[Neurogenesis and gliogenesis modulation in cerebral ischemia by CDK5 RNAi-based therapy].

INTRODUCTION: Cerebral ischemia is the third cause of death risk in Colombia and the first cause of physical disability worldwide. Different studies on the silencing of the cyclin-dependent kinase 5 (CDK5) have shown that reducing its activity is beneficial in ischemic contexts. However, its effect on neural cell production after cerebral ischemia has not been well studied yet. OBJECTIVE: To evaluate CDK5 silencing on the production of neurons and astrocytes after a focal cerebral ischemia in rats. MATERIALS AND METHODS: We used 40 eight-week-old male Wistar rats. Both sham and ischemia groups were transduced at CA1 hippocampal region with an adeno-associated viral vector using a noninterfering (shSCRmiR) and an interfering sequence for CDK5 (shCDK5miR). We injected 50 mg/kg of bromodeoxyuridine intraperitoneally from hour 24 to day 7 post-ischemia. We assessed the neurological abilities during the next 15 days and we measured the immunoreactivity of bromodeoxyuridine (BrdU), doublecortin (DCX), NeuN, and glial fibrillary acid protein (GFAP) from day 15 to day 30 post-ischemia. RESULTS: Our findings showed that CDK5miR-treated ischemic animals improved their neurological score and presented increased BrdU+ cells 15 days after ischemia, which correlated with higher DCX and lower GFAP fluorescence intensities, and, although mature neurons populations did not change, GFAP immunoreactivity was still significantly reduced at 30 days post-ischemia in comparison with untreated ischemic groups. CONCLUSION: CDK5miR therapy generated the neurological recovery of ischemic rats associated with the induction of immature neurons proliferation and the reduction of GFAP reactivity at short and longterm post-ischemia.

Neurogenesis and gliogenesis modulation in cerebral ischemia by CDK5 RNAi-based therapy / Modulación de la neurogénesis y la gliogénesis en la isquemia cerebral mediante una terapia basada en la interferencia de CDK5

Abstract Introduction: Cerebral ischemia is the third cause of death risk in Colombia and the first cause of physical disability worldwide. Different studies on the silencing of the cyclin-dependent kinase 5 (CDK5) have shown that reducing its activity is beneficial in ischemic contexts. However, its effect on neural cell production after cerebral ischemia has not been well studied yet. Objective: To evaluate CDK5 silencing on the production of neurons and astrocytes after a focal cerebral ischemia in rats. Materials and methods: We used 40 eight-week-old male Wistar rats. Both sham and ischemia groups were transduced at CA1 hippocampal region with an adeno-associated viral vector using a noninterfering (shSCRmiR) and an interfering sequence for CDK5 (shCDK5miR). We injected 50 mg/kg of bromodeoxyuridine intraperitoneally from hour 24 to day 7 post-ischemia. We assessed the neurological abilities during the next 15 days and we measured the immunoreactivity of bromodeoxyuridine (BrdU), doublecortin (DCX), NeuN, and glial fibrillary acid protein (GFAP) from day 15 to day 30 post-ischemia. Results: Our findings showed that CDK5miR-treated ischemic animals improved their neurological score and presented increased BrdU+ cells 15 days after ischemia, which correlated with higher DCX and lower GFAP fluorescence intensities, and, although mature neurons populations did not change, GFAP immunoreactivity was still significantly reduced at 30 days post-ischemia in comparison with untreated ischemic groups. Conclusion: CDK5miR therapy generated the neurological recovery of ischemic rats associated with the induction of immature neurons proliferation and the reduction of GFAP reactivity at short and longterm post-ischemia.

