Kir4.1 potassium channel regulation via miR-205 in astrocytes exposed to hyperglycemic conditions.

Abstract

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 miR-205 to the Kir4.1 3’-UTR 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 q-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 6 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 up-regulated 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.

Introduction

Hyperglycemia or high blood glucose affects patients who suffer from diabetes. Diabetes mellitus is a metabolic disorder that also affects the central nervous system (CNS) by raising brain glucose levels. These higher levels of glucose in the CNS result in glucose neurotoxicity that can lead to abnormal brain function and trauma [1]. Diabetes can be classified as type I or type II, both leading to higher levels of glucose in the blood hence affecting all organs in the body, significantly reducing their function and ultimately leading to their failure.

Astrocytes contribute a variety of functions and support to the brain, including homeostasis, synapse formation, plasticity and metabolism and disruption of such normal functions can contribute to and exacerbate brain injury. In normal conditions, astrocytes have a major role in the CNS by maintaining extracellular homeostasis of neuroactive substances such as K⁺, H⁺, GABA and glutamate: functions that may be depressed in multiple CNS diseases [2]. A more hyperpolarized membrane potential compared to neurons can be found in astrocytes in physiological conditions. This hyperpolarized membrane potential provides the necessary driving force for K⁺ spatial buffering and glutamate transport [3,4]. When astrocytes fail to take up excess K⁺ and glutamate from the extracellular space [3], it can result in excitotoxic neuronal cell death [2,4].

Among several potassium channels expressed in astrocytes, the major ion channel is the inwardly rectifying potassium channel Kir4.1 (encoded by the gene KCNJ10) [2,5]. It is expressed in astrocytes, radial glia, oligodendrocytes and NG2+ oligodendrocyte precursor cells [2,5]. The Kir4.1 potassium channel is not only a key player in efficient uptake of K⁺ released by neurons during axon potential propagation [4,6], but these channels also influence the ability of glial glutamate transporters to clear glutamate from the synaptic space [3]. Astrocytes are sensitive to hyperglycemia and we demonstrated recently that astrocytes grown in hyperglycemic conditions have a 50% reduction in Kir4.1 protein expression resulting in a decrease in two of their main homeostatic functions: potassium uptake and glutamate clearance [7]. Such reduction may result in a neurotoxic environment for the neurons but the specific mechanism of Kir4.1 regulation under hyperglycemic conditions remains unknown.

Previous studies performed in human corneal epithelial cell injury demonstrated a mechanism of regulation of Kir4.1 expression via microRNAs, specifically miR-205 which binds to the 3’UTR portion of the Kir4.1 mRNA sequence and inhibits expression of Kir4.1 [8]. MicroRNAs (miRNAs) are short single stranded endogenously-initiated non-coding RNAs that post-transcriptionally control gene expression either by translational repression or by degradation of mRNA. The length of a miRNA is about ~22nt long and its regulatory function has been described not only in cell development but also in cell proliferation, cell differentiation and apoptosis as well as in tumorigenesis [9]. miR-205 has been shown to regulate the migration and proliferation of cancer cells such as breast cancer cells [9]. In a corneal epithelial cell wound model, miR-205 inhibits Kir4.1 by binding at the 3’ UTR region resulting in a reduction of K⁺ membrane permeability increasing cell proliferation thereby repairing the wounded cells [8]. The aim of this study is to test 1) if astrocytes express miR-205 and 2) if miR-205 regulates Kir4.1 expression of astrocytes grown in hyperglycemic conditions. The results of this study will give us a mechanistic understanding of how hyperglycemia affects the normal function of astrocytes.

Methods

Astrocyte Primary Cultures

Primary cultures of astrocytes were prepared from neocortex of 1–2 day old rats as previously described [7,10] and in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC). Briefly, brains were removed after decapitation and the meninges stripped away to minimize fibroblast contamination. The forebrain cortices were collected and dissociated using the stomacher blender method. The cell suspension was then allowed to filter by gravity through a #60 sieve and then through a #100 sieve. After centrifugation, the cells were suspended in 2 groups. Both groups were plated in uncoated 75 cm² flasks at a density of 300,000 cells/cm². One group was plated in high glucose Dulbecco’s Modified Eagle Medium (DMEM) containing 25 mM glucose, 2 mM glutamine, 1 mM pyruvate and 10% fetal bovine serum, 100 iU/ml penicillin/100 μg/ml streptomycin. The second group was plated in normal glucose DMEM where the glucose concentration was 5 mM. The medium was exchanged with the appropriate fresh culture medium about every 4 days. At confluence (about 12 days), the mixed glial cultures were treated with 50 −75 mM leucine methylester (pH 7.4) for 10–20 min to kill microglia. Cultures were then allowed to recover for at least one day in growth medium prior to experimentation. Astrocytes were dissociated by trypsinization and reseeded onto appropriate plates for the experiments.

