Spinal cord injury causes a wide-spread, persistent loss of Kir4.1 and glutamate transporter 1: benefit of 17 beta-oestradiol treatment

Abstract

During neuronal activity astrocytes function to remove extracellular increases in potassium, which are largely mediated by the inwardly-rectifying potassium channel Kir4.1, and to take up excess glutamate via glutamate transporter 1, a glial-specific glutamate transporter. Here we demonstrate that expression of both of these proteins is reduced by nearly 80% following a crush spinal cord injury in adult male rats, 7 days post-injury. This loss extended to spinal segments several millimetres rostral and caudal to the lesion epicentre, and persisted at 4 weeks post-injury. Importantly, we demonstrate that loss of these two proteins is not a direct result of astrocyte loss, as immunohistochemistry at 7 days and western blots at 4 weeks demonstrate a marked up-regulation in glial fibrillary acidic protein expression. Kir4.1 and glutamate transporter 1 expression were partially rescued by post-spinal cord injury administration of physiological levels of 17beta-oestradiol (0.08 mg/kg/day) in vivo. Utilizing an in vitro culture system we demonstrate that 17beta-oestradiol treatment (50 nM) is sufficient to increase glutamate transporter 1 protein expression in spinal cord astrocytes. This increase in glutamate transporter 1 protein expression was reversed and Kir4.1 expression reduced in the presence of an oestrogen receptor antagonist, Fulvestrant 182,780 suggesting a direct translational regulation of Kir4.1 and glutamate transporter 1 via genomic oestrogen receptors. Using whole-cell patch-clamp recordings in cultured spinal cord astrocytes, we show that changes in protein expression following oestrogen application led to functional changes in Kir4.1 mediated currents. These findings suggest that the neuroprotective benefits previously seen with 17beta-oestradiol after spinal cord injury may be in part due to increased Kir4.1 and glutamate transporter 1 expression in astrocytes leading to improved potassium and glutamate homeostasis.

Figure 1

A representative Western blot. Western blotting demonstrates significant decreases in Kir4.1 and GLT-1 seven days post-SCI. Representative western blot obtained from an injured and control animal probed with anti-Kir4.1 demonstrate significant decreases in immunoreactivity of a band at ∼200 kDa that corresponds to a tetramer and a band at ∼50 kDa corresponding to a monomer of Kir4.1. Significant decreases were detected for GLT-1. For a loading control this blot was probed with anti-actin.

Figure 2

Immunohistochemistry confirms a decrease in Kir4.1 and GLT-1 in the injured spinal cord. (A) Kir4.1 and GLT-1 demonstrate a similar staining pattern in the adult male spinal cord as demonstrated by the merged image (Kir4.1 = green, GLT-1 = red in merged image, scale bar = 50 µm). (B) Kir4.1 immunoreactivity demarcates neuronal cell bodies which are shown in the middle panel by Neu-N labelling (a neuronal cell body marker) (Kir4.1 = green, Neu-N = red in merged image, scale bar = 50 µm). (C) Low power transverse sections demonstrate widespread loss of both Kir4.1 and GLT-1 in the injured spinal cord. (D) This loss is reiterated in high power confocal images of Kir4.1 (green) and GLT-11 (green) overlayed with GFAP (red) (inset is secondary alone, scale bar = 50 µm). GFAP immunoreactivity is increased in injured tissue.

Figure 3

Kir4.1 and GLT-1 remain low 4 weeks post-injury. (A) Western blot with tissue from three control and two injured animals demonstrates at 4 weeks post-SCI there is still a significant loss of GLT-1 and Kir4.1 when compared to glyceraldehyde 3-phosphate dehydrogenase (gapDH) as a loading control. (B) A second western blot demonstrating similar results to those seen in (A), but also demonstrating a marked increase in GFAP, suggesting the loss of Kir4.1 and GLT-1 is not due to a lack of astrocytes in the region we examined.

Figure 4

Kir4.1 and GLT-1 loss extends longitudinally following SCI. (A) A cartoon depicting the distance rostral and caudal to the lesion used for western blotting. (B) This western blot from several animals 28 days post-SCI indicates Kir4.1 is significantly decreased as far as several millimetres in either direction from the epicentre of the lesion when compared to a loading control (glyceraldehyde 3-phosphate dehydrogenase; gapDH). This blot was subsequently stripped and blotted with anti-GLT-1 which also shows a significant loss that extends from the epicentre of the lesion. Human embryonic kidney (HEK) cells were used as a negative control.

