Elevated Cortical Extracellular Fluid Glutamate in Transgenic Mice Expressing Human Mutant (G93A) Cu/Zn Superoxide Dismutase

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Abstract
Transgenic mice expressing a mutated (G93A) human Cu/Zn superoxide dismutase (SOD1) develop motor neuron pathology and clinical symptoms similar to those seen in patients with amyotrophic lateral sclerosis. Loss of motor neurons is most prominent in lumbar, followed by cervical cord and then brainstem. No significant cell death has been reported in motor cortex. The integrity of the cortical glutamate reuptake systems was evaluated using intracerebral microdialysis and western immunoblot assays for the glutamate transporters GLT‐1, GLAST, and EAAC1. The basal extracellular fluid levels of aspartate, glutamate, glutamine, 3,4‐dihydroxyphenylacetic acid, and 5‐hydroxyindole‐3‐acetic acid were evaluated by HPLC. The extraction fraction of L‐[3H]glutamate, corrected with [14C]mannitol, was also evaluated. GLT‐1, EAAC1, and GLAST protein levels were determined by semiquantitative chemiluminescence immunoblot of proteins from membrane‐enriched fractions. The relative optical density of film was translated into relative protein level by comparison with a standard control mouse. The SOD1 mutant mice demonstrated a significant (p < 0.05) increase in basal levels of extracellular aspartate and glutamate. In addition, when the glutamate extraction fraction was challenged with exogenous unlabeled glutamate (500 μM) by reversed microdialysis, the glutamate extraction fraction in the mutant SOD1 mice was decreased significantly from control levels. The SOD1 mutant mice demonstrated no difference in the cortical protein levels of the glutamate transporter subtypes. This study demonstrates that in areas of no visible pathology and no loss of glutamate transporter proteins, SOD1 mutant mice have elevated extracellular fluid aspartate and glutamate levels and a decreased capacity to clear glutamate from the extracellular space.

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