Resumen Introducción. La isquemia cerebral es la tercera causa de riesgo de muerte en Colombia y la primera causa de discapacidad física en el mundo. En diversos estudios en los que se silenció la cinasa 5 dependiente de la ciclina (CDK5) se ha demostrado que la reducción de su actividad es beneficiosa frente a la isquemia. Sin embargo, su efecto sobre la neurogénesis después de la isquemia no se ha dilucidado suficientemente. Objetivo. Evaluar el silenciamiento de la CDK5 en la neurogénesis y la gliogénesis después de la isquemia cerebral focal en ratas. Materiales y métodos. Se usaron 40 machos de rata Wistar de ocho semanas de edad. Los grupos de control y los isquémicos sometidos a transducción en la región del hipocampo CA1, se inyectaron intraperitonealmente por estereotaxia con 50 mg/kg de bromodesoxiuridina (BrdU) a partir de las 24 horas y hasta el día 7 después de la isquemia, con un vector viral asociado a adenovirus usando una secuencia no interferente (SCRmiR) y una interferente (CDK5miR). Se evaluó la capacidad neurológica durante los quince días siguientes y se detectó la capacidad de inmunorreacción para la BrdU, la proteína doblecortina (DCX), los núcleos neuronales (NeuN), y la proteína fibrilar acídica de la glía (Glial Fibrillary Acidic Protein, GFAP) a los 15 y 30 días de la isquemia. Resultados. Los animales isquémicos tratados con CDK5miR mejoraron su puntuación neurológica y presentaron un incremento de la BrdU+ a los 15 días de la isquemia, lo cual se correlacionó con una mayor intensidad de la DCX+ y una menor de la GFAP+. No hubo modificación de los NeuN+, pero sí una reducción significativa de la GFAP+ a los 30 días de la isquemia en los animales tratados comparados con los animales isquémicos no tratados. Conclusión. La terapia con CDK5miR generó la recuperación neurológica de ratas isquémicas asociada con la inducción de la neurogénesis y el control de la capacidad de reacción de la proteína GFAP a corto y largo plazo después de la isquemia.

Quercetin ameliorates inflammation in CA1 hippocampal region in aged triple transgenic Alzheimer´s disease mice model / La quercetina disminuye la inflamación en la región CA1 del hipocampo en un modelo de ratón triple transgénico para la enfermedad de Alzheimer

Introduction: Alzheimer's disease is the most common form of dementia. It is characterized by histopathological hallmarks such as senile plaques and neurofibrillary tangles, as well as a concomitant activation of microglial cells and astrocytes that release pro-inflammatory mediators such as IL-1ß, iNOS, and COX-2, leading to neuronal dysfunction and death. Objective: To evaluate the effect of quercetin on the inflammatory response in the CA1 area of the hippocampus in a 3xTg-AD male and female mice model. Materials and methods: Animals were injected intraperitoneally with quercetin every 48 hours during three months, and we conducted histological and biochemical studies. Results: We found that in quercetin-treated 3xTg-AD mice, reactive microglia and fluorescence intensity of Aß aggregates significantly decreased. GFAP, iNOS, and COX-2 immunoreactivity also decreased and we observed a clear tendency in the reduction of IL-1ß in hippocampal lysates. Conclusion: Our work suggests an anti-inflammatory effect of quercetin in the CA1 hippocampal region of aged triple transgenic Alzheimer's disease mice.

Introducción. La enfermedad de Alzheimer es la forma más común de demencia; se caracteriza por la presencia de marcadores histopatológicos, como las placas seniles y los ovillos neurofibrilares, así como por una activación concomitante de células microgliales y astrocitos que liberan mediadores proinflamatorios, como IL-1ß, iNOS y COX-2, lo cual conduce a la disfunción y la muerte neuronal. Objetivo. Evaluar el efecto de la quercetina sobre la reacción inflamatoria en el área CA1 del hipocampo en un modelo de ratones 3xTg-AD. Materiales y métodos. Los animales se inyectaron intraperitonealmente con quercetina cada 48 horas durante tres meses, y se hicieron estudios histológicos y bioquímicos. Resultados. Se encontró que en los animales 3xTg-AD tratados con quercetina, la microglía reactiva y la intensidad de fluorescencia de los agregados Aß disminuyeron significativamente, y que hubo una menor reacción de GFAP, iNOS y COX-2, así como una clara tendencia a la reducción de la IL-1 ß en lisados de hipocampo. Conclusión. Los resultados del estudio sugieren un efecto antiinflamatorio de la quercetina en la región CA1 del hipocampo en un modelo en ratón triple trasgénico para la enfermedad de Alzheimer.

