Margaret L. Sutherland - US grants

DESCRIPTION (provided by applicant): Glutamate neurotoxicity has been proposed as a common secondary pathway to cell death in many neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS). In support of this hypothesis, recent evidence demonstrates that in the spinal cord, brainstem and motor cortex of roughly 65% of patients with sporadic forms of ALS, protein levels for the EAAT2 sub-type of glutamate transporter are greatly reduced. These changes in EAAT2 protein levels are correlated with aberrant splicing of the EAAT2 mRNA, resulting in proteins that either undergo rapid degradation and/or produce a dominant negative effect on normal EAAT2 trafficking. The net result of these changes is a reduction in glutamate uptake and a mechanism for increasing extracellular glutamate concentrations to neurotoxic levels. To address the role of glutamate transport in excitotoxic cell-death, we have recently generated a transgenic model of EAAT2 overexpression. Transgene expression is driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter and results in a 3-5 fold increase in synaptosomal, hippocampal, cerebrocortical and spinal cord D-aspartate uptake. In a kainic acid model of excitotoxic cell death, EAAT2 transgenic mice demonstrate a significant decrease in hippocampal cell death, which correlates with attenuation of the c-Jun immediate early gene stress response pathway. In transgenic models of motor neuron degeneration, EAAT2 protein levels are also diminished at either disease onset and/or end stage disease. Therefore, to test the hypothesis that excitotoxicity contributes to motor neuron cell death in these models, we have crossed EAAT2 transgenic mice with SOD1 G93A transgenics. Disease onset in the double-transgenic mice is delayed by 30 days, compared with clinical onset in SOD1 G93A littermates. The purpose of this grant is to further test the hypothesis that excitotoxicity is a component of motor neurodegeneration through the characterization of EAAT2/SOD1 G93A double transgenics. Using a non-SOD1 model of motor neuron loss, which also exhibits decreased glutamate uptake with disease onset, we will test the hypothesis that enhanced glutamate uptake plays a neuroprotective role in chronic neurodegenerative disease processes. To investigate mechanisms that lead to, or contribute to, motor neuron cell death, we propose to study the roles of astrocytic differentiation and gliosis in the regulation of EAAT2 mRNA and protein levels, and EAAT2 cellular distribution in spinal cord organotypic and primary culture.

DESCRIPTION (provided by applicant): Our long term goal is to characterize the cellular and molecular mechanisms regulated by glutamate transport activity under both normal and pathophysiological conditions. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, and as such plays a key role in neurological diseases involving hyperexcitability and excitotoxic cell death. Glutamate transport maintains extracellular glutamate concentrations below neurotoxic levels and loss of transporter protein is associated with several neurodegenerative disorders. We have tested the hypothesis that glutamate transporters play a key role in preventing cell death in limbic seizures, by generating a transgenic mouse model of astrocytic EAAT2 (Glt-1) overexpression. The EAAT2 protein is tagged with GFP (green fluorescent protein) and expression is driven by the astrocyte-specific GFAP promoter. Overexpression of the EAAT2 transgenic protein results in a 2-3 fold increase in hippocampal and cerebrocortical synaptosomal D-aspartate uptake. In a kainic acid (KA) model of temporal lobe epilepsy, increased glutamate transport results in an 80 percent decrease in hippocampal cell death compared to the level of cell death following KA-induced seizures in a wild-type age-matched animals. Surprisingly, we also found that increased glutamate uptake in the EAAT2 transgenic blunted network excitability and immediate early gene responses. These data, taken together with recent findings from other investigators, indicate that in addition to maintaining low steady-state concentrations of glutamate around the synaptic cleft, transporters may mediate a more rapid control of synaptic efficacy. Since glutamate is the major excitatory neurotransmitter in the CNS, we hypothesize that increased glutamate transport will blunt network excitability in most, if not all, CNS models of seizure-related hyperexcitability. To test this central hypothesis, we will use three models of status epilepticus (KA, pilocarpine and kindling). To extend our preliminary studies we will examine the acute actions of the glutamate analogue kainic acid using two in vitro slice preparations (hippocampal and piriform cortex) to determine if increased glutamate transport alters the threshold, frequency or duration of epileptiform activity. We will also inject KA, NMDA or AMPA into the hippocampus and record EEG activity to elucidate differences in seizure onset, frequency and duration in the presence of increased glutamate uptake (Specific Aim 1). To determine if the effects of increased glutamate transporter expression are generalized or limited to convulsants acting directly through a glutamate receptor pathway, we will use pilocarpine, that acts through muscarinic receptors, to generate seizure activity in vivo and in an in vitro hippocampal slice preparation to compare wild-type to EAAT2 transgenic responses (Specific Aim 2). Finally we will use two kindling models of status epilepticus to determine if increased glutamate transport activity lowers the threshold, rate of kindling acquisition, molecular plasticity or degree of cell death in PAAT2 transgenic mice compared with age-matched wild-type animals Specific Aim 3).