Helena Biasibetti

Cytoskeleton of cortical astrocytes as a target to proline through oxidative stress mechanisms

Hyperprolinemia is an inherited disorder of proline (Pro) metabolism and patients affected by this disease may present neurological manifestations. However, the mechanisms of neural excitotoxicity elicited by hyperprolinemia are far from being understood. Considering the pivotal role of cytoskeletal remodeling in several neurodegenerative pathologies and the potential links between cytoskeleton, reactive oxygen species production and cell death, the aim of the present work was to study the effects of Pro on astrocyte and neuron cytoskeletal remodeling and the possible oxidative stress involvement. Pro induced a shift of actin cytoskeleton in stress fibers together with increased RhoA immunocontent and ERK1/2 phosphorylation/activation in cortical astrocytes. Unlike astrocytes, results evidenced little susceptibility of neuron cytoskeleton remodeling, since Pro-treated neurons presented unaltered neuritogenesis. We observed increased hydrogen peroxide production characterizing oxidative stress together with decreased superoxide dismutase (SOD) and catalase (CAT) activities in cortical astrocytes after Pro treatment, while glutathione peroxidase (GSHPx) activity remained unaltered. However, coincubation with Pro and Trolox/melatonin prevented decreased SOD and CAT activities in Pro-treated astrocytes. Accordingly, these antioxidants were able to prevent the remodeling of the actin cytoskeleton, RhoA increased levels and ERK1/2 phosphorylation in response to high Pro exposure. Taken together, these findings indicated that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Pro and such effects could be mediated, at least in part, by redox imbalance, RhoA and ERK1/2 signaling pathways. The vulnerability of astrocyte cytoskeleton may have important implications for understanding the effects of Pro in the neurotoxicity linked to inborn errors of Pro metabolism.

Quinolinic acid neurotoxicity: Differential roles of astrocytes and microglia via FGF-2-mediated signaling in redox-linked cytoskeletal changes.

QUIN is a glutamate agonist playing a role in the misregulation of the cytoskeleton, which is associated with neurodegeneration in rats. In this study, we focused on microglial activation, FGF2/Erk signaling, gap junctions (GJs), inflammatory parameters and redox imbalance acting on cytoskeletal dynamics of the in QUIN-treated neural cells of rat striatum. FGF-2/Erk signaling was not altered in QUIN-treated primary astrocytes or neurons, however cytoskeleton was disrupted. In co-cultured astrocytes and neurons, QUIN-activated FGF2/Erk signaling prevented the cytoskeleton from remodeling. In mixed cultures (astrocyte, neuron, microglia), QUIN-induced FGF-2 increased level failed to activate Erk and promoted cytoskeletal destabilization. The effects of QUIN in mixed cultures involved redox imbalance upstream of Erk activation. Decreased connexin 43 (Cx43) immunocontent and functional GJs, was also coincident with disruption of the cytoskeleton in primary astrocytes and mixed cultures. We postulate that in interacting astrocytes and neurons the cytoskeleton is preserved against the insult of QUIN by activation of FGF-2/Erk signaling and proper cell-cell interaction through GJs. In mixed cultures, the FGF-2/Erk signaling is blocked by the redox imbalance associated with microglial activation and disturbed cell communication, disrupting the cytoskeleton. Thus, QUIN signal activates differential mechanisms that could stabilize or destabilize the cytoskeleton of striatal astrocytes and neurons in culture, and glial cells play a pivotal role in these responses preserving or disrupting a combination of signaling pathways and cell-cell interactions. Taken together, our findings shed light into the complex role of the active interaction of astrocytes, neurons and microglia in the neurotoxicity of QUIN.

Intracerebroventricular D-galactose administration impairs memory and alters activity and expression of acetylcholinesterase in the rat.

