexto, PAX7 ha sido descrito como un factor de transcripción fundamental para la función de las CS, ya que su expresión promueve la mantención/readquisición del estado troncal, mientras que la reducción de sus niveles proteicos, permite la progresión miogénica y diferenciación terminal. La ubiquitin ligasa NEDD4-1 es capaz de ubiquitinar a PAX7, promoviendo su degradación vía proteosoma. Otros blancos moleculares de NEDD4-1 han sido descritos en diversos procesos celulares, más su función en distintas vías asociadas a la regeneración muscular ha sido poco explorada. Recientemente se ha descrito que la autofagia favorece la regeneración muscular promoviendo la mantención de la troncalidad de las CS y la diferenciación de mioblastos a miotubos y miofibras. Investigaciones previas muestran que el silenciamiento de la expresión de NEDD4-1 inhibe el flujo autofágico a través de la desregulación de los niveles de p62 y LC3, proteínas claves en la selección de cargos y formación de los autofagosomas, respectivamente. Además, el silenciamiento de NEDD4-1 resulta en la acumulación de mitocondrias con formas aberrantes. Estas observaciones sugieren que NEDD4-1 podría tener funciones más complejas durante la progresión miogénica, ya que además de participar en la regulación de la troncalidad de las CS, podría participar en el control de la autofagia general o específica (ej. mitofagia). En base a estos antecedentes, en esta tesis doctoral se buscó entender cómo NEDD4-1 participa de la regulación de la mitofagia en células miogénicas, en el marco de los procesos de proliferación y diferenciación miogénica, junto a la regeneración muscular. Las estrategias experimentales principales que fueron utilizadas son: i) la pérdida de función de NEDD4-1 y ii) la modulación farmacológica de la autofagia. Metodológicamente, i) se abordó in vitro mediante silenciamiento (siRNA) e in vivo mediante un modelo de recombinación genética célula-específica; ii) se abordó principalmente in vitro (cultivos celulares). Tanto en i) como en ii) se midieron marcadores de regeneración (PAX7,MYOG,MHC), autofagia (LC3,p62) y masa mitocondrial (mtHSP70). Los resultados obtenidos en esta tesis doctoral indican que la ausencia de NEDD4-1 resulta en una regeneración muscular deficiente y la caída en marcadores miogénicos claves en el proceso regenerativo, a su vez se observa una población mitocondrial con fenotipo fragmentado, una caída de los niveles de marcadores de masa mitocondrial y de flujo autofágico. Estos resultados apoyan la hipótesis de que NEDD4-1 es una proteína importante dentro de la regulación de la homeostasis mitocondrial y flujo autofágico, ambos procesos que han sido demostrados como fundamentales para una correcta regeneración muscular.