Recovery of Neurovascular Unit Integrity by CDK5-KD Astrocyte Transplantation in a Global Cerebral Ischemia Model.

Astrocytes play metabolic and structural support roles and contribute to the integrity of the blood-brain barrier (BBB), linking communication between neurons and the endothelium. Cyclin-dependent kinase 5 (CDK5) likely exerts a dual effect on the endothelium and astrocytes due to its involvement in migration and angiogenesis; the overactivation of CDK5 is associated with dysfunction in glutamate recapture and hypoxia. Recently, we proposed that CDK5-targeted astrocytes facilitate the recovery of neurological and motor function in transplanted ischemic rats. In the current study, we treated cerebral ischemic rats and endothelial cells exposed to glutamate toxicity with CDK5 knock-down (CDK5-KD) astrocytes to determine the role of CDK5 in neurovascular integrity. We found that the effects of CDK5-KD were sustained for 4 months, preventing neuronal and astrocyte loss, facilitating the recovery of the BBB via the production of BDNF by endogenous astrocytes (GFP-) surrounding vessels in the motor cortex and the corpus callosum of global ischemic rats, and improving neurological performance. These findings were supported by the in vitro findings of increased transendothelial resistance, p120-ctn+ adhesion and reduced intercellular gaps induced by a CDK5 inhibitor (roscovitine) in bEnd.3 cells in a glutamate-toxicity model. Additionally, CDK5-KD astrocytes in co-culture protected the endothelial cell viability, increased BDNF release from astrocytes, increased BDNF immunoreactivity in neighboring astrocytes and endothelial cells and enhanced cell adhesion in a glutamate-toxicity model. Altogether, these findings suggest that a CDK5 reduction in astrocytes protects the endothelium, which promotes BDNF release, endothelial adhesion, and the recovery of neurovascular unit integrity and brain function in ischemic rats.

Atorvastatin Modulates Regulatory T Cells and Attenuates Cerebral Damage in a Model of Transient Middle Cerebral Artery Occlusion in Rats.

Regulatory T cells (Tregs) inhibit the activation of the immune response which could down-regulate the systemic and focal activation observed during ischemic stroke. In fact, in animal models, Tregs infiltrate the infarcted brain and reduce the pro-inflammatory cytokine production and infarct volume, mainly in late stages of ischemia. Recently, an expansion and greater suppressive capacity of circulating Tregs after treatment with statins was observed, in addition to their cardio- and neuroprotective actions demonstrated previously. Thus, to determine whether Treg modulation mediated by statins can also be beneficial during stroke, cerebral ischemia was artificially induced in Wistar rats by transient middle cerebral artery occlusion (tMCAO) during 60 minutes with subsequent reperfusion for 7 days. Six hours after surgery, some animals were treated with atorvastatin (ATV, 10 mg/kg) or carboxymethylcellulose as vehicle at the same concentration every other day during 7 days. Some animals were sham operated as control group of surgery. Interestingly, ATV treatment prevented the development of infarct volume, reduced the neurological deficits, and the circulating and cervical lymph node CD25+FoxP3+ Treg population. Moreover, there was a reduction of glial cell activation, which correlated with decreased circulating Tregs. Remarkably, treatment with ATV induced an increase in the frequency of CD4+CD25+ T cells, in particular of those expressing CTLA-4, in brain samples. Together, these results suggest that ATV can modulate Tregs in peripheral tissue and favor their accumulation in the brain, where they can exert neuroprotective actions maybe by the reduction of glial cell activation.

Linalool reverses neuropathological and behavioral impairments in old triple transgenic Alzheimer's mice.

