No abstract

Epilepsy, characterized by recurrent seizures, affects 1% of the general population. Interestingly, 25% of diabetics develop seizures with a yet unknown mechanism. Hyperglycemia downregulates inwardly rectifying potassium channel 4.1 (Kir4.1) in cultured astrocytes. Therefore, the present study aims to determine if downregulation of functional astrocytic Kir4.1 channels occurs in brains of type 2 diabetic mice and could influence hippocampal neuronal hyperexcitability. Using whole-cell patch clamp recording in hippocampal brain slices from male mice, we determined the electrophysiological properties of stratum radiatum astrocytes and CA1 pyramidal neurons. In diabetic mice, astrocytic Kir4.1 channels were functionally downregulated as evidenced by multiple characteristics including depolarized membrane potential, reduced barium-sensitive Kir currents and impaired potassium uptake capabilities of hippocampal astrocytes. Furthermore, CA1 pyramidal neurons from diabetic mice displayed increased spontaneous activity: action potential frequency was ≈9 times higher in diabetic compared with non-diabetic mice and small EPSC event frequency was significantly higher in CA1 pyramidal cells of diabetics compared to non-diabetics. These differences were apparent in control conditions and largely pronounced in response to the pro-convulsant 4-aminopyridine. Our data suggest that astrocytic dysfunction due to downregulation of Kir4.1 channels may increase seizure susceptibility by impairing astrocytic ability to maintain proper extracellular homeostasis.

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.

Protecting neurons from neurotoxicity is a job mainly performed by astrocytes through glutamate uptake and potassium buffering. These functions are aided principally by the Kir4.1 inwardly rectifying potassium channels located in the membrane of astrocytes. Astrocytes grown in hyperglycemic conditions have decreased levels of Kir4.1 potassium channels as well as impaired potassium and glutamate uptake. Previous studies performed in a human corneal epithelial cell injury model demonstrated a mechanism of regulation of Kir4.1 expression via the binding of microRNA-250 (miR-205) to the Kir4.1 3´ untranslated region. Our purpose is to test if astrocytes express miR-205 and elucidate its role in regulating Kir4.1 expression in astrocytes grown in hyperglycemic conditions. We used quantitative-PCR to assess the levels of miR-205 in astrocytes grown in high glucose (25 mM) medium compared to astrocytes grown in normal glucose (5 mM). We found that not only was miR-205 expressed in astrocytes grown in normal glucose, but its expression was increased up to six-fold in astrocytes grown in hyperglycemic conditions. Transfection of miR-205 mimic or inhibitor was performed to alter the levels of miR-205 in astrocytes followed by western blot to assess Kir4.1 channel levels in these cells. Astrocytes treated with miR-205 mimic had a 38.6% reduction of Kir4.1 protein levels compared to control (mock-transfected) cells. In contrast, astrocytes transfected with miR-205 inhibitor were significantly upregulated compared to mock by 47.4%. Taken together, our data indicate that miR-205 negatively regulates the expression of Kir4.1 in astrocytes grown in hyperglycemic conditions.

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