Nordihydroguaiaretic acid increases glutamate uptake in vitro and in vivo: therapeutic implications for amyotrophic lateral sclerosis.

Synaptic accumulation of glutamate causes neuronal death in many neurodegenerative pathologies including amyotrophic lateral sclerosis. Drugs capable of increasing glutamate uptake could therefore be therapeutically effective. We screened in a cell-based assay a library of 1040 FDA-approved drugs and nutrients for compounds that could enhance glutamate uptake. Nordihydroguaiaretic acid (NDGA), an anti-inflammatory drug that inhibits lipoxygensases, potently enhanced glutamate uptake in MN-1 cells. Given subcutaneously at 1 mg/day for 30 days in mice, NDGA increased glutamate uptake in spinal cord synaptosomes persistently throughout the treatment. However, when administered following the same regimen to the SOD1-G93A transgenic mouse model of ALS at disease onset, NDGA did not extend survival of these mice. We found that NDGA failed to sustain increased glutamate uptake in the SOD1-G93A mice despite an initial upregulation measured during the first 10 days of treatment. SOD1-G93A mice displayed a progressive increase in spinal cord expression levels of the efflux transporter P-glycoprotein beginning at disease onset. This increase was not mediated by the NDGA treatment because it was measured in untreated SOD1-G93A mice. Since P-glycoproteins control the extrusion of a broad range of toxins and xenobiotics and are responsible for drug resistance in many diseases including cancer and brain diseases such as epilepsy, we propose that the failure of NDGA in maintaining glutamate uptake upregulated in SOD1-G93A mice and its therapeutic inefficacy are due to acquired pharmacoresistance mediated by the increased expression of P-glycoprotein.

A caspase-3-cleaved fragment of the glial glutamate transporter EAAT2 is sumoylated and targeted to promyelocytic leukemia nuclear bodies in mutant SOD1-linked amyotrophic lateral sclerosis.

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS.

Caspase-3 cleaves and inactivates the glutamate transporter EAAT2.

EAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS.