Efecto patológico de la expansión de poliglutaminas de hTBP en la enfermedad neurodegenerativa SCA17 en Drosophila melanogaster

Las ataxias espinocerebelosas (SCAs) son trastornos progresivos en los que el cerebelo y el tronco cerebral se degeneran lentamente. El síntoma principal de las diversas SCAs que existen es el patrón de neurodegeneración cerebelar que los pacientes exhiben. La ataxia espinocerebelosa 17 (SCA17) también conocida como enfermedad de Huntington-4 (HDL4), es causada por una expansión del trinucleótido CAG/CAA en las regiones polyQ del gen que codifica al factor transcripcional TBP humano (hTBP). El umbral fisiológico de las glutaminas totales presentes en hTBP es de alrededor de 30 a 40 repeticiones y un número mayor de 40 causa la patología. En esta tesis analizamos el efecto neuropatológico de hTBP con la región polyQ expandida (80Q) modelado en D. melanogaster. La estrategia experimental consistió en la generación de las moscas transgénicas para dirigir la expresión específica de hTBP34Q y 80Q en el cerebro y en los ojos de Drosophila. La agregación de proteínas en el cerebro de las moscas se determinó mediante inmunofluorescencia y la muerte celular fue evaluada utilizando el ensayo TUNEL. Para determinar el estado de la marcha de las moscas que expresaban hTBP34Q y 80Q en el cerebro se realizó la prueba de escalada en moscas adultas. Los resultados obtenidos mostraron exitosamente la expresión especifica de tejido de hTBP 34 y 80Q en el cerebro y ojos de Drosophila usando los promotores ELAV y GMR respectivamente. La expresión de hTBP34 y 80Q causó fenotipos aberrantes en el ojo de Drosophila. Así mismo, la expresión de hTBP80Q en el cerebro mostró ser neurotóxica acortando el tiempo de vida de las moscas. Las imágenes confocales mostraron que la agregación de hTBP34Q y 80Q en el cerebro de Drosophila es dependiente de la edad de las moscas y del tamaño de la expansión, donde los agregados de hTBP80Q mostraron un incremento de los 2 a los 20 días. La expresión de hTBP 34 y 80Q provocó muerte neuronal dependiente de la edad y las moscas hTBP80Q fueron severamente afectadas en las pruebas de escalada. Estos resultados permitieron concluir claramente el efecto neurotóxico de hTBP80Q en el cerebro de la mosca que acortó su tiempo de vida y capacidad locomotriz debido a la formación de los agregados proteicos y muerte celular por lo que abren la posibilidad para evaluar el tipo de muerte celular producida en las neuronas afectadas y el comportamiento molecular de estas expansiones polyQs en las patologías neurodegenerativas como SCA17. SUMMARY Spinocerebellar ataxias (SCAs) are progressive disorders in which the cerebellum and the brain stem slowly degenerate. We modeled spinocerebellar ataxia 17 (SCA17) in Drosophila melanogaster, also known as Huntington-4-like disease (HDL4). SCA17 is caused by an expansion of the CAG / CAA trinucleotide in the gene encoding polyQ regions in the human TBP protein (hTBP). The physiological threshold of the total glutamines present in non-pathological TBP is around 30 to 40 repetitions and a greater number of 40 causes the pathology. To analyze the neuropathological effect of hTBP with the extended polyQ (80Q) compared with wild type hTBP (34Q) using D. melanogaster as a model. Our experimental strategy consisted of addressing specific expression of hTBP with polyQ expansions of 34Q and 80Q using the eye driver GMR-GAL4 to evaluate eyephenotype toxicity due to these polyQ expansions. Then, we used ELAV-GAL4 to address hTBP34 and 80Q to the flies’ brain and measure protein aggregation, cell death and climbing ability. Protein aggregation in the flies’ brain was measured by immunofluorescence; cell death was evaluated using TUNEL and climbing test in adult flies were compared between 34Q and 80Q. Our results, showed that hTBP34 and 80Q expressed in the flies’ eyes caused an aberrant phenotype which was polyQ lengthdependent as well as age-dependent. hTBP80Q expression in the flies’ brain shortened its life span when compared with the control groups. Confocal images of the flies’ brains expressing hTBP34 and 80Q, showed that protein aggregation from hTBP80Q occurred from day 2 to day 20, and, hTBP34Q started aggregating at day 50. Expression of hTBP34 and 80Q caused neurodegeneration, hTBP80Q triggered more neuronal death than hTBP34Q. When testing climbing ability, hTBP80Q flies were severely affected when compared with the rest of the groups in the RING test. These results let us conclude that hTBP34 and 80Q are neurotoxic, form protein aggregates, cause neurodegeneration and affect the climbing ability of Drosophila melanogaster. We expect to complement these results with further experiments and analyze these polyQ expansions in additional models

