Efecto de la minociclina y de la sulfadiazina sobre las alteraciones del sistema somatostatinérgico cerebral de la rata inducidas por el péptido [beta]-amiloide
- Autores: Emma Burgos Ramos
- Directores de la Tesis: Eduardo Arilla Ferreiro (dir. tes.), Lilian Puebla Jiménez (dir. tes.)
- Lectura: En la Universidad de Alcalá ( España ) en 2006
- Idioma: español
- Tribunal Calificador de la Tesis: Miguel Angel Pérez Albarsanz (presid.), Juan José López Fando Castro (secret.), Eulalia Rodriguez Martin (voc.), Mariano Betés de Toro (voc.), Antonio Gómez Pan (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: e_Buah
- Resumen
- español
La enfermedad de Alzheimer (EA) es un trastorno neurodegenerativo irreversible del sistema nervioso central, que se caracteriza clínicamente por una demencia progresiva y pérdida de la memoria. El principal agente patogénico de la EA es el péptido b-amiloide (Ab) . El fragmento Ab(25-35) es el dominio neurotóxico del péptido nativo Ab(1-40) o Ab(1-42). En la EA disminuyen los niveles de somatostatina (SRIF). La SRIF es un neurotrasmisor encargado de modular procesos de aprendizaje y memoria, alterados en la EA. La administración intracerebroventricular del Ab(25-35) disminuye la actividad del sistema somatostatinérgico en el hipocampo y en la corteza frontal, parietal y temporal de la rata de una manera dependiente del tiempo, siendo su efecto más acusado después de su infusión continua que de la administración aguda. El Ab(25-35) provoca una reducción del número de receptores de SRIF en las distintas áreas corticales estudiadas pero no en el hipocampo. Asimismo, la disminución del contenido de SRIF inmunorreactiva (SRIF-IR) es mayor en la corteza temporal que en el resto de áreas cerebrales, por lo que los efectos del Ab(25-35) muestran una especificidad regional. En el hipocampo, el Ab(25-35) provoca una disminución de la actividad enzimática adenilato ciclasa (AC), consecuencia de una menor expresión de la isoforma AC I. Esta reducción explica la menor fosforilación de la proteína de unión al elemento de respuesta a AMPc (CREB), conduciendo así a la disminución de los niveles de SRIF-IR. En la corteza frontal, parietal y temporal de la rata el Ab(25-35) disminuye el efecto inhibidor de la SRIF sobre la actividad AC. Este menor efecto es consecuencia de la disminución del número de receptores de SRIF. En la corteza temporal la infusión crónica del Ab(25-35) produce una disminución selectiva de la expresión del receptor de SRIF sst2, tanto a nivel proteico como transcripcional. Asimismo, esta reducción también se atribuye a un aumento de la desensibilización e internación del sst2, mediada por la enzima GRK2.
Por otro lado la administración de minociclina previene la disminución del contenido de neprilisina, principal enzima encargada de degradar el Ab en el cerebro, vía aumento de SRIF.
Dado que el Ab(25-35) deteriora los procesos de aprendizaje y memoria mientras que la SRIF los facilita, los presentes resultados demuestran que la disminución de la actividad del sistema somatostatinérgico en el hipocampo y corteza cerebral de la rata podría estar implicada en las alteraciones cognitivas descritas en el modelo animal de EA. Además, el hecho de que la minociclina y la sulfadiazina previenen las alteraciones del sistema somatostatinérgico inducidas por dicho péptido en la corteza temporal sugiere que ambos fármacos podrían tener un efecto beneficioso sobre los trastornos cognitivos presentes en la EA.
- English
Alzheimer´s disease (AD) is an irreversible neurodegenerative disorder characterized clinically by a progressive cognitive and memory deterioration, with a devastating impact on the whole society. The prevalence of this disease increases logarithmically with age, affecting mainly people over 65 years old. In Spain, it has been calculated to affect around 400,000 people. There is accumulating evidence that the production and accumulation of amyloid-beta (Aβ) is central to the pathogenesis of AD. This peptide accumulates in the brain parenchyma, causing the formation of senile neuritic plaques. It has recently been reported that the hydrophobic 11-aminoacid fragment Aβ (25-35) is the most toxic portion of the full-length peptide. In addition, several studies suggest that this fragment could be produced in humans by racemization of the Aβ (1-40) or Aβ (1-42) peptides. At the neurochemical level, an impairment of several neurotransmitter signal transduction pathways have been reported, including the cholinergic, adrenergic and dopaminergic systems. These alterations affect the associative brain areas and part of the limbic system, thus leading to the cognitive, behavioural and emotional manifestations characteristic of the disease. Several authors have also described a significant reduction in the somatostatin (SRIF) content in the post-mortem human brain as well as in the cerebrospinal fluid of AD patients. However, it is presently unknown whether this decrease is caused directly by Aβ.