SDS-PAGE and Western Blotting Analysis

Astrocytes from primary cortical cultures were harvested, pelleted and resuspended in homogenization buffer for SDS-PAGE as previously described [9] using an anti-Kir4.1 guinea pig polyclonal antibody (1:800, Alomone, Jerusalem, Israel). Final detection was performed with enhanced chemiluminescence methodology (SuperSignal® West Dura Extended Duration Substrate; Pierce, Rockford, IL) as described by the manufacturer, and the intensity of the signal measured in a gel documentation system (ChemiDoc, BioRad, Hercules, California, USA). The intensity of the chemiluminescence signal was corrected for minor differences in protein content after densitometry analysis of the India ink stained membrane [11].

Real Time RT-PCR

For miRNA analysis, miRNA was isolated from astrocytes grown in normal and high glucose medium using an miRNeasy Kit (Qiagen, Valencia, California, USA, cat. no. 217004). Because miRNAs are very small and it has been reported to be very difficult to amplify them using normal primers, we used a stem-loop primer for mouse miR-205 (Qiagen, cat. no. MS000028833). A range between 10ng-2μg of sample was used for reverse transcription by Qiagen miScript II RT Kit (Qiagen, cat. no. 218160). Typical reverse transcription reactions (20 μL) contained 5x miScript HiSpec buffer, 10x miScript nucleics mix and miScript reverse transcriptase mix. The reaction was performed as suggested by the manufacturer’s protocol: 60 min incubation at 37°C followed by an inactivation step for 5 min at 95°C. The cDNA was processed for qPCR using the miScript SYBR Green PCR kit protocol (Qiagen, cat. no. 218073) for qPCR and iQcycler (BioRad). Typical amplification reactions (25 μL) contained 2x Quantitect SYBR Green PCR master mix, 10x miScript Universal Primers, 10x miScript Primer Assay (for either miR-205 or the SNORD 72 housekeeping gene for data normalization). After the 15 min activation step at 95°C, the amplification began with a 15 s denaturation step at 94°C, followed by 40 cycles of annealing at 55°C for 30 s and extension at 70°C for 30 s. Data were analyzed using my iQ software (BioRad).

Transfection

Astrocytes were cultured in either normal glucose (5 mM) or high glucose (25 mM) DMEM depending upon the experiment. Before transfection, 100,000 to 200,000 astrocytes were seeded in medium size petri dishes (Falcon cat. no. 35046) in 1.5ml of DMEM containing FBS and antibiotics and the appropriate concentration of glucose. Cells were transfected using HiPerfect (Qiagen, Cat. No. 301705) and the fast-forward protocol for transfection of adherent cells recommended by the manufacturer. Astrocytes grown in high glucose DMEM were treated with the miRNA205 inhibitor (Qigaen Cat. No. MIN0000878) or HiPerfect alone (mock-transfected), whereas astrocytes grown in normal glucose DMEM were treated with the miR-205 mimic (Qiagen, Cat. No. MSY0000878) or HiPerfect alone (mock-transfected). Briefly, 10 μl of 20 μM miR-205 inhibitor and 12μL of HiPerfect or 10 μl of 20 μM miR-205 mimic and 24μl of HiPerfect were diluted in 400μL of culture medium without serum. The mix was incubated for 10 minutes at room temperature to allow the formation of transfection complexes. The complexes were then added to the cells in a drop-wise fashion giving a final volume of 2mL and a final concentration of 100nM for the mimic and inhibitor. The plate was gently swirled to evenly distribute the transfection complexes and cells were incubated at 37°C for three days.

Data Analysis

Data from Western Blot and RT-PCR experiments were analyzed using GraphPad Prism (GraphPad, San Diego, CA) using a Student’s paired t-test. A value of P<0.05 was considered significant.

Results

We used RT-qPCR first to identify if miR-205 was present in primary rat cortical astrocyte cultures and second to determine if the levels of miR-205 were altered in astrocytes grown in hyperglycemic conditions. As shown in figure 1A, we found not only was miR-205 present in astrocytes but miR-205 was also significantly up-regulated (by about 6-fold) in astrocytes grown in high glucose DMEM compared to normal glucose control (6.2 ± 0.9, n=3). The relative expression of miR-205 was determined by the 2^−ΔΔct method with SNORD72 (small nucleolar RNA, C/D box 72) used as a reference gene to compensate for loading and pipetting differences. In this study, the reference gene, as shown in fig 1B, was expressed consistently in both the normal and high glucose groups.

