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Post-mitotic neuronal cells are more sensitive to the concentration of reactive oxygen species (ROS) in their surroundings than other cells. To remove excess amounts of deleterious ROS, neurons rely on oxidative phosphorylation and antioxidants such as glutathione (GSH). Previous studies have observed reduced levels of GSH in neuronal cells during neurological disorders, cognitive impairments and aging. However, the question of when GSH reduction happens (before, during or after neurological impairment) still remains unanswered. The main reason behind this gap in knowledge is due to the lack of an experiential model that truly represents a natural neuronal environment with low GSH levels.

Fernandez-Fernandez et al.2018, sought to find answers to the question of when do the reduced levels of GSH in neuronal cells happen during a neurological impairment. The researchers genetically engineered a mouse model to mimic an in vivo cellular environment with low GSH. To do this, they decided to knock down the catalytic subunit of Glutamate-Cysteine Ligase (GCL), which is the rate-limiting enzyme of cellular GSH production. The genetically engineered GCL knocked down mouse (small hairpin RNA against GCL unswitched flox GCL; shGCLUFL) is capable of mildly down regulating GSH levels in neurons, allowing the researchers to characterize oxidative stress and neuronal and behavioral responses.

Initially, western blotting and biochemical assays were performed to confirm that the shGCLUFL mouse exhibits reduced levels of GSH and thiol and increased levels of oxidized GSH and ROS. After verifying the low levels of GSH in brain, 2D electrophoresis was performed to detect oxidized protein carbonyls which further confirm GCL knockdown causes protein oxidation in vivo. Detection of oxidized protein carbonyls is a symbol of redox stress. In the hippocampal region, thirteen oxidized protein carbonyls were discovered by 2D electrophoresis. These proteins were purified and identified by mass spectroscopy; thus, confirming the appearance of redox stress and protein oxidation under mild GSH concentration in hippocampus.

Next, immunohistochemistry was performed on CK2a-Cre/shGCLSFL mice brains. The goal of the immunohistochemistry test is to see if any morphological changes or disruption to the neuronal network are seen due to redox stress. Antibodies for Microtubule-associated protein-2 (MAP2), GCL, CK2, and GSH (A001-50UG from Arbor Assays) were used to assess general abundance and colocalizations across the hippocampus, while an antibody against neuron-specific Class III β-tubulin (TUJ1) was used to assess specific dendritic disruption. Immunostaining showed disruptions of dendritesand neuronal inflammation in hippocampal region compared to control mice models. No morphological signs for neuronal loss such as a change in cell density or the length of cortex layers was seen. Finally, to assess how GCL knock down in CK2a-Cre/shGCLSFL mice impacts cognitive functions, a set of blind-behavioral experiments were performed. Results showed age dependent alterations in coordination, short-term memory and general cognitive deterioration.

In conclusion, the authors showed that mild down regulation of GSH in hippocampal neurons causes oxidative stress, damaging dendritic cells and altering brain functions. These results confirm that a large quantity of GSH is necessary for hippocampal neurons to prevent dendrite disruptionand maintain cognitive function.

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