Somatostatin (SRIF) is a tetradecapeptide widely distributed throughout the central nervous system (CNS) and peripheral tissues. Several studies have revealed that it plays a pivotal role as a neuromodulator and neurotransmitter, participating in the control of learning and memory processes.
SRIF exerts its biological actions via binding to specific high-affinity membrane receptors which belong to the G-protein-coupled receptor superfamily. To date, the genes for five SRIF receptor subtypes designated sst1-sst5 have been cloned. All five sst subtypes couple negatively to adenylyl cyclase (AC) via Gi proteins, leading to a reduction in the levels of the second messenger cyclic AMP (cAMP). In addition, some sst couple to calcium channels, potassium channels, secretion vesicles, mitogen-activated protein kinases (MAPK), phospholipase C (PLC) or phospho-tyrosine phosphatases.
At present, it is unknown whether Aβ is capable of altering the SRIF receptor-effector system in the rat brain. Hence, the present study was undertaken to analyze the effects of Aβ (25-35) on this system in the rat hippocampus as well as the rat frontal, parietal and temporal cortices. With this aim, we measured SRIF-like immunoreactivity (SRIF-LI) content by radioimmunoassay (RIA) and SRIF gene expression by measuring SRIF mRNA levels, using the reverse transcription-polymerase chain reaction (RT-PCR), as well as the protein levels of phosporylated-CREB (p-CREB) by western blotting, in control rats and Aβ (25-35)-treated rats. In order to evaluate the functionality of the somatostatinergic system, we measured the binding of 125I-Tyr11-SRIF-14 to SRIF receptors, basal and forskolin-stimulated AC activity and the capacity of SRIF to inhibit both activities in membranes derived from the above-mentioned brain areas in control and Aβ (25-35)-treated rats. In addition, the protein levels of sst1-sst4, of the Gi protein subunits Giα1, Giα2 and Giα3, as well as those of the G-protein-coupled receptor kinase GRK2 were determined by western blotting using specific antibodies. Finally, the sst2 mRNA levels were also determined by RT-PCR in these experimental groups.
Tetracyclines have been demonstrated not only to inhibit the formation of Aβ aggregates in vitro but to disassemble preformed fibrils as well. Consequently, it was of interest to analyze whether minocycline, a semisynthetic second-generation tetracycline, is capable of protecting the somatostatinergic system in vivo from Aβ toxicity, given its anti-amyloidogenic activity. On the other hand, administration of the antibiotic sulphadiazine to the elderly has been reported to produce a psychic and physical amelioration. In view of this fact, we proposed to analyze whether sulphadiazine could prevent the Aβ (25-35)-induced effects on the somatostatinergic system in the rat temporal cortex. We thus examined the effects of both drugs on the above-mentioned parameters in control rats and rats treated chronically with Aβ (25-35).
In order to partially reproduce AD in experimental animals, male Wistar rats weighing 200-250 g were used in the study. Aβ (25-35) was dissolved in 1% acetic acid and administered intracerebroventricularly (icv) to the rat in a single dose (10 μg) or continuously via an osmotic minipump implanted in the left brain ventricle (300 pmol/day) for 14 days. Minocycline was dissolved in saline and was injected intraperitoneally twice daily (45 mg/Kg) 24 hours prior to Aβ (25-35) administration. An additional dose of 22,5 mg/kg/day was administered during the last two days of the Aβ (25-35) infusion. In another group of rats, the same experimental model was used except that sulphadiazine (instead of minocycline) was administered intragastrically at a dose of 160 mg/Kg twice daily. A second dose of 80 mg/Kg/day was administered during the last two days of the Aβ (25-35) infusion. All rats were sacrificed by decapitation 24 hours following the last injection.
In conclusion, the present results demonstrate for the first time that Aβ (25-35) decreases the activity of the somatostatinergic system in the rat hippocampus, frontal cortex, parietal cortex and temporal cortex. In addition, it reveals that both minocycline and sulphadiazine treatments prevent the Aβ (25-35)-induced impairment of the somatostatinergic system in the rat temporal cortex. Consequently, these results suggest that minocycline and sulphadiazine might have a beneficial effect in AD reatment in humans.
- español