Figure 1. miR-205 expression and regulation of Kir4.1 potassium channels in astrocytes grown in high glucose DMEM vs astrocytes grown in normal glucose DMEM.

A. Using quantitative RT-PCR for miR-205 from astrocytic miRNA obtained from primary rat cortical astrocytes two weeks after culturing in either high or normal glucose DMEM. We found a 6-fold up-regulation of miR-205 in astrocytes grown in DMEM containing high glucose compared to astrocytes grown in normal glucose DMEM (6.2 ± 0.9%). (*p<.05; n=3 in both groups). B. Using quantitative RT-PCR for SNORD72 from astrocytic miRNA obtained from primary rat cortical astrocytes two weeks after culturing in either high or normal glucose DMEM. We found no significant difference between normal glucose DMEM (22.58 ± 3.1, n=3) with high glucose DMEM (22.63 ± 2.7, n=3). Making SNORD 72 a good housekeeping gene for our normalization with miR-205.

To test if Kir4.1 expression is down-regulated by miR-205 in astrocytes, we used a miR-205 mimic to increase the expression of miR-205 in astrocytes grown in normal glucose DMEM and then measured Kir4.1 protein expression levels. As summarized in figure 2A, after transfection with the mimic there was a significant down-regulation of Kir4.1 protein expression (61.4± 6.6%, n=5) in astrocytes grown in normal glucose DMEM. In figure 2B, representative western blots for mock transfected and miR-205 mimic transfected astrocytes are shown as well as the corresponding India ink stained membrane used to correct for minor differences in protein content between samples.

Figure2. miR-205 expression and regulation of Kir4.1 potassium channels in astrocytes grown in normal glucose DMEM and transfected with miRNA-205 mimic.

A. Protein levels of Kir4.1 measured by Western blot in astrocytes grown in DMEM containing normal glucose and transfected with miRNA-205 mimic (mimic 205) were significantly down-regulated compared to mock-transfected astrocytes (61.4 ± 6.62%; n=5; *p<0.05). B. Representative picture of the membrane blotting after Kir4.1 antibody incubation with is corresponding India ink stained membrane for loading control calculations. In B the molecular weight of Kir4.1 was detected at ~43kDa.

To further verify the connection between miR-205 and Kir4.1 expression, we treated astrocytes grown in high glucose DMEM (with reduced Kir4.1 levels) with a miR-205 inhibitor to determine if reduction of miR-205 would result in an increase of Kir4.1 potassium channel protein expression. As shown in figure 3A, after transfection with the inhibitor there was a significant up-regulation of Kir4.1 protein expression (147.4 ± 8.6%; n=5) in astrocytes grown in high glucose DMEM. In figure3B, representative western blots for mock transfected and miR-205 inhibitor transfected astrocytes are shown as well as the corresponding India ink stained membrane.

Figure 3. miR-205 expression and regulation of Kir4.1 potassium channels in astrocytes grown in high glucose DMEM and transfected with miRNA-205 inhibitor.

A. Proteins levels of Kir.4.1 measured by Western blot in astrocytes grown in DMEM containing high glucose and transfected with miRNA-205 inhibitor (Inhibitor 205) were significantly up-regulated compare to mock-transfected astrocytes (147.4 ± 8.6%; n=5, *p<0.05). B. Representative picture of the membrane blotting after Kir4.1 antibody incubation with is corresponding India ink stained membrane for loading control calculations. In B the molecular weight of Kir4.1 was detected at ~43kDa.

Discussion

miRNAs (microRNAs) are small molecules that negatively regulate mRNA expression by binding complementarily to their targets. Many studies have indicated the changes in expression of miRNAs occur in diseases such as cancer, brain disorders, and diabetes [12]. Specifically, miR-205 in corneal epithelial cells [8] as well as miR-5096 in glioblastoma U87 and U251 cell lines [13] inhibit expression of Kir4.1. Kir4.1 downregulation in glioblastoma cells was achieved by (i) transfection with miR-5096, (ii) knockdown of Kir4.1 by siRNA or (iii) blockade of Kir4.1 by barium and each ultimately promoted the release of extracellular vesicles, increased the outgrowth of filopodia and promoted invasion [13]. On the other hand, in a corneal epithelial cell wound model, miR-205 inhibits Kir4.1 by binding to the 3’-UTR region which results in a reduction of K⁺ membrane permeability and increased cell proliferation helping to heal the damaged area of the cornea [8].

Since downregulation of Kir4.1 channels in astrocytes has been linked to hyperglycemia [7], we sought to identify a potential miRNA candidate which may regulate expression of Kir4.1 potassium channels in astrocytes exposed to hyperglycemic conditions. Our initial experiments identified miR-205 as the most likely candidate. We confirmed the presence of miR-205 in primary rat cortical astrocyte cultures by performing RT-qPCR and found that expression of miR-205 was increased when astrocytes were grown in a hyperglycemic environment.

