Korean J Physiol Pharmacol.  2021 Nov;25(6):565-574. 10.4196/kjpp.2021.25.6.565.

Kir4.1 is coexpressed with stemness markers in activated astrocytes in the injured brain and a Kir4.1 inhibitor BaCl ₂ negatively regulates neurosphere formation in culture

• Kwon JK
• Choi DJ
• Yang H
• Ko DW
• Jou I
• Park SM
• Joe EH

• KMID: 2521482
• DOI: http://doi.org/10.4196/kjpp.2021.25.6.565

Abstract

Astrocytes are activated in response to brain damage. Here, we found that expression of Kir4.1, a major potassium channel in astrocytes, is increased in activated astrocytes in the injured brain together with upregulation of the neural stem cell markers, Sox2 and Nestin. Expression of Kir4.1 was also increased together with that of Nestin and Sox2 in neurospheres formed from dissociated P7 mouse brains. Using the Kir4.1 blocker BaCl₂ to determine whether Kir4.1 is involved in acquisition of stemness, we found that inhibition of Kir4.1 activity caused a concentration-dependent increase in sphere size and Sox2 levels, but had little effect on Nestin levels. Moreover, induction of differentiation of cultured neural stem cells by withdrawing epidermal growth factor and fibroblast growth factor from the culture medium caused a sharp initial increase in Kir4.1 expression followed by a decrease, whereas Sox2 and Nestin levels continuously decreased. Inhibition of Kir4.1 had no effect on expression levels of Sox2 or Nestin, or the astrocyte and neuron markers glial fibrillary acidic protein and β-tubulin III, respectively. Taken together, these results indicate that Kir4.1 may control gain of stemness but not differentiation of stem cells.

Keyword

Kir4.1; Reactive astrocyte; Sox2; Stem cells

Figure

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  Fig. 1 Kir4.1 expression is increased in the injured brain. Brain damage was induced by stereotaxic injection of ATP (400 nM) into the striatum. (A) Proteins were prepared at the indicated times after ATP injection, and expression levels of Kir4.1 and glial fibrillary acidic protein (GFAP) were analyzed by Western blotting and quantified using Image J. GADPH was used as a loading control. Values are presented as means ± SEMs of three samples (**p < 0.001, ***p < 0.0001). (B–D) Brain sections were prepared at the indicated times after ATP injection and stained with antibodies for Kir4.1 (B), Kir4.1/Nestin/GFAP (C), or Kir4.1/Sox2/GFAP (D). Penumbra regions adjacent to the damage core (*) and farther from the core are designated (a, c) and (b, d), respectively. Scale bars: 100 μm (B, C, and D, upper panel) and 20 μm (C and D, lower panel).

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  Fig. 2 The Kir4.1 blocker, BaCl2, has little effect on self-renewal of stem cells or expression of Sox2 and Nestin. Cells from E13.5 mouse brains were cultured with epidermal growth factor (EGF) (20 ng/ml)/fibroblast growth factor (FGF) (20 ng/ml) and BaCl2 as indicated. (A, B) Size of spheres was measured. Scale bars: 100 μm. (A, C) mRNA levels of the indicated factors were analyzed. Values are means ± SEMs of three samples (*p < 0.05, **p < 0.001, ***p < 0.0001).

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  Fig. 3 BaCl2 enhances self-renewal of stem cells and increases Sox2 expression. Cells from P7 mouse brains were cultured with epidermal growth factor (EGF)/fibroblast growth factor (FGF) and BaCl2 as indicated. (A–C) Size of spheres was measured. Scale bars: 100 μm. (A, D, E) mRNA (A, D) and protein (E) levels of the indicated factors were analyzed. GAPDH was used as a loading control (D). Values are means ± SEMs of three samples (*p < 0.05, **p < 0.001, ***p < 0.0001).

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  Fig. 4 Kir4.1 expression and effect of Kir4.1 inhibition on differentiation of neural stem cells. NSCs were prepared from E13.5 mouse embryo brains and cultured with or without BaCl2. Differentiation was induced by withdrawal of epidermal growth factor (EGF) and fibroblast growth factor (FGF) from the culture medium. (A) mRNA levels of Nestin, glial fibrillary acidic protein (GFAP), Tubb3, and Kir4.1 at the indicated times after induction of differentiation were analyzed using qPCR. au, arbitrary unit. (B, C) Protein levels of Sox2, Nestin, GFAP, Tubb3 (Tuj-1 antibody), and Kir4.1 were analyzed by Western blotting (left panel) and quantified using Image J (right panel). GAPDH was used as a loading control. Values are means ± SEMs of three samples (*p < 0.05, **p < 0.001, ***p < 0.0001).

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