Als2-deficient mice exhibit disturbances in endosome trafficking associated with motor behavioral abnormalities.

ALS2 is an autosomal recessive form of spastic paraparesis (motor neuron disease) with juvenile onset and slow progression caused by loss of function of alsin, an activator of Rac1 and Rab5 small GTPases. To establish an animal model of ALS2 and derive insights into the pathogenesis of this illness, we have generated alsin-null mice. Cytosol from brains of Als2(-/-) mice shows marked diminution of Rab5-dependent endosome fusion activity. Furthermore, primary neurons from Als2(-/-) mice show a disturbance in endosomal transport of insulin-like growth factor 1 (IGF1) and BDNF receptors, whereas neuronal viability and endocytosis of transferrin and dextran seem unaltered. There is a significant decrease in the size of cortical motor neurons, and Als2(-/-) mice are mildly hypoactive. Altered trophic receptor trafficking in neurons of Als2(-/-) mice may underlie the histopathological and behavioral changes observed and the pathogenesis of ALS2.

Effect of LTP-reinforcing paradigms on neurotransmitter release in the dentate gyrus of young and aged rats.

Long-term potentiation (LTP) is considered a cellular correlate of memory processing. A short-lasting early-LTP can be prolonged into a late-L TP (>4h) by stimulation of the basolateral amygdala (BLA) or motivational behavioral stimuli in young, but not in aged, cognitively impaired rats. We measured the changes in transmitter release-induced by BLA or behavioral reinforcement-in young and aged cognitively impaired rats, after implanting a microdialysis cannula at the dentate gyrus. Samples were taken under baseline conditions and during stimulation of BLA. Rats were water deprived and tested again next day, taking samples after allowing access to water. Higher concentrations of choline, HIAA, aspartate, glutamate, and glycine were found in baseline samples from young animals compared to aged. In young animals, BLA stimulation increased the levels of ACh and reduced norepinephrine and serotonine, while behavioral reinforcement reduced the levels of glutamate and glycine. These effects were absent among aged rats, suggesting that this reduced neurochemical response might be linked to the impaired LTP-reinforcement reported previously.

[In vitro viability and glutathione levels in mesencephalic neurons after seven days hibernation]. / Viabilidad in vitro y niveles de glutati n en neuronas mesencef licas hibernadas durante siete d as.

In embryonic mesencephalic transplant in patients with Parkinson s disease dopaminergic survival is low (5 10%), and for this reason the use of multiple donors has been considered. The difficulty of obtaining more tissue determines the need for a procedure that enables human nigral tissue to be stored for a time without affecting its physiological state in any significant way. This study was designed to determine whether hibernation of tissue fragments has any influence on viability, how the viability of the mesencephalic cells behaves after 7 days hibernation and the glutathione levels in the hibernated tissue (HT). The viability of the HT in pieces (82.37 2.12) was found to be higher than the value for the whole mesencephalon (70.29 3.43). Viability of the HT, seven days at 4 C, at different post dissociation times, did not differ significantly. Despite the significant differences found between hibernated and fresh tissue at t= 0, this procedure does not seem to affect the mesencephalic tissue in any significant way, as it conserved a 94% viability after hibernation. No evidence was found of increased glutathione content as an antioxidizing response to the damage that might be caused by hibernation. These results suggest that since hibernation does not have any significant effect on the state of the cells it could be considered a useful procedure for conserving tissue to be used in clinical transplants. Moreover, further research is needed on survival and functionality of hibernated cells after being transplanted into animal models in order to evaluate their potential for use in cell therapy.

Viabilidad in vitro y niveles de glutatión en neuronas mesencefálicas hibernadas durante siete días / In vitro viability and glutathione levels in mesencephalic neurons after seven days' hibernation

En el trasplante de mesencéfalo embrionario en pacientes con enfermedad de Parkinson, la sobrevivencia dopaminérgica es baja (510 por ciento); de ahí que se considere el empleo de múltiples donantes. La dificultad en obtener más tejido determina la necesidad de un procedimiento para almacenar tejido nigral humano por un tiempo, sin afectar significativamente el estado fisiológico del mismo. Este estudio se diseñó para determinar si la hibernación del tejido en fragmentos influye sobre la viabilidad, cómo se comporta la viabilidad de las células mesencefálicas después de siete días de hibernación y los niveles de glutatión en el tejido hibernado (TH). La viabilidad del TH en piezas (82,37 ñ 2,12), resultó superior al valor del mesencéfalo completo (70,29 ñ 3,43). La viabilidad del TH durante siete días a 4 °C, a diferentes tiempos posdisociación, no difirió significativamente. Se encontraron diferencias significativas entre TH y tejido fresco a t= 0, a pesar de lo cual este procedimiento no parece afectar el tejido mesencefálico de forma significativa, ya que se conservó una viabilidad del 94 por ciento después de hibernadas. No se evidenció un aumento del contenido de glutatión como respuesta antioxidante ante los daños que pudiera provocar la hibernación. Estos resultados sugieren que la hibernación, al no afectar significativamente el estado de las células, podría considerarse un procedimiento útil para su aplicación en la conservación del tejido para trasplante clínico. Además, se requiere del estudio de la sobrevivencia y funcionalidad de las células hibernadas después de trasplantadas en modelos animales, para evaluar su potencialidad en la terapia celular (AU)