Since miR-205 is upregulated and Kir4.1 is down-regulated in astrocytes grown in high glucose conditions, we explored whether there was a direct connection between miR-205 levels and Kir4.1 potassium channel expression in astrocytes. Indeed, we found that overexpression of miR-205 caused a reduction in Kir4.1 potassium channel protein levels in astrocytes, whereas inhibition of miR-205 led to increased Kir4.1 protein levels. Taken together, these data suggest that miR-205 is directly capable of regulating Kir4.1 potassium channel levels in astrocytes in response to changes in brain glucose concentrations. Although our astrocyte cultures are highly enriched with less then 8% non-GFAP⁺ cells [10], we cannot completely rule out that paracrine signaling from a few remaining oligodendrocytes can play a role in the downregulation of astrocytic Kir4.1 by miRNA-205. Nevertheless, such a contribution is likely to be minimal and would not alter the finding that miRNA-205 down-regulates Kir4.1 expression in astrocytes.

Furthermore, it is known that astrocytes grow faster in high glucose containing medium and this is one of the main reasons why most groups culture astrocytes in such conditions. In the miRNA-205 mimic and inhibitor experiments of the present study, we have minimized any possible effects of the different culture conditions influencing Kir4.1 expression. The miRNA-205 mimic experiments were performed using astrocytes grown in normal glucose conditions that were either mock-transfected or transfected with the miRNA-205 mimic. There were no apparent differences in the growth kinetics of these cells, but Kir4.1 protein was down-regulated in the cells transfected with the mimic. Similarly, the miRNA-205 inhibitor experiments were performed using astrocytes grown in high glucose conditions and while there were no apparent differences in the growth kinetics of these mock- and inhibitor-transfected astrocytes, Kir4.1 protein was up-regulated in those cells transfected with the inhibitor. These data indicate that levels of miRNA-205 and not the differences in culture conditions are responsible for the alterations in Kir4.1 protein expression.

It is important to highlight that previous work has demonstrated the binding of miR205 at the 3’-UTR regions of the Kir4.1 mRNA (KCNJ10). This was done specifically with the use of luciferase reporter assay where the KCNJ10 3’-UTR region was inserted in a vector containing the reporter gene (Luciferase) with either no mutation in the sequence (control) and or with the mutated KCNJ10 3’-UTR sequence. The study determined that miR-205 binds directly to the KCNJ10 3’-UTR region to regulate Kir4.1 expression [8].

It has also been demonstrated that protein levels of Kir4.1 and K⁺ inward current are decreased leading to a higher osmotic stress to the Müller glial cells in retina of streptozotocin treated diabetic rats [14]. In cultured Müller cells, hyperglycemia causes a reduction in Kir4.1 protein expression which could be reversed by administration of pigment epithelium-derived factor [15]. Although the mechanism by which Kir4.1 was reduced in Müller glial cells exposed to hyperglycemic conditions both in vivo and in vitro is unknown, it is interesting to speculate that a similar mechanism involving up-regulation of miR-205 may occur in retina. Indeed, miRNAs have recently been shown to regulate retinal function [16].

In addition to its role in regulating Kir4.1, miR-205 has been implicated as an oncogene or tumor suppressor depending on the target they regulate [17]. When miR-205 was overexpressed in prostate cancer cells, these cells showed a reduction in invasion and cell locomotion [18]. Other target genes of miR-205 are: N-chimaerin, ERBB3, E2F1 and 5, ZEB1 and 2, Cepsilon, VEGF-A, IL32 and 24 and CCNJ. Of these, ERBB3, E2F1 and 5, ZEB1 and 2, Cepsilon, VEGF-A and IL32 are present in astrocytes [19–24]. Therefore, evaluation of the possible miR-205 effect on the expression of these genes in astrocytes could reveal possible affected pathways during hyperglycemia conditions hence affecting astrocytic function.

Based on the experiments described above, we now provide evidence that miR-205 downregulates Kir4.1 expression in astrocytes exposed to hyperglycemic conditions and hyperglycemia up-regulates miR-205 expression in astrocytes. Furthermore, as previously shown in corneal epithelial cells, miR-205 negatively regulates Kir4.1 potassium channel expression in astrocytes. Taken together, it seems likely that hyperglycemia down-regulates Kir4.1 potassium channel expression in astrocytes directly via miR-205. If Kir4.1 in astrocytes is regulated similarly in vivo, potassium and glutamate clearance of astrocytes may be affected perhaps contributing to the cognitive impairment [5] observed in diabetics.