Estudio del estrés oxidativo hepático asociado a la enfermedad de Alzheimer. Efecto del tratamiento con bexaroteno y/o genisteína

La enfermedad de Alzheimer (EA) no tiene una etiología perfectamente definida. Una de las teorías que se barajan como causa de la EA es la del péptido β-amiloide (βA). Esta molécula es neurotóxica y es capaz de producir estrés oxidativo. Se forma principalmente en el cerebro y se acumula formando placas extracelulares. Se ha sugerido que el βA es el eje principal de la EA, y por tanto que a partir de esta molécula se desencadenan todos los procesos asociados a la EA, lo que se ha llamado la cascada amiloidea. Otra de las hipótesis es la que la relaciona con el estrés oxidativo. Este factor sería el agente que realmente provoca toda la serie de cambios descritos en los tejidos nerviosos, a través de las moléculas relacionadas con la EA, como el βA. La acumulación progresiva del daño oxidativo a lo largo de los años podría explicar que sea una enfermedad asociada al envejecimiento. El hígado tiene, entre otras, la función de servir como un agente detoxificante de la sangre, por lo que será muy importante para eliminar restos nocivos que se encuentren en el sistema sanguíneo, como el βA característico de esta patología neurológica. El βA puede llegar a la sangre por mediación del transportador LRP-1, que lo saca del cerebro a la sangre, y desde aquí lo introduce en el hígado. El transportador RAGE tiene la particularidad de que es capaz de sacarlo de la sangre e introducirlo de nuevo en el cerebro. Por evidentes razones éticas no es posible realizar ciertos estudios en humanos, como el análisis de órganos, por lo que se emplean modelos animales. El modelo elegido por nuestro grupo de investigación es el ratón transgénico APPswe/PS1dE9 para la EA, porque es un modelo que desarrolla particularidades de la patología asociada a la EA en humanos, como es el depósito y posterior aparición de las placas de βA en el ratón. El objetivo principal de la presente tesis es estudiar el estrés oxidativo hepático y los efectos del tratamiento con bexaroteno y/o genisteína asociados a la enfermedad de Alzheimer en un modelo animal. En la 1ª parte de esta tesis hemos analizado la relación entre el cerebro y el hígado. Trabajamos con machos y hembras wild type y APP/PS1 de diferentes edades, 3-5 meses, 10-13 meses y más de 20 meses, para ver la evolución de la patología asociada. Hemos analizado parámetros de estrés oxidativo en hígado, como la tasa de producción de peróxido de hidrógeno mitocondrial, oxidación proteica (medida como carbonilación), y lipoperoxidación (medida como malondialdehído). Para determinar esta relación medimos los transportadores LRP1, tanto en hígado como en cerebro, y RAGE solo en cerebro. También hemos determinado la concentración de βA en sangre. Los resultados muestran cómo en los ratones APP/PS1 disminuyen los diferentes parámetros de estrés oxidativo en hígado con la edad, mientras que la concentración de βA en sangre aumenta. Trabajamos con la hipótesis de que el hígado está detoxificando el βA y en este proceso está generando radicales libres. Esta capacidad de degradación disminuiría progresivamente con la edad, lo que explicaría por qué hay menos estrés oxidativo y más βA. Los resultados muestran que el transportador RAGE no cambia entre los diferentes grupos. Respecto al LRP1, disminuye con la edad en cerebro en ratones wild type y APP/PS1. En hígado disminuye con la edad solo en APP/PS1, y vemos que hay más en el ratón APP/PS1 que en el wild type en las edades de 3-5 meses y 10-13 meses. En resumen, vemos que el βA se forma en el cerebro, que es capaz de salir a la circulación sistémica por mediación del LRP1, en mayor medida en edades jóvenes. A pesar de esto vemos que hay un mayor nivel en sangre en el animal viejo. Puede volver al cerebro por mediación del RAGE, pero no parece ser importante ya que no hay cambios en las edades estudiadas. La diferencia fundamental viene en el flujo hacia el hígado. En el animal joven hay un flujo mucho mayor que en el viejo, al haber más βA, también se degradará en mayor medida y provocará los cambios en el estrés oxidativo. El estudio del mecanismo de aclaramiento cerebral del βA nos llevó a interesarnos por elementos que sean capaces de contribuir a este proceso. Al disminuir la patología amiloidea asociada a la EA, se espera contribuir a la mejora de los síntomas de la enfermedad. Recientemente se ha publicado un innovador tratamiento para la EA, que parece tener un efecto positivo al menos en modelos animales. Este tratamiento consiste en la administración de bexaroteno, que es un medicamento usado en el tratamiento del linfoma cutáneo. La particularidad que lo relaciona con la EA se da por 2 vías. Por un lado incrementa la fagocitosis del βA por la microglía. Por otro se une al dímero RXR-PPARg, lo que va a hacer que se sobreexprese la apolipoproteína E (apoE), que mediará la degradación del péptido del cerebro. Hace unos años nuestro grupo publicó que la genisteína incrementa los niveles de PPARg. Por tanto, pensamos que si la administración de este fitoestrógeno aumenta la disponibilidad del factor de transcripción puede incrementar la eficacia del fármaco. A parte de este efecto, la genisteína por sí sola puede tener efectos antioxidantes y neuroprotectores muy beneficiosos para la EA. En la 2ª parte de esta tesis, para probar un nuevo tratamiento para la EA diseñamos un experimento en el que hemos usado hembras ovariectomizadas, ya que la EA tiene una incidencia mayor en hembras a partir de la menopausia. Solo hemos usado APP/PS1 porque lo que nos interesa es el posible aclaramiento cerebral de βA. Usamos ratones de 8 meses porque a esta edad ya presentan placas de βA. Hemos determinado los niveles de βA en cerebro y sangre, las placas amiloideas en cerebro, y los niveles de PPARg y apoE en cerebro. También los niveles de transaminasas en sangre. Se ovariectomizó los animales al principio del experimento. Los tratamientos administrados son los siguientes: • Control con agua. • Genisteína durante todo el experimento a la concentración de 0,022 mg/kg/día. • Bexaroteno durante 3 días a la concentración de 100 mg/kg/día. • Tratamiento conjunto, primero con genisteína durante 3 días para que sobreexprese PPARg, y durante 3 días más con bexaroteno. Al medir los niveles de βA en cerebro se ve que los tratamientos reducen la patología amiloidea. Incluso se aprecia un cierto efecto sinérgico en el tratamiento conjunto. En conclusión podemos decir que el transportador LRP1 disminuye con el