Behavioral and biochemical effects of glutathione depletion in the rat brain.

Glutathione serves the function of providing reducing equivalents for the maintenance of oxidant homeostasis, and besides it plays roles in intra- and intercellular signaling in the brain. Our purpose was to test the effects of depleting tissue glutathione by diethylmaleate (5.3 mmol/kg, intraperitoneal) on brain antioxidant metabolism, nerve growth factor levels, and cognitive performance in rats. Six hours after the treatment, glutathione level in the hippocampus dropped down to 30% of the mean value of vehicle-treated animals and glutathione peroxidase activity also declined. Twenty-four hours after the injection the values had been partially restored. Moreover, the hippocampal and cortical levels of nerve growth factor protein did not change in response to diethylmaleate treatment. Glutathione depletion did not influence the performance of animals in the step-through passive avoidance test, but impairs acquisition in the Morris water maze when given before training. However, when diethylmaleate was administered after acquisition in the same paradigm, it did not affect the retention tested at the following day. Our results suggest that glutathione status is important during acquisition, but not for retention, of spatial memory in maze tasks and they support the hypothesis of the oxidant/antioxidant equilibrium as a key piece acting in the regulation of brain function.

Purification and characterization of murine beta-nerve growth factor.

Beta-nerve growth factor (beta-NGF) is a trophic factor in the nervous system. We aimed to isolate and characterize this protein in view of its potential therapeutic use in neurodegenerative diseases. For purification a two-step ion-exchange procedure was followed. The characterization was performed using separation and immunological techniques, as well as a biological assay. These studies showed that the obtained protein consisted of a mixture of beta-NGF molecules, intact at their NH2-terminal extreme, and molecules which have lost the NH2-terminal octapeptide and exhibit modifications increasing its hydrophobicity. All these molecular species were recognized immunologically and showed biological activity.

Synaptic plasticity is impaired in rats with a low glutathione content.

Long-term potentiation (LTP) is a sustained increase in the efficacy of synaptic transmission, based on functional changes involving pre- and postsynaptic mechanisms, and has been considered a cellular model for learning and memory. The sulphurated tripeptide glutathione acts as a powerful antioxidant agent within the nervous system. Recent in vitro studies suggest that the cellular redox status might influence the mechanisms involved in synaptic plasticity. It is not known, however, how glutathione depletion might affect LTP. In the present study, we evaluated the input-output relationships, LTP, and paired-pulse interactions in rats with low glutathione levels induced by systemic injection of diethylmaleate. Our results in anesthetized rats show that the basic synaptic transmission between the perforant pathway and the dentate gyrus granule cells was not affected by glutathione depletion. However, in the same synapses it was not possible to induce prolonged changes in synaptic efficacy (LTP). Paired-pulse facilitation was also absent in the treated animals, suggesting an impairment of short-term synaptic interactions. These findings indicate that low content of glutathione can impair short-term and long-term mechanisms of synaptic plasticity and stress the importance of the redox balance in the normal function of brain circuitry.

Quinolinic acid lesion induces changes in rat striatal glutathione metabolism.

Although the involvement of oxidative mechanisms in the cytotoxicity of excitatory amino acids has been well documented, it is not known whether the intrastriatal injection of quinolinic acid (QA) induces changes in glutathione (GSH) metabolism. In this work, the activities of the enzymes GSH reductase (GRD), GSH peroxidase (GPX), and GSH S-transferase (GST), as well as the GSH content, were studied in the striatum, hippocampus, and frontal cortex of rats 1 and 6 weeks following the intrastriatal injection of QA (225 nmol). One group of animals remained untreated. This lesion resulted in a 20% decrease in striatal GRD activity at both the 1- and 6-week postlesion times, whereas GST exhibited a 30% activity increase in the lesioned striatum observable only 6 weeks after the lesion. GPX activity remained unchanged. In addition, the QA injection elicited a 30% fall in GSH level at the 1-week postlesion time. GSH related enzyme activities and GSH content from other areas outside the lesioned striatum were not affected. GST activation could represent a beneficial compensatory response to neutralize some of the oxidant agents generated by the lesion. However, this effect together with the reduction in GRD activity could be the cause or a contributing factor to the observed QA-induced deficit in GSH availability and, consequently, further disrupt the oxidant homeostasis of the injured striatal tissue. Therefore, these results provide evidence that the in vivo excitotoxic injury to the brain might affect oxidant/antioxidant equilibrium by eliciting changes in glutathione metabolism.