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder. Several types of treatments have been tested to block or delay the onset of the disease, but none have been completely successful. Diet, lifestyle and natural products are currently the main scientific focuses. Here, we evaluate the effects of oral administration of the monoterpene linalool (25 mg/kg), every 48 h for 3 months, on aged (21-24 months old) mice with a triple transgenic model of AD (3xTg-AD) mice. Linalool-treated 3xTg-AD mice showed improved learning and spatial memory and greater risk assessment behavior during the elevated plus maze. Hippocampi and amygdalae from linalool-treated 3xTg-AD mice exhibited a significant reduction in extracellular ß-amyloidosis, tauopathy, astrogliosis and microgliosis as well as a significant reduction in the levels of the pro-inflammatory markers p38 MAPK, NOS2, COX2 and IL-1ß. Together, our findings suggest that linalool reverses the histopathological hallmarks of AD and restores cognitive and emotional functions via an anti-inflammatory effect. Thus, linalool may be an AD prevention candidate for preclinical studies.

CDK5 knockdown in astrocytes provide neuroprotection as a trophic source via Rac1.

Astrocytes perform metabolic and structural support functions in the brain and contribute to the integrity of the blood-brain barrier. Astrocytes influence neuronal survival and prevent gliotoxicity by capturing glutamate (Glu), reactive oxygen species, and nutrients. During these processes, astrocytic morphological changes are supported by actin cytoskeleton remodeling and require the involvement of Rho GTPases, such as Rac1. The protein cyclin-dependent kinase 5 (CDK5) may have a dual effect on astrocytes because it has been shown to be involved in migration, senescence, and the dysfunction of glutamate recapture; however, its role in astrocytes remains unclear. Treating a possible deregulation of CDK5 with RNAi is a strategy that has been proposed as a therapy for neurodegenerative diseases. Models of glutamate gliotoxicity in the C6 astroglioma cell line, primary cultures of astrocytes, and co-cultures with neurons were used to analyze the effects of CDK5 RNAi in astrocytes and the role of Rac1 in neuronal viability. In C6 cells and primary astrocytes, CDK5 RNAi prevented the cell death generated by glutamate-induced gliotoxicity, and this finding was corroborated by pharmacological inhibition with roscovitine. This effect was associated with the appearance of lamellipodia, protrusions, increased cell area, stellation, Rac1 activation, BDNF release, and astrocytic protection in neurons that were exposed to glutamate excitotoxicity. Interestingly, Rac1 inhibition in astrocytes blocked BDNF upregulation and the astrocyte-mediated neuroprotection. Actin cytoskeleton remodeling and stellation may be a functional phenotype for BDNF release that promotes neuroprotection. In summary, our findings suggest that CDK5- knockdown in astrocytes acts as a trophic source for neuronal protection in a Rac1-dependent manner.

p35 and Rac1 underlie the neuroprotection and cognitive improvement induced by CDK5 silencing.

CDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles (NFT) and prevented neuronal loss in triple transgenic Alzheimer's mice. Here, we report that CDK5 RNAi protected against glutamate-mediated excitotoxicity using primary hippocampal neurons transduced with adeno-associated virus 2.5 viral vector eGFP-tagged scrambled or CDK5 shRNA-miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down-stream Rho GTPase activity. Furthermore, p35 over-expression and constitutively active Rac1 mimicked CDK5 silencing-induced neuroprotection. In addition, 3xTg-Alzheimer's disease mice (24 months old) were injected in the hippocampus with scrambled or CDK5 shRNA-miR, and spatial learning and memory were performed 3 weeks post-injection using 'Morris' water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimer's mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi.

The flavonoid quercetin ameliorates Alzheimer's disease pathology and protects cognitive and emotional function in aged triple transgenic Alzheimer's disease model mice.

