Activation of Glutamate Transporter-1 (GLT-1) Confers Sex-Dependent Neuroprotection in Brain Ischemia

Stroke is one of the leading causes of long-term disability. During ischemic stroke, glutamate is released, reuptake processes are impaired, and glutamate promotes excitotoxic neuronal death. Astrocytic glutamate transporter 1 (GLT-1) is the major transporter responsible for removing excess glutamate from the extracellular space. A translational activator of GLT-1, LDN/OSU 0212320 (LDN) has been previously developed with beneficial outcomes in epileptic animal models but has never been tested as a potential therapeutic for ischemic strokes. The present study evaluated the effects of LDN on stroke-associated brain injury. Male and female mice received LDN or vehicle 24 h before or 2 h after focal ischemia was induced in the sensorimotor cortex. Sensorimotor performance was determined using the Rung Ladder Walk and infarct area was assessed using triphenyltetrazolium chloride staining. Males treated with LDN exhibited upregulated GLT-1 protein levels, significantly smaller infarct size, and displayed better sensorimotor performance in comparison to those treated with vehicle only. In contrast, there was no upregulation of GLT-1 protein levels and no difference in infarct size or sensorimotor performance between vehicle- and LDN-treated females. Taken together, our results indicate that the GLT-1 translational activator LDN improved stroke outcomes in young adult male, but not female mice

Up-Regulation of TREK-2 Potassium Channels in Cultured Astrocytes Requires <i>De Novo</i> Protein Synthesis: Relevance to Localization of TREK-2 Channels in Astrocytes after Transient Cerebral Ischemia

<div><p>Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K⁺ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.</p></div

TREK-2 expression is increased in the astrocytic membrane and cytoplasm after ischemia.

<p>Cortical astrocytes in culture were exposed to hypoxic/hypoglycemic conditions for 24 hours and then processed using a cell surface biotinylation assay. The graph displays the quantification of the relative chemiluminescence intensity ± standard error of the mean (SEM) of TREK-2 protein in membrane and cytoplasmic fractions obtained from control astrocytes and astrocytes subjected to ischemia (representative Western blots shown below the graph). TREK-2 was detected as a band of around 60kDa which is consistent with the predicted molecular weight of 59.6kDa. The results of 4 separate experiments using different astrocyte cultures are shown. The asterisks indicate a significant difference from control (t-test; p<0.05). Data are expressed relative to control.</p

TREK-2 up-regulation in response to ischemia is due to De novo protein synthesis.

<p>Cortical astrocytes were treated with different protein synthesis inhibitors (1μg/mL cycloheximide, 300nM emetine, 5μg/mL puromycin) or without treatment (vehicle) for 24 hours. The cells were then exposed to control or hypoxia/hypoglycemic conditions for 24 hours still in the presence of the inhibitors. Afterwards, cells were harvested and TREK-2 protein levels determined by Western blot. The graph summarizes the effect of three different protein synthesis inhibitors on TREK-2 up-regulation during ischemia. The data are expressed as relative chemiluminescence intensity ± SEM. The results of 6 separate experiments using different astrocyte cultures are shown. The asterisk indicates significant difference from the corresponding control (ANOVA followed by Tukey’s test; p<0.05).</p

TREK-2 protein degradation pathway(s) are not altered after ischemia.

<p>Cortical astrocyte in cultures exposed to (A) control or (B) hypoxia/hypoglycemic conditions for 24 hours were treated with either 10 μM chloroquine, 50 μM calpeptin or 10 μM MG132 for 8 hours. Astrocytes were initially exposed to hypoxia/hypoglycemic conditions for 16 hours. The cells were then treated with the degradative pathway inhibitors or without treatment and returned to hypoxia/hypoglycemic conditions for an additional 8 hours. TREK-2 expression in astrocytes was determined by Western blot. The results of 3 separate experiments using different astrocyte cultures are shown. Data are expressed relative to control.</p