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.

Polyamines (PAs) are polycationic biomolecules containing multiple amino groups. Patients with HIV-associated neurocognitive disorder (HAND) have high concentrations of the polyamine N-acetylated spermine in their brain and cerebral spinal fluid (CSF) and have increased PA release from astrocytes. These effects are due to the exposure to HIV-Tat. In healthy adult brain, PAs are accumulated but not synthesized in astrocytes, suggesting that PAs must enter astrocytes to be N-acetylated and released. Therefore, we tested if Cx43 hemichannels (Cx43-HCs) are pathways for PA flux in control and HIV-Tat-treated astrocytes. We used biotinylated spermine (b-SPM) to examine polyamine uptake. We found that control astrocytes and those treated with siRNA-Cx43 took up b-SPM, similarly suggesting that PA uptake is via a transporter/channel other than Cx43-HCs. Surprisingly, astrocytes pretreated with both HIV-Tat and siRNA-Cx43 showed increased accumulation of b-SPM. Using a novel polyamine transport inhibitor (PTI), trimer 44NMe, we blocked b-SPM uptake, showing that PA uptake is via a PTI-sensitive transport mechanism such as organic cation transporter. Our data suggest that Cx43 HCs are not a major pathway for b-SPM uptake in the condition of normal extracellular calcium concentration but may be involved in the release of PAs to the extracellular space during viral infection.

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.

Background/Objectives: Diabetes mellitus is a metabolic disorder, and hyperglycemia results in abnormal brain function. Since glycolysis is the main energy pathway in glial cells, astrocytes possess a more developed glyoxalase (Glo) system than neurons and exhibit better survival. Glycolysis helps to protect glia from (i) dicarbonyl stress and (ii) formation of advanced glycation end products (AGEs). Since aminoguanidine (AG) is an inhibitor of AGE production, the purpose of this study was to determine the role of AG in crucial astrocytic proteins, such as Kir4.1, Glo1, and Glo2, in hyperglycemic conditions. Methods: We cultured astrocytes in normal (5 mM)- and high (25 mM)-glucose conditions. After two weeks, we seeded the cells in six-well plates, with 300,000 cells/well, and then treated them with 9 mM of AG for 24 h. Results: Expression of the glyoxalases Glo1 and Glo2, and of Kir4.1, is decreased in hyperglycemic conditions; however, treatment with AG recovers the expression of the Kir4.1 protein as well as the inward currents of hyperglycemic astrocytes. Conclusion: We demonstrated that regulation of the glyoxalase system via AG or another scavenger of carbonyl and aldehydes containing polyamine groups can contribute to the recovery of astrocyte function in diabetic patients.

Polyamine (PA) spermidine (SPD) plays a crucial role in aging. Since SPD accumulates in glial cells, particularly in Müller retinal cells (MCs), the expression of the SPD-synthesizing enzyme spermidine synthase (SpdS) in Müller glia and age-dependent SpdS activity are not known. We used immunocytochemistry, Western blot (WB), and image analysis on rat retinae at postnatal days 3, 21, and 120. The anti-glutamine synthetase (GS) antibody was used to identify glial cells. In the neonatal retina (postnatal day 3 (P3)), SpdS was expressed in almost all progenitor cells in the neuroblast. However, by day 21 (P21), the SpdS label was pronouncedly expressed in multiple neurons, while GS labels were observed only in radial Müller glial cells. During early cell adulthood, at postnatal day 120 (P120), SpdS was observed solely in ganglion cells and a few other neurons. Western blot and semi-quantitative analyses of SpdS labeling showed a dramatic decrease in SpdS at P21 and P120 compared to P3. In conclusion, the redistribution of SpdS with aging indicates that SPD is first synthesized in all progenitor cells and then later in neurons, but not in glia. However, MCs take up and accumulate SPD, regardless of the age-associated decrease in SPD synthesis in neurons.