Tissue accumulation of galactose is a hallmark in classical galactosemia. Cognitive deficit is a symptom of this disease which is poorly understood. The aim of this study was to investigate the effects of intracerebroventricular administration of galactose on memory (inhibitory avoidance and novel object recognition tasks) of adult rats. We also investigated the effects of galactose on acetylcholinesterase (AChE) activity, immunocontent and gene expression in hippocampus and cerebral cortex. Wistar rats received a single injection of galactose (4 mM) or saline (control). For behavioral parameters, galactose was injected 1 h or 24 h previously to the testing. For biochemical assessment, animals were decapitated 1 h, 3 h or 24 h after galactose or saline injection; hippocampus and cerebral cortex were dissected. Results showed that galactose impairs the memory formation process in aversive memory (inhibitory avoidance task) and recognition memory (novel object recognition task) in rats. The activity of AChE was increased, whereas the gene expression of this enzyme was decreased in hippocampus, but not in cerebral cortex. These findings suggest that these changes in AChE may, at least in part, to lead to memory impairment caused by galactose. Taken together, our results can help understand the etiopathology of classical galactosemia.

Toxic Synergism Between Quinolinic Acid and Glutaric Acid in Neuronal Cells Is Mediated by Oxidative Stress: Insights to a New Toxic Model

It has been shown that synergistic toxic effects of quinolinic acid (QUIN) and glutaric acid (GA), both in isolated nerve endings and in vivo conditions, suggest the contribution of these metabolites to neurodegeneration. However, this synergism still requires a detailed characterization of the mechanisms involved in cell damage during its occurrence. In this study, the effects of subtoxic concentrations of QUIN and/or GA were tested in neuronal cultures, co-cultures (neuronal cells + astrocytes), and mixed cultures (neuronal cells + astrocytes + microglia) from rat cortex and striatum. The exposure of different cortical and striatal cell cultures to QUIN + GA resulted in cell death and stimulated different markers of oxidative stress, including reactive oxygen species (ROS) formation; changes in the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; and depletion of endogenous antioxidants such as -SH groups and glutathione. The co-incubation of neuronal cultures with QUIN + GA plus the N-methyl-d-aspartate antagonist MK-801 prevented cell death but not ROS formation, whereas the antioxidant melatonin reduced both parameters. Our results demonstrated that QUIN and GA can create synergistic scenarios, inducing toxic effects on some parameters of cell viability via the stimulation of oxidative damage. Therefore, it is likely that oxidative stress may play a major causative role in the synergistic actions exerted by QUIN + GA in a variety of cell culture conditions involving the interaction of different neural types.

Effects of previous physical exercise to chronic stress on long-term aversive memory and oxidative stress in amygdala and hippocampus of rats

Since stressful situations are considered risk factors for the development of depression and there are few studies evaluating prevention therapies for this disease, in the present study we evaluated the effect of previous physical exercise in animals subjected to chronic variable stress (CVS), an animal model of depression, on behavior tasks. We also investigated some parameters of oxidative stress and Na+, K+‐ATPase activity, immunocontent and gene expression of alpha subunits in amygdala and hippocampus of rats. Young male rats were randomized into four study groups (control, exercised, stressed, exercised + stressed). The animals were subjected to controlled exercise treadmill for 20 min,three times a week, for two months prior to submission to the CVS (40 days). Results show that CVS impaired performance in inhibitory avoidance at 24 h and 7 days after training session. CVS induced oxidative stress, increasing reactive species, lipoperoxidation and protein damage, and decreasing the activity of antioxidant enzymes. The activity of Na+, K+‐ATPase was decreased, but the immunocontents and gene expression of catalytic subunits were not altered. The previous physical exercise was able to improve performance in inhibitory avoidance at 24 h after training; additionally, exercise prevented oxidative damage, but was unable to reverse completely the changes observed on the enzymatic activities. Our findings suggest that physical exercise during the developmental period may protect against aversive memory impairment and brain oxidative damage caused by chronic stress exposure later in life.