Fornix volumetric increase during aging associates to microglia activation leading to defective cognitive performance

Background: Ageing displays a low-grade pro-inflammatory profile in blood and brain. It has been documented proinflammatory cytokines accumulation leading to neuroinflammation during aging. Aged brains integrate pro inflammatory cytokines accumulation, active microglia and volumetric changes which correlates with defective cognitive performance and neurodegeneration. Methods: Mice from 2-,12- and 20-months-old of age were submitted to different memory tests: Y-maze, Barnes maze, object location test and object location test. Afterwards, we performed structural MRI to evaluate macrostructural changes related to memory and learning regions. Following this, we also evaluated in peripheral blood and in brain tissue the presence of pro-inflammatory cytokines using the BioPlex platform. We also evaluated the presence of microglia and its morphology. Results: We found a progressive memory loss in an age-dependent manner among in the 12- and 20-months-old mice when compared with the 2-month-old mice. Regarding the MRI, it demonstrated that the fornix volume increased the most and, the left medial entorhinal cortex showed the most volume loss. Microglia number was augmented in fornix and decreased in medial entorhinal cortex which correlated with volume gain or loss, respectively. Microglia morphology was dystrophic and activated in fornix and in a “surveillance” phenotype in the medial entorhinal cortex. We found these phenotypes to be correlated to those volume changes we found in fornix and left medial entorhinal cortex. Conclusions: Here, we selectively identified an age-dependent proinflammatory profile and microglia activation favoring major volumetric brain changes in selective regions associated to cognitive decline in aged mice

Drosophila en el estudio de las interacciones proteicas de hTBP en el desarrollo y modelaje de SCA17

Antecedentes: Las interacciones proteicas participan en una gran cantidad de mecanismos moleculares que rigen los procesos celulares. La proteína de unión a la caja TATA humana (hTBP) interacciona con Antennapedia (Antp) a través de su extremo N-terminal, específicamente a través de sus homopéptidos de glutaminas. Esta región PolyQ sirve como sitio de unión a factores de transcripción en condiciones normales, pero cuando se expande genera la ataxia espinal cerebelosa 17 (SCA17), cuyos agregados proteicos en el cerebro impiden su funcionamiento correcto. Objetivo: Determinar si la región rica en glutaminas de hTBP interviene en su interacción con homeoproteínas y el papel que tiene en la formación de agregados proteicos en SCA17. Material y métodos: Se caracterizó la interacción de hTBP con otras homeoproteínas usando BiFC y se modeló SCA17 en Drosophila melanogaster dirigiendo hTBPQ80 al cerebro de las moscas usando UAS/GAL4. Resultados: Existió interacción de hTBP con homeoproteínas a través de su región rica en glutaminas. Los agregados proteicos de hTBP con las glutaminas expandidas afectaron la capacidad locomotriz de las moscas. Conclusiones: El estudio de las interacciones de hTBP abre la posibilidad para la búsqueda de nuevas estrategias terapéuticas en patologías neurodegenerativas como SCA17

Drosophila en el estudio de las interacciones proteicas de hTBP en el desarrollo y modelaje de SCA17