Alzheimer's disease (AD) is the most common senile dementia in the world. Although important progress has been made in understanding the pathogenesis of AD, current therapeutic approaches provide only modest symptomatic relief. In this study, we evaluated the neuroprotective effect of quercetin (25 mg/kg) administration via i.p. injection every 48 h for 3 months on aged (21-24 months old) triple transgenic AD model (3xTg-AD) mice. Our data show that quercetin decreases extracellular ß-amyloidosis, tauopathy, astrogliosis and microgliosis in the hippocampus and the amygdala. These results were supported by a significant reduction in the paired helical filament (PHF), ß-amyloid (ßA) 1-40 and ßA 1-42 levels and a decrease in BACE1-mediated cleavage of APP (into CTFß). Additionally, quercetin induced improved performance on learning and spatial memory tasks and greater risk assessment behavior based on the elevated plus maze test. Together, these findings suggest that quercetin reverses histological hallmarks of AD and protects cognitive and emotional function in aged 3xTg-AD mice.

Cyclin-Dependent kinase 5 targeting prevents ß-Amyloid aggregation involving glycogen synthase kinase 3ß and phosphatases.

Inappropriate activation of cyclin-dependent kinase 5 (CDK5) resulting from proteolytic release of the activator fragment p25 from the membrane contributes to the formation of neurofibrillary tangles, ß-amyloid (ßA) aggregation, and chronic neurodegeneration. At 18 months of age, 3× Tg-AD mice were sacrificed after either 3 weeks (short term) or 1 year (long term) of CDK5 knockdown. In short-term-treated animals, CDK5 knockdown reversed ßA aggregation in the hippocampi via inhibitory phosphorylation of glycogen synthase kinase 3ß Ser9 and activation of phosphatase PP2A. In long-term-treated animals, CDK5 knockdown induced a persistent reduction in CDK5 and prevented ßA aggregation, but the effect on amyloid precursor protein processing was reduced, suggesting that yearly booster therapy would be required. These findings further validate CDK5 as a target for preventing or blocking amyloidosis in older transgenic mice.

Nuevas aproximaciones para la comprensión de la enfermedad de alzheimer / New approaches to understanding alzheimer disease

La enfermedad de Alzheimer es la causa de demencia senil más común en el mundo. Los marcadores histopatológicos asociados a la enfermedad son la acumulación extracelular de los péptidos β-amiloides y la intracelular de la proteína tau hiperfosforilada. La fosforilación de la proteína tau es regulada por múltiples quinasas y fosfatasas, y del equilibrio entre éstas depende su adecuada función o su agregación. La quinasa dependiente de ciclina 5 es una de las principales quinasas implicadas en la fosforilación de la proteína tau; tiene una acción directa sobre diversos residuos y participa en la regulación de diferentes sustratos para el funcionamiento correcto de la neurona; sin embargo, en condiciones alteradas puede desencadenar la enfermedad de Alzheimer. Así mismo, alteraciones que lleven a la agregación de proteínas o fallas en las vías de degradación de éstas en la célula, como el sistemaubicuitina-proteasoma y la autofagia, pueden facilitar el desarrollo de la enfermedad. La búsqueda de estrategias terapéuticas eficaces para los pacientes con la enfermedad de Alzheimer debe intentar unificar los mecanismos patogénicos de la enfermedad desde la complejidad que representa un proceso crónico y multifactorial.

Alzheimer disease is the most common cause of dementia in the world. The major histopathologicalmarkers associated with the disease are the accumulation of extracellular amyloid β-peptides and the accumulation of intracellular hyperphosphorylated tau protein. Multiple kinases and phosphatases can regulate tau phosphorylation, and the adequate function or aggregation depends on the balance between these enzymes. Cyclin-dependent kinase 5 is one of the major kinases involved in tau phosphorylation.This kinase has a direct action by phosphorylating various residues, and participates in the regulation of a variety of substrates for proper neuron function; however, dysregulation conditions can trigger Alzheimer disease. As well, changes in the cell that leading to protein aggregation or failure in their degradation pathways, such as the ubiquitin-proteasome system and autophagy, can facilitate the development of the disease. The search of effective treatment strategies for patients with Alzheimer disease should try to unify the pathogenic mechanisms from within complexity of this chronic and multifactorial condition.

