Desarrollo de un modelo experimental de estrés oxidativo in vivo

Abstract

The deleterious effects of reactive oxygen species (ROS) occur during adulthood, and it has been suggested that excess ROS -oxidative stress- may be a contributing factor in neurodegenerative processes. Glutathione (GSH) is one of the most abundant antioxidants and, in neurological diseases such as Parkinson's disease or mental disorders, GSH deficiency is the earliest known biochemical indicator of neuronal degeneration.This observation has led to the suggestion that oxidative stress may be behind the causes of neuronal dysfunction associated with these neurological disorders. Unfortunately, due to the lack of a sufficiently robust tool, the specific effect of GSH deficiency in the pathogenesis of neurological diseases has never been shown in vivo, thus the actual role of GSH loss-mediated oxidative stress in these disorders still remains elusive. Glutathione is a tripeptide (g-glutamylcysteinilglycine) synthesized by two consecutive ATP-dependent reactions. Glutamate-cysteine ligase (GCL or g-glutamylcysteine synthetase; EC 6.3.2.2) catalyzes the first -and rate-limiting- step, forming g-glutamylcysteine from glutamate and cysteine. This is followed by the glutathione synthetase (EC 6.3.2.3)-catalyzed reaction, which binds glycine to g-glutamylcysteine, forming glutathione. GCL is a heterodimeric enzyme composed of a catalytic (heavy; 73 kDa) and a modulatory (light; 27.7 kDa) subunit. Studies performed with purified GCL have shown that the active site resides at the catalytic subunit, whereas the modulatory subunit increases the affinity of the catalytic subunit for glutamate and decreases the sensitivity to feedback inhibition by GSH . In the brain, where -to the best of our knowledge- the enzyme has never been purified, GCL activity is very weak, although it is higher in astrocytes when compared with neurons.This contributes to the higher resistance of astrocytes, when compared with neurons, against oxidative stress. Astrocytes co-operate with neurons for neuronal antioxidant GSH biosynthesis by supplying GSH precursors. Thus, limiting either the supply of precursors, or the ability of neurons to use them, triggers oxidative stress in neurons leading to neurodegeneration, at least in culture. This has led us to hypothesize that neuronal-specific and temporally-controlled knockdown of GCL in vivo may lead to spontaneous neurological dysfunction, thus possibly mimicking the neurological problems associated with Parkinson's or mental diseases and potentially useful for the identification of novel redox-sensitive proteins involved in neurological disorders.

The existing in vivo models for studying GCL, catalytic subunit, deficiency in the brain are scarce and failed. Homozygous knockout mice against GCL, catalytic subunit, are not viable beyond embryonic day 8th, and the heterozygous ones display compensation mechanisms such as increased ascorbate biosynthesis. In addition, this available genetic system does not knockout GCL tissue specifically or temporally controlled, thus being unsuitable to investigate the role of oxidative stress in central nervous system during adulthood. We had previously identified a small hairpin RNA (shRNA) targeted against GCL, catalytic subunit that, in cultured neurons, triggered spontaneous oxidative stress. We have implemented RNAi strategy in vivo to produce a double-conditional mouse expressing the GCL shRNA in the cells of the central nervous system, particularly hippocampal neurons, to recreate oxidative stress in vivo t tissue specific and inducible way. We used Cre-LoxP technology to create mice expressing shGCL in neurons in vivo, and they were characterized biochemically, immuno-histologically and behaviorally. We found a decrease in GCL, and an increase in oxidative markers. We also found sex-dependent effects in behavioural characterization for anxiety, motor ability and memory tasks.

In conclusion, here we describe a novel strategy for studying oxidative stress in vivo in a tissue specific and time-controlled manner. This model may open new possibilities to study the involvement of elevated ROS in mental illnesses, such as Alzheimer's disease or anxiety, which are intrinsic to a number of psychiatric disorders including depression, panic attacks, phobias, obsessive-compulsive disorder and post-traumatic stress; however, these diseases currently lack of appropriate in vivo models for research on new therapeutic strategies. Furthermore, since we designed the genetic tool with two unique restriction sites flanking the shRNA sequence, new transgenic mice models could be straightforward generated to knockdown any other protein tissue-specifically in vivo. We believe that the transgenic mouse model herein described may be useful for a better understanding of the consequences of oxidative stress in specific cells in vivo, as well as for assessing novel pharmacological approaches.