Antecedentes: Las interacciones proteicas participan en una gran cantidad de mecanismos moleculares que rigen los procesos celulares. La proteína de unión a la caja TATA humana (hTBP) interacciona con Antennapedia (Antp) a través de su extremo N-terminal, específicamente a través de sus homopéptidos de glutaminas. Esta región PolyQ sirve como sitio de unión a factores de transcripción en condiciones normales, pero cuando se expande genera la ataxia espinal cerebelosa 17 (SCA17), cuyos agregados proteicos en el cerebro impiden su funcionamiento correcto. Objetivo: Determinar si la región rica en glutaminas de hTBP interviene en su interacción con homeoproteínas y el papel que tiene en la formación de agregados proteicos en SCA17. Material y métodos: Se caracterizó la interacción de hTBP con otras homeoproteínas usando BiFC y se modeló SCA17 en Drosophila melanogaster dirigiendo hTBPQ80 al cerebro de las moscas usando UAS/GAL4. Resultados: Existió interacción de hTBP con homeoproteínas a través de su región rica en glutaminas. Los agregados proteicos de hTBP con las glutaminas expandidas afectaron la capacidad locomotriz de las moscas. Conclusiones: El estudio de las interacciones de hTBP abre la posibilidad para la búsqueda de nuevas estrategias terapéuticas en patologías neurodegenerativas como SCA1

Maternal Overnutrition Programs Central Inflammation and Addiction-Like Behavior in Offspring

Obesity or maternal overnutrition during pregnancy and lactation might have long-term consequences in offspring health. Fetal programming is characterized by adaptive responses to specific environmental conditions during early life stages. Programming alters gene expression through epigenetic modifications leading to a transgenerational effect of behavioral phenotypes in the offspring. Maternal intake of hypercaloric diets during fetal development programs aberrant behaviors resembling addiction in offspring. Programming by hypercaloric surplus sets a gene expression pattern modulating axonal pruning, synaptic signaling, and synaptic plasticity in selective regions of the reward system. Likewise, fetal programming can promote an inflammatory phenotype in peripheral and central sites through different cell types such as microglia and T and B cells, which contribute to disrupted energy sensing and behavioral pathways. The molecular mechanism that regulates the central and peripheral immune cross-talk during fetal programming and its relevance on offspring’s addictive behavior susceptibility is still unclear. Here, we review the most relevant scientific reports about the impact of hypercaloric nutritional fetal programming on central and peripheral inflammation and its effects on addictive behavior of the offspring

Priming of Hypothalamic Ghrelin Signaling and Microglia Activation Exacerbate Feeding in Rats’ Offspring Following Maternal Overnutrition

Maternal overnutrition during pregnancy leads to metabolic alterations, including obesity, hyperphagia, and inflammation in the offspring. Nutritional priming of central inflammation and its role in ghrelin sensitivity during fed and fasted states have not been analyzed. The current study aims to identify the effect of maternal programming on microglia activation and ghrelin-induced activation of hypothalamic neurons leading to food intake response. We employed a nutritional programming model exposing female Wistar rats to a cafeteria diet (CAF) from pre-pregnancy to weaning. Food intake in male offspring was determined daily after fasting and subcutaneous injection of ghrelin. Hypothalamic ghrelin sensitivity and microglia activation was evaluated using immunodetection for Iba-1 and c-Fos markers, and Western blot for TBK1 signaling. Release of TNF-alpha, IL-6, and IL-1β after stimulation with palmitic, oleic, linoleic acid, or C6 ceramide in primary microglia culture were quantified using ELISA. We found that programmed offspring by CAF diet exhibits overfeeding after fasting and peripheral ghrelin administration, which correlates with an increase in the hypothalamic Iba-1 microglia marker and c-Fos cell activation. Additionally, in contrast to oleic, linoleic, or C6 ceramide stimulation in primary microglia culture, stimulation with palmitic acid for 24 h promotes TNF-alpha, IL-6, and IL-1β release and TBK1 activation. Notably, intracerebroventricular (i.c.v.) palmitic acid or LPS inoculation for five days promotes daily increase in food intake and food consumption after ghrelin administration. Finally, we found that i.c.v. palmitic acid substantially activates hypothalamic Iba-1 microglia marker and c-Fos. Together, our results suggest that maternal nutritional programing primes ghrelin sensitivity and microglia activation, which potentially might mirror hypothalamic administration of the saturated palmitic acid