Glycogen synthase kinase-3ß/ß-catenin signaling in the rat hypothalamus during the estrous cycle.

During the estrous cycle, a remodeling of synapses on somas and dendritic spines occurs in the rat hypothalamic arcuate nucleus. The synaptic remodeling is known to be induced by estradiol, but the molecular mechanisms involved still have not been fully clarified. ß-catenin is known to regulate synaptic plasticity, so we have assessed possible modifications of ß-catenin in the rat mediobasal hypothalamus during the estrous cycle. Our findings indicate that ß-catenin expression is increased during proestrus and estrus in comparison with diestrus day. This increase was accompanied by an enhanced phosphorylation of Akt in Ser473 and of glycogen synthase kinase-3ß (GSK3ß) in Ser9. Also, the association of ß-catenin with the synaptic protein PSD95 was increased during these same stages of the estrous cycle, whereas the levels of synapsin I were significantly decreased in proestrus. These findings suggest that Akt/GSK3ß/ß-catenin signaling is involved in the synaptic modifications that occur in the basal hypothalamus during the estrous cycle.

Silencing of CDK5 reduces neurofibrillary tangles in transgenic alzheimer's mice.

Alzheimer's disease is a major cause of dementia for which treatments remain unsatisfactory. Cyclin-dependent kinase 5 (CDK5) is a relevant kinase that has been hypothesized to contribute to the tau pathology. Several classes of chemical inhibitors for CDK5 have been developed, but they generally lack the specificity to distinguish among various ATP-dependent kinases. Therefore, the efficacy of these compounds when tested in animal models cannot definitively be attributed to an effect on CDK5. However, RNA interference (RNAi) targeting of CDK5 is specific and can be used to validate CDK5 as a possible treatment target. We delivered a CDK5 RNAi by lentiviral or adenoassociated viral vectors and analyzed the results in vitro and in vivo. Silencing of CDK5 reduces the phosphorylation of tau in primary neuronal cultures and in the brain of wild-type C57BL/6 mice. Furthermore, the knockdown of CDK5 strongly decreased the number of neurofibrillary tangles in the hippocampi of triple-transgenic mice (3×Tg-AD mice). Our data suggest that this downregulation may be attributable to the reduction of the CDK5 availability in the tissue, without affecting the CDK5 kinase activity. In summary, our findings validate CDK5 as a reasonable therapeutic target for ameliorating tau pathology.

El DHEAS incrementa la expresión de GluR2/3 y GLUR2 del receptor AMPA en el hipocampo de ratones C57/BL6 / DHEAS increases levels of GluR2/3 and GluR2, AMPA receptor subunits, in C57BL/6 mice hippocampus

El DHEAS es un neuroesteroide con efecto neuromodulador de la transmisión sináptica y en la neuroprotección, sin embargo las vías moleculares a través de las cuales se inducen estos cambiosno están completamente claras. Como varios de los neuroesteroides actúan a través de los recetores ionotrópicos de glutamato, se evaluó el efecto del DHEAS en las subunidades GluR2 y GluR3 del receptor AMPA para esclarecer sus efectos. Con este fin se administró DHEAS o una sustancia control durante 7 días a ratones C57/BL6. La expresión de las subunidades se evaluó por Westernblotting.Los resultados presentados muestran que la administración prolongada de 40mg/kg/día de DHEAS a ratones C57/BL6 produce un incremento en los niveles de proteína de las subunidades GluR2/3 yGluR2 del receptor AMPA en el hipocampo. Dado el papel específico que juega la subunidad GluR2 del receptor AMPA en el control de la entrada de calcio durante los procesos de muerte celular y de plasticidad sináptica, este hallazgo contribuye al estudio de los neuroesteroides como una estrategia terapéutica relevante en enfermedades neurodegenerativas y eventos cerebrovasculares.