Synaptic plasticity is impaired in rats with a low glutathione content

Long-term potentiation (LTP) is a sustained increase in the efficacy of synaptic transmission, based on functional changes involving pre- and postsynaptic mechanisms, and has been considered a cellular model for learning and memory. The sulphurated tripeptide glutathione acts as a poweful antioxidant agent within the nervous system. Recent in vitro studies suggest that the cellular redox status might influence the mechanisms involved in synaptic plasticity. It is not known, however, how glutathione depletion might affect LTP. In the present study, we evaluated the imput-output relationships, LTP, and paired-pulse interactions in rats with low glutathione levels induced by sistemic injection of diethylmaleate. Our results in anesthetized rats show that the basic synaptic transmission between the perforant pathway and the dentate gyrus granule cells was not affected by glutathione depletion. However, in the same synapses it was not possible to induce prolonged changes in synaptic efficacy (LTP). Paired-pulse facilitation was also absent in the treated animals, suggesting an impairment of short-term synaptic intections. These findings indicate that low content of gluthatione can impair short-term and long-term mechanisms of synaptic plasticity and stress the importance of the redox balance in the normal funcion of brain circuitry(AU)

[Nerve growth factor: possibilities and limitations of its clinical application]. / Factor de crecimiento nervioso: posibilidades y limitaciones de su aplicación clínica.

INTRODUCTION: The use of neurotrophic factors for the treatment of degenerative disorders of the nervous system opens up promising new perspectives. DEVELOPMENT: Nerve growth factor (NGF) represents the most known and studied trophic factor, which acts on sensory and sympathetic neurons of the peripheral nervous system, and on basal forebrain and striatal cholinergic neurons of the central nervous system. The specificity and trophic actions of NGF on these neuronal populations and its efficacy at preventing neurodegeneration have led to its proposal of evaluation in the treatment of neurological diseases such as: Alzheimer's disease, diabetic neuropathies and Huntington's diseases. Preclinical and clinical studies carried out in animal models and patients with diagnosis of these diseases have revealed satisfactory results. The difficulties of the NGF central chronic infusion, and the NGF detrimental effects arising from the stimulation of other sensitive neuronal population have stimulated active efforts for the development of more efficacious delivery strategies. Besides, it has also promoted further studies on the relation between the neuropathological stage, the dose and the effects of NGF administration. CONCLUSION: The NGF is a potential therapeutic agent in the treatment of neurodegenerative diseases.

NGF prevents changes in rat brain glutathione-related enzymes following transection of the septohippocampal pathway.

The activities of the enzymes glutathione reductase (GRD), glutathione peroxidase (GPX), and glutathione S-transferase (GST) were studied in several rat brain areas following the aspirative transection of the septohippocampal pathway (fimbria fornix) and the administration of nerve growth factor (NGF) or cytochrome c. One group of animals remained untreated. This lesion resulted in a decreased hippocampal GRD and septal GST activities, as well as, in an increase in GPX activity from the frontal cortex, striatum, and septum. NGF prevented the lesion-induced changes in hippocampal GRD and septal GPX. These findings show that the insult resulting from the aspiration of the fimbria fornix bundle involves modifications in glutathione-related enzymes, and, therefore, in the antioxidant status of brain tissue. These changes in glutathione metabolism could be a consequence of the oxidative damage to GRD and GST proteins or represent a compensatory response of GPX to the oxidative threat The restoring effects of NGF on altered enzyme activities are possibly linked to its known neuroprotective action.