envejecimiento en cerebro e hígado. Ello hace que el flujo de βA que entra al hígado sea menor, y por tanto se degradará en menor medida y disminuirá el estrés oxidativo hepático con la edad. Los tratamientos con bexaroteno y/o genisteína son efectivos para reducir la patología amiloidea, en el modelo animal APP/PS1 para la enfermedad de Alzheimer.The etiology of Alzheimer's disease (AD) is not clearly defined, but one theory under consideration is that the cause of AD is amyloid β peptide (AB). This molecule is neurotoxic and is able to produce oxidative stress. It is produced mainly in the brain and forms extracellular plaques. It has been suggested that AB is the main cause of AD, that this molecule triggers all the processes associated with the AD; this has been called the amyloid cascade hypothesis. Another hypothesis is related to oxidative stress. This factor could be the agent that actually causes the entire series of changes described in the nervous tissues, through related molecules like AB. Progressive accumulation of oxidative damage over the years could explain why this disease is associated with aging. The liver has, inter alia, the function of serving as a detoxifying agent of the blood. It is very important to remove harmful residues that are in the bloodstream, such as the AB characteristic from this neurological pathology. It can reach the bloodstream by means of transporter LRP-1, driving it out of the brain into the blood and from there it enters the liver. The transporter RAGE is able to get AB out of the blood and put it back into the brain. For obvious ethical reasons it is not possible to perform certain human studies, such as analysis of organs, so we used an animal model. The model chosen by our research group was AD transgenic mice APPswe/PS1dE9, because it develops characteristics of the pathology associated with AD in humans, such as the deposition and subsequent emergence of AB plaques. The main objective of this thesis is to study hepatic oxidative stress and the effects of treatment with bexarotene and/or genistein associated with Alzheimer’s disease in an animal model. In the first part of this thesis we analyzed the relationship between the brain and the liver. We worked with male and female wild type and APP/PS1 mice of different ages, 3-5 months, 10-13 months, and over 20 months to see the evolution of the associated pathology. We analyzed parameters of oxidative stress in the liver, like the mitochondrial rate of hydrogen peroxide production, protein oxidation (measured as carbonylation) and lipid peroxidation (measured as malondialdehyde). To determine this relationship we measured transporter LRP1 in both liver and brain, and RAGE only in brain. We also determined the concentration of blood AB. The results show how in APP/PS1 mice the different oxidative stress parameters decrease in liver with age, whereas AB concentration in blood increases. We work with the hypothesis that the liver detoxifies AB and in this process generates free radicals. This ability to degrade may progressively decrease with age, which could explain why there is less oxidative stress and more AB. The brain results show that RAGE does not change in the different groups, but LRP1 decreases with age in wild type and APP/PS1 mice. In liver it decreases with age only in APP/PS1 mice, and we saw more in the APP/PS1 than in the wild type mice at the age of 3-5 months and 10-13 months. In summary, we found that AB is formed in the brain, and can enter the systemic circulation by means of LRP1, more so at younger ages. Despite this we saw that there is an increased blood level in the older animal. AB can return to the brain by means of RAGE, but this may not be important since there was no change at any of the ages studied. The fundamental difference is in the AB flow to the liver. In the young animal there is a much greater flow than in the old, as it has more AB and undergoes further degradation thereby causing the changes in oxidative stress. The study of the brain AB clearance mechanism led us to be interested in items that may contribute to this process. Decreasing the amyloid pathology associated with AD was expected to contribute to the improvement of the symptoms of the disease. There is a recent publication for an innovative treatment for AD, which seems to have a positive effect, at least in animal models. This treatment involves the administration of bexarotene, a drug used in the treatment of cutaneous lymphoma. The characteristic that relates it to AD is manifested in two ways. On the one hand it enhances AB phagocytosis by microglia. On the other side it binds to the RXR-PPARg dimer, which will make overexpressing apolipoprotein E (apoE), which mediates the degradation of AB. A few years ago our group reported that genistein increased PPARg levels. Therefore, we believe that if this phytoestrogen administration increases the availability of the transcription factor, it can increase the effectiveness of the drug. Apart from this effect, genistein alone may have antioxidant and neuroprotective effects on AD. In the second part of this thesis, in order to test a new treatment for AD, we designed an experiment in which we used ovariectomized females, because there is a higher incidence of AD in females after menopause. We have only used APP/PS1 because what interests us is the possible brain AB clearance. We used eight-month-old mice because at this age they already have AB plaques. We determined AB levels in brain and blood, amyloid plaques in the brain, and PPARg and apoE levels in brain, as well as transaminase levels in blood. Ovariectomy was performed at the beginning of the experiment. The treatment provided was: • Control group with water. • Genistein group throughout the experiment at a concentration of 0.022 mg/kg/day. • Bexarotene group for 3 days at the concentration of 100 mg/kg/day. • Combination treatment group, first with genistein for 3 days to overexpress PPARg, and then bexarotene for 3 days, at the same concentration that the other groups. AB levels in brain showed that the treatment was effective reducing amyloid pathology. We even saw a synergistic effect in the combination treatment group. In conclusion, we can say that transporter LRP1 decreases with aging in brain and liver. This makes the AB flow that enters the liver decrease with aging, and therefore there is less degradation and reduces hepatic oxidative stress with aging. Treatment with bexarotene and/or genistein is effective in reducing amyloid pathology in the APP/PS1 animal model for AD