Dehydroepiandrosterone sulfate (DHEA-S) is a neurosteroid that has effects such as neuromodulator of synaptic transmission and neuroprotection. The specific signaling pathways for these effects are not elucidated yet. Given that, some neurosteroids act through the activation of ionotropic glutamate receptors, therefore the effect of DHEA-S on the subunits GluR2 and GluR3of the AMPA receptor was evaluated. Either DHEA-S or a control substance was administered to C57/BL6 mice. Subunit expression of the AMPA receptor was analyzed by Western blotting. Results show that long-term DHEA-S administration toC57/BL6 mice, increases the protein levels of the subunits GluR2 and GluR2/3 of the AMPA receptors located in the hippocampus. Due to the role of AMPA receptor, specifically GluR2subunit in the regulation of intracellular calcium levels, cellular apoptosis, and synaptic plasticity, the study of neurosteroids as a therapeutic strategy in neurodegenerative diseases and cerebrovascular events is very relevant.

Decrease of Tau hyperphosphorylation by 17ß estradiol requires sphingosine kinase in a glutamate toxicity model.

Several studies have linked estrogens with sphingosine kinase (SphK) activity, enzyme responsible of sphingosine-1-phosphate synthesis (S-1P), however their possible interaction in the nervous system is not documented yet. In the present study, we developed a glutamate toxicity model in SH-SY5Y cells to evaluate the possible effect of the inhibition of SphK activity on the protective capability of 17ß-estradiol (E2). Glutamate induced cytoskeletal actin changes associated to cytotoxic stress, significant increase of apoptotic-like nuclear fragmentation, Tau hyperphosphorylation and increase of p25/p35 cleavage. These effects were prevented by E2 pre-treatment during 24 h. Although the inhibition of SphK did not block this protective effect, significantly increased Tau hyperphosphorylation by glutamate, in a way that was not reverted by E2. Our results suggest that the decrease of glutamate-induced Tau hyperphosphorylation by 17ß-estradiol requires SphK.

El 17β - estradiol disminuye la expresión y asociación de quinasas responsables de la hiperfosforilación de Tau / 17 β - estradiol decreases the expression and association of kinases responsible of Tau hyperphosphorylation

Introducción: Un componente molecular predominante en el estudio de las enfermedades neurodegenerativas es la presencia del complejo Tau-GSK3β y su asociación con agregados proteicos al interior de la célula. Evidencias considerables muestran que GSK3β es el principal causante de la hiperfosforilación de Tau. Sin embargo, son poco claros los eventos moleculares que gobiernan este complejo. Objetivo: Determinar el efecto del 17 β-estradiol en la expresión y asociación de las quinasas responsables de la hiperfosforilación de Tau. Métodos: Se realizaron tratamientos con 17 β-estradiol en hipocampo de rata Wistar adulta ovariectomizada y en cultivos primarios de hipocampo de rata tratados con b-amiloide. Se evaluó la asociación de complejos proteicos por co-inmunoprecipitación, ensayo de toxicidad por liberación LDH y cambios morfológicos celulares por microscopía confocal. Resultados: Este estudio mostró evidencias de que el estradiol disocia complejos macromoleculares como Tau/GSK3β, Tau/GluR2/3, Tau/FAK, Tau/Fyn en hipocampo de rata adulta. Ademßs, disminuyó la expresión de GSK3β-ptyr por el tratamiento hormonal y éste reguló la defosforilación de Tau en un modelo de excitoxicidad poráβ-amiloide. Conclusiones: Lo anterior sugiere, nuevos blancos que contribuyen al estudio de la neuroprotección y plasticidad neuronal mediada por el estrógeno.