Figure 5

Kir4.1 and GLT-1 loss is attenuated by 17β-oestradiol treatment. (A) Kir 4.1 and GLT-1 immunoreactivity levels are higher when animals are treated with 17β-oestradiol (E₂). In this western blot from lysates obtained 7 days post-SCI, increasing the oestrogen dose led to increased levels of Kir4.1 and GLT-1 when compared to a loading control. (B) Averaged data from five independent groups of animals (3 laminectomy + E₂) experiments show Kir 4.1 is reduced nearly 85% following a crush injury and physiological doses (0.5 mg/kg) of time-release 17β-oestradiol increase Kir4.1 in injured animals over 50%. There is no significant difference between laminectomy treated animals and injured animals treated with (0.5 mg/kg) of 17β-oestradiol or laminectomy treated animals treat with 17β-oestradiol. (C) Similar results were observed for GLT-1, approximately an 85% reduction in immunoreactivity following a crush injury and 17β-oestradiol increase GLT-1 in injured animals over 50%. (D, E) The alpha and beta oestrogen receptors are expressed in adult male spinal cord before and after injury at variable levels. Western blots from these animals were probed with antibodies to the oestrogen receptors α and β. Immunoreactivity demonstrates both receptors were present in all lysates. Uterus and ovary tissue from a post-partum rat were used as a positive control for oestrogen receptor-α and MCF7 cells, a breast cancer cell line, were used as a positive control for oestrogen receptor-β.

Figure 6

Increased Kir4.1 and GLT-1 expression in 17β-oestradiol-treated injured animals is transient. (A) A representative blot from three groups of animals demonstrates that there is a significant loss of Kir4.1 at 28 days in injured animals that is unchanged following oestrogen treatment. (B) Densitometry normalizing Kir4.1 levels to glyceraldehyde 3-phosphate dehydrogenase from at least five animals per condition demonstrate that Kir4.1 levels are similar in injured animals treated with a placebo to those treated with 0.5 mg/kg of 17β-oestradiol. (C) A representative western blot from the same group of three animals as in A shows GLT-1 levels are similar between injured animals and injured animals treated with 17β-oestradiol. (D) Mean data from five animals per group demonstrate an increase in overall protein by 4 weeks post-injury relative to the 7-day time point and with little difference between injured animals and those injured animals treated with 17β-oestradiol.

Figure 7

Acute slice recording from adult controls and injured spinal cord astrocytes demonstrates a high degree of variability of Kir4.1 currents in injured animals. (A) Representative Ba²⁺-sensitive Kir4.1 currents recordings in response to a voltage step protocol (inset) from each group of animals is depicted. In the injured animals, there was a high degree of variability in the amplitude of the Ba²⁺ subtracted traces; therefore we show several examples for this group of cells. (B) Data from all cells in which we obtained a barium subtraction are depicted in this scatter plot.

Figure 8

Kir4.1 and GLT-1 expression in cultured spinal cord astrocytes are modulated via oestrogen receptor signalling. (A) A western blot with spinal cord astrocytes treated for 24 or 48 h with 17β-oestradiol (50 nM) shows a decrease in Kir4.1 expression when treated with 17β-oestradiol (50 nM) + the oestrogen receptor antagonist ICI 182 780 (1 µM) when compared to a loading control. GLT-1 demonstrated an up-regulation in expression levels at 24 and 48 h when treated with 17β-oestradiol and values returned back to control levels when treated with 17β-oestradiol (50 nM) + the oestrogen receptor antagonist ICI 182 780 (1 µM). (B) Representative Ba²⁺-sensitive Kir4.1 currents recordings in response to a voltage step protocol (shown in inset) demonstrate a significant decrease in current amplitude in cells treated with 17β-oestradiol + the oestrogen receptor antagonist ICI 182 780 for 48 h. (C) Mean data from cells treated for 24 or 48 h with 17β-oestradiol (50 nM) or 17β-oestradiol (50 nM) + the oestrogen receptor antagonist ICI 182 780 (1 µM) demonstrate a significant (P < 0.01 at 24 and P < 0.05 at 48 h) decrease in Kir4.1 barium-sensitive currents at each time point. E₂ = 17β-oestradiol.