Factor de crecimiento nervioso: posibilidades y limitaciones de su aplicación clínica / Nerve growth factor

Introducción. El uso de factores neurológicos en el tratamiento de las enfermedades degenerativas del sistema nervioso abre nuevas y prometedoras perspectivas. Desarrollo. El factor de crecimiento nervioso (NGF) representa el factor trófico mejor conocido y estudiado, actuando sobre las neuronas sensitivas y simpáticas del sistema nervioso periférico y sobre el cerebro basal frontal y neuronas colinérgicas estriadas del sistema nervioso central. La especificidad y acciones tróficas del NGF sobre estas poblaciones neuronales y su eficacia en prevenir la neurodegeneración han conducido a la propuesta de valorar su uso en el tratamiento de enfermedades neurológicas tales como la enfermedad de Alzheimer, la neuropatía diabética y la enfermedad de Huntington. Estudios preclínicos y clínicos llevados a cabo en modelos animales y en enfermos con diagnósticos de estas enfermedades han puesto de manifiesto resultados satisfactorios. Las dificultades para la infusión central de NGF originados por la estimulación de otras poblaciones neuronales sensibles han estimulado la iniciativa para desarrollar estrategias de entrega más eficaces. Además, esto ha dado lugar a otros estudios sobre la relación entre la etapa neuropatológica, la dosis y los efectos de la administración de NGF. Conclusión. El NGF es un agente terapéutico potencial en el tratamiento de las enfermedades neurodegerativas(AU)

Effects of nerve growth factor on brain glutathione-related enzymes from aged rats.

Neurotrophins, like the nerve growth factor (NGF), trigger a variety of biological effects in their targets. Stimulating effects on antioxidant defenses have been postulated to underlie neurotrophic influence on neuron survival and maintenance. To test whether NGF is capable of inducing changes in glutathione-related enzymes in the aged cognitively impaired brain, glutathione reductase (GRD), glutathione S-transferase (GST) and total glutathione peroxidase (GPX) activities were measured in the striatum, septum, hippocampus and frontal cortex of four Sprague-Dawley rat groups: young (2 months old), aged (20 months old) untreated, aged cytochrome c-treated, and aged NGF-treated (icv delivery, 34 micrograms during 28 days). All the aged rats utilized in the study were memory impaired according to their performance in the Morris water maze test. These aged rats showed increases in the activities of septal and hippocampal GST, as well as, in the hippocampal, striatal and cortical GPX. These increases could be interpreted as compensatory responses to cope with the oxidative damage that has been accumulated by the aged brain. The increases in hippocampal and cortical GPX activity were attenuated by NGF treatment, whereas the neurotrophin induced an increase in GRD activity in the striatum of aged rats. These results point out GRD and GPX as possible targets of the neurotrophic effects.

Effects of nerve growth factor on brain glutathione-related enzymes from aged rats

Neurotrophins, like the nerve growth factor (NGF), trigger a variety of biological effects in their targets. Stimulating effects on antioxidant defenses have been postulated to underlie neurotrophic influence on neuron survival and maintenance. To test whether NGF is capable of inducing changes in glutathione-related enzymes in the aged cognitively impaired brain, glutathione reductase (GRD), glutathione S-transferase (GST) and total glutathione peroxidase (GPX) activities were measured in the striatum, septum, hippocampus and fraontal cortex of four Sprague-Dawley rat groups: young (2 months old), aged (20 months old) untreated, aged cytochrome c-treated, and aged NGF-treated (icv delivery, 34 æg during 28 days). All the aged rats utilized in the study were memory impaired according to their performance in the Morris water maze test. These aged rats showed increases in the activities of septal and hippocampal GST, as well as, in the hippocampal, striatal and cortical GPX. These increases could be interpreted as compensatory responses to cope with the oxidative damage that has been accumulated by the aged brain. The increases in hippocampal and corical GPX activity were attenuated by NGF treatment, whereas the neurotrophin induced an increase in GRD activity in the striatum of aged rats. These resultas point out GRD and GPX as possible targets of the neurotrophic effects

Effects of nerve growth factor on brain glutathione-related enzymes from aged rats

Neurotrophins, like the nerve growth factor (NGF), trigger a variety of biological effects in their targets. Stimulating effects on antioxidant defenses have been postulated to underlie neurotrophic influence on neuron survival and maintenance. To test whether NGF is capable of inducing changes in glutathione-related enzymes in the aged cognitively impaired brain, glutathione reductase (GRD), glutathione S-transferase (GST) and total glutathione peroxidase (GPX) activities were measured in the striatum, septum, hippocampus and fraontal cortex of four Sprague-Dawley rat groups: young (2 months old), aged (20 months old) untreated, aged cytochrome c-treated, and aged NGF-treated (icv delivery, 34 æg during 28 days). All the aged rats utilized in the study were memory impaired according to their performance in the Morris water maze test. These aged rats showed increases in the activities of septal and hippocampal GST, as well as, in the hippocampal, striatal and cortical GPX. These increases could be interpreted as compensatory responses to cope with the oxidative damage that has been accumulated by the aged brain. The increases in hippocampal and corical GPX activity were attenuated by NGF treatment, whereas the neurotrophin induced an increase in GRD activity in the striatum of aged rats. These resultas point out GRD and GPX as possible targets of the neurotrophic effects