Introduction: A predominant molecular component analyzed in the study of neurodegenerative diseases is the presence of the Tau-GSK3β complex and its association with protein aggregation into the cell. Several evidences show that GSK3β has an important role in abnormal pattern of the phosphorylation of Tau. However, the molecular events that are governing this complex are unknown. Aim: To determine the effect of 17 β-estradiol treatment on the expression and association of Tau hyperphosphorylation responsible kinases. Methods: 17 β-estradiol treatments were realized in the hippocampus of ovariectomized adult wistar rats and in hippocampal primary cultures treated with β-amiloid. Protein complex association was assessed by co-immunoprecipitation, toxicity assay by LDH release and cell morphologic changes by confocal microscopy. Results: Our results show that 17β-estradiol produced dissociation of macromolecular complexes like Tau/GSK3β, Tau /GluR2/3, Tau/FAK, and Tau/Fyn in hippocampus of adult rat. In addition the expression of GSK3β-ptyr was decreased by the hormonal treatment and this one regulated the defosforilation of Tau in an excitotoxicity model by β-amiloid. Conclusions: It suggests new targets that will contribute to neuroprotection and neuronal plasticity studies mediated by the estrogen.

Estrogen dissociates Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit in postischemic hippocampus.

During cerebral ischemia, part of the damage associated with the hyperactivation of glutamate receptors results from the hyperphosphorylation of the microtubule-associated protein Tau. Previous studies have shown that estradiol treatment reduces neural damage after cerebral ischemia. Here, we show that transient occlusion of the middle cerebral artery results in the hyperphosphorylation of Tau and in a significant increase in the association of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type glutamate receptor subunits 2/3 in the hippocampus. Estradiol treatment decreased hippocampal injury, inhibited glycogen synthase kinase-3beta and decreased the hyperphosphorylation of Tau and the interaction of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. These findings suggest that ischemia produces a strong association between Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, and estradiol can exert at least part of its neuroprotective activity through inhibition of glycogen synthase kinase-3beta.

Synergistic interaction of estradiol and insulin-like growth factor-I in the activation of PI3K/Akt signaling in the adult rat hypothalamus.

Estradiol and insulin-like growth factor-I (IGF-I) interact in the hypothalamus to regulate neuronal function, synaptic plasticity and neuroendocrine events. However, the molecular mechanisms involved in these interactions are still unknown. In the present study, the effect of estradiol on the signaling pathways of IGF-I receptor has been assessed in the hypothalamus of young adult ovariectomized rats, using specific antibodies for the phosphorylated forms of extracellular-signal regulated kinase (ERK) 1 and ERK2 and Akt/protein kinase B (Akt/PKB). Estradiol treatment resulted, between 6 and 24 h after systemic administration, in dose-dependent effects on the phosphorylation of ERK and Akt/PKB. Estradiol did not modify the level of ERK phosphorylation induced by intracerebroventricular administration of IGF-I. However, both hormones had a synergistic effect on the phosphorylation of Akt/PKB. These findings suggest that estrogen effects in the hypothalamus may be mediated in part by the activation of the signaling pathways of the IGF-I receptor.

Interactions of estrogen and insulin-like growth factor-I in the brain: molecular mechanisms and functional implications.

In the brain, as in other tissues, estradiol interacts with growth factors. One of the growth factors that is involved in the neural actions of estradiol is insulin-like growth factor-I (IGF-I). Estradiol and IGF-I cooperate in the central nervous system to regulate neuronal development, neural plasticity, neuroendocrine events and the response of neural tissue to injury. The precise molecular mechanisms involved in these interactions are still not well understood. In the central nervous system there is abundant co-expression of estrogen receptors (ERs) and IGF-I receptors (IGF-IRs) in the same cells. Furthermore, the expression of estrogen receptors and IGF-I receptors in the brain is cross-regulated. In addition, using specific antibodies for the phosphorylated forms of extracellular-signal regulated kinase (ERK) 1 and ERK2 and Akt/protein kinase B (Akt/PKB) it has been shown that estradiol affects IGF-I signaling pathways in the brain. Estradiol treatment results in a dose-dependent increase in the phosphorylation of ERK and Akt/PKB in the brain of adult ovariectomized rats. In addition, estradiol and IGF-I have a synergistic effects on the activation of Akt/PKB in the adult rat brain. These findings suggest that estrogen effects in the brain may be mediated in part by the activation of the signaling pathways of the IGF-I receptor.