PTEN Mediates the Antioxidant Effect of Resveratrol at Nutritionally Relevant Concentrations

Introduction. Antioxidant properties of resveratrol have been intensively studied for the last years, both in vivo and in vitro. Its bioavailability after an oral dose is very low and therefore it is very important to make sure that plasma concentrations of free resveratrol are sufficient enough to be active as antioxidant. Aims. In the present study, using nutritionally relevant concentrations of resveratrol, we aim to confirm its antioxidant capacity on reducing peroxide levels and look for the molecular pathway involved in this antioxidant effect. Methods. We used mammary gland tumor cells (MCF-7), which were pretreated with different concentrations of resveratrol for 48 h, and/or a PTEN inhibitor (bpV: bipy). Hydrogen peroxide levels were determined by fluorimetry, PTEN levels and Akt phosphorylation by Western Blotting, and mRNA expression of antioxidant genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. Resveratrol treatment for 48 h lowered peroxide levels in MCF-7, even at low nutritional concentrations (1 nM). This effect was mediated by the activation of PTEN/Akt pathway, which resulted in an upregulation of catalase and MnSOD mRNA levels. Conclusion. Resveratrol acts as an antioxidant at nutritionally relevant concentrations by inducing the expression of antioxidant enzymes, through a mechanism involving PTEN/Akt signaling pathway

Physical exercise neuroprotects ovariectomized 3xTg-AD mice through BDNF mechanisms

Postmenopausal women may be more vulnerable to cognitive loss and Alzheimer's disease (AD) than premenopausal women because of their deficiency in estrogens, in addition to their usually older age. Aerobic physical exercise has been proposed as a therapeutic approach for maintaining health and well-being in postmenopausal women, and for improving brain health and plasticity in populations at high risk for AD. To study the neuroprotective mechanisms of physical exercise in a postmenopausal animal model, we submitted previously ovariectomized, six-month old non-transgenic and 3xTg-AD mice to three months of voluntary exercise in a running wheel. At nine months of age, we observed lower grip strength and some exacerbation of the behavioral and psychological symptoms of dementia (BPSD)-like involving active exploratory activities. A similar major cognitive impairment was observed of ovariectomized 3xTg-AD mice in comparison with sham-operated 3xTg-AD mice. A reduction of bodily fitness and lack of retention of memory were observed in the ovariectomized non-transgenic mice. Physical exercise protected against all deleterious behaviors and normalized learning and memory. It also protected against body frailty, as expected. Analyses of hippocampal key markers of antioxidant and neuroplasticity signaling pathways, showed that ovariectomy impairs the activation of CREB through physical exercise. Furthermore, molecular and behavioral correlates suggested a central role of BDNF in the neuroprotection mediated by physical exercise therapy against apathy and memory loss induced by ovariectomy and the AD-genotype. © 2014 Elsevier Ltd.This study was supported by grants: SAF2009-13093-C02-02, SAF2010-19498, SAF2012-39852-C02-02 and CSD2010-00045 from the Spanish MINECO; 2009/SGR/214 from the Generalitat and 062931 from the Fundació La Marató de TV3, of Catalonia; and 35NEURO GentxGent. Yoelvis García-Mesa acknowledges support received from the Fundació La Marató de TV3Peer Reviewe