Diabetics are at risk for a number of serious health complications including an increased incidence of epilepsy and poorer recovery after ischemic stroke. Astrocytes play a critical role in protecting neurons by maintaining extracellular homeostasis and preventing neurotoxicity through glutamate uptake and potassium buffering. These functions are aided by the presence of potassium channels, such as Kir4.1 inwardly rectifying potassium channels, in the membranes of astrocytic glial cells. The purpose of the present study was to determine if hyperglycemia alters Kir4.1 potassium channel expression and homeostatic functions of astrocytes. We used q-PCR, Western blot, patch-clamp electrophysiology studying voltage and potassium step responses and a colorimetric glutamate clearance assay to assess Kir4.1 channel levels and homeostatic functions of astrocytes grown in normal and high glucose conditions. We found that astrocytes grown in high glucose (25 mM) had an approximately 50% reduction in Kir4.1 mRNA and protein expression as compared with those grown in normal glucose (5 mM). These reductions occurred within 4 to 7 days of exposure to hyperglycemia, whereas reversal occurred between 7 to 14 days after return to normal glucose. The decrease in functional Kir channels in the astrocytic membrane was confirmed using barium to block Kir channels. In the presence of 100 μm barium, the currents recorded from astrocytes in response to voltage steps were reduced by 45%. Furthermore, inward currents induced by stepping extracellular [K+]o from 3 to 10 mM (reflecting potassium uptake) were 50% reduced in astrocytes grown in high glucose. In addition, glutamate clearance by astrocytes grown in high glucose was significantly impaired. Taken together, our results suggest that down-regulation of astrocytic Kir4.1 channels by elevated glucose may contribute to the underlying pathophysiology of diabetes-induced CNS disorders and contribute to the poor prognosis after stroke.

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.

A-kinase-anchoring proteins, AKAPs, are scaffolding proteins that associate with kinases and phosphatases, and direct them to a specific submembrane site to coordinate signaling events. AKAP150, a rodent ortholog of human AKAP79, has been extensively studied in neurons, but very little is known about the localization and function of AKAP150 in astrocytes, the major cell type in brain. Thus, in this study, we assessed the localization of AKAP150 in astrocytes and elucidated its role during physiological and ischemic conditions. Herein, we demonstrate that AKAP150 is localized in astrocytes and is up-regulated during ischemia both in vitro and in vivo. Knock-down of AKAP150 by RNAi depolarizes the astrocytic membrane potential and substantially reduces by 80% the ability of astrocytes to take up extracellular potassium during ischemic conditions. Therefore, upregulation of AKAP150 during ischemia preserves potassium conductance and the associated hyperpolarized membrane potential of astrocytes; properties of astrocytes needed to maintain extracellular brain homeostasis. Taken together, these data suggest that AKAP150 may play a pivotal role in the neuroprotective mechanism of astrocytes during pathological conditions.

Glioblastomas, the most malignant form of gliomas, harbor multiple cell types. In particular, microglial cells can contribute up to 30% of a brain tumor mass, and can promote glioma cell growth and dispersal. The purpose of the present study was to test the hypothesis that glioma cells recruit nearby microglia through an MCP-1-mediated mechanism and enhance their production of MCP-1 in the tumor microenvironment. We evaluated the role of MCP-1 on glioma cell proliferation and invasion. Consistent with previous studies in rat models (Platten et al., 2003), we found that U-87 and A-172 human glioma cells promote microglial migration towards the glioma environment using a modified Boyden chamber migration assay. Furthermore, this effect was decreased after immunoneutralization of MCP-1 released from glioma cells. Additionally, using an antibody array we found that U-87 or A-172 glioma cell conditioned medium stimulates a 7-10 fold increase of MCP-1 release by microglia by 24 hours. We next analyzed the consequences of elevated MCP-1 on the invasiveness and proliferation of glioma cells. Using standard invasion assays with or without microglia in a lower compartment, we demonstrated that microglia significantly increased invasion of glioma cells, but this effect was not blocked by MCP-1 immunoneutralization. This suggests that the stimulatory effect of microglia on glioma invasion was due to some other factor such as release of matrix metalloproteinases (MMPs). In contrast, microglia increased U-87 and A-172 glioma cell proliferation within 72 hours and MCP-1 immunoneutralization reversed this proliferative effect of microglia in both cell lines. Based on these results, we propose that glioma cells release low levels of MCP-1 to recruit nearby microglia and enhance the production of MCP-1. This increased secretion of MCP-1 from microglial cells recruits more microglial cells into the tumor and stimulate glioma progression. A variety of substances released by microglia in tumor microenvironment, including cytokines, growth factors, proteases and extracellular matrix components could lead to increased proliferation and invasiveness of glioma cells. Supported by G11 HD052352, G12 RR03035, 8G12MD007583-27, U54 NS039408 and by the UCC Pilot Project Program Citation Format: Lilia Y. Kucheryavykh, Aixa F. Rivera-Pagán, Kimberleve Rolón-Reyes, Serguey N. Skatchkov, Misty J. Eaton. Role of monocyte chemotactic protein-1 (MCP-1) in the tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1429. doi:10.1158/1538-7445.AM2013-1429