Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury

Abstract

Bestrophin-3, a potential candidate for a calcium-activated chloride channel, recently was suggested to have cell-protective functions. We studied the expression and alternative splicing of bestrophin-3 in neonatal mouse brain and after hypoxic-ischemic (HI) injury and in human neonatal brain samples. HI brain injury was induced in 9-day old mice by unilateral permanent common carotid artery occlusion in combination with exposure to 10% oxygen for 50 min. Endoplasmic reticulum stress was induced by thapsigargin treatment in primary culture of mouse brain astrocytes. We also investigated expression of bestrophin-3 protein in a sample of human neonatal brain tissue. Bestrophin-3 protein expression was detected with immunohistochemical methods and western blot; mRNA expression and splicing were analyzed by RT-PCR. HI induced a brain tissue infarct and a pronounced increase in the endoplasmic reticulum-associated marker CHOP. Three days after HI a population of astrocytes co-expressed bestrophin-3 and nestin in a penumbra-like area of the injured hemisphere. However, total levels of Bestrophin-3 protein in mouse cortex were reduced after injury. Mouse astrocytes in primary culture also expressed bestrophin-3 protein, the amount of which was reduced by endoplasmic reticulum stress. Bestrophin-3 protein was detected in astrocytes in the hippocampal region of the human neonatal brain which had patchy white matter gliosis and neuronal loss in the Sommer's sector of the Ammon's horn (CA1). Analysis of bestrophin-3 mRNA in mouse brain with and without injury showed the presence of two truncated spliced variants, but no full-length mRNA. Total amount of bestrophin-3 mRNA increased after HI, but showed only minor injury-related change. However, the splice variants of bestrophin-3 mRNA were differentially regulated after HI depending on the presence of tissue injury. Our results show that bestrophin-3 is expressed in neonatal mouse brain after injury and in the human neonatal brain with pathology. In mouse brain bestrophin-3 protein is upregulated in a specific astrocyte population after injury and is co-expressed with nestin. Splice variants of bestrophin-3 mRNA respond differently to HI, which might indicate their different roles in tissue injury.

Keywords: alternative splicing; astroglia; bestrophin-3 (Best3); endoplasmic reticulum (ER) stress; hypoxia-ischemia.

FIGURE 1

Immunohistochemical evidence for Best3 expression in newborn mouse and human brain after injury. Best3 is expressed in mouse brain cortex after HI (a–d), in mouse primary astrocyte culture (e,f) and in brain of newborn child with birth pathology (g,h). Best3 was expressed in the injured mouse brain 3 days after HI (a) in a subpopulation of GFAP-positive cells (b,d). Some cells expressed both Best3 and GFAP (b, red arrows), while other GFAP-positive cells did not express Best3 (b, green arrows). Best3-positive cells also expressed the intermediate filament nestin and were localized mostly in cortex and hippocampus in a penumbra-like area of the injury (a,c). Confocal image of the penumbra-like area (d) shows Best3 intracellularly in the perinuclear area. Similar to the brain tissue astrocytes, some, but not all GFAP-positive astrocytes in a primary culture were expressing Best3 (e). These cells also expressed the intermediate filament nestin (f). Best3 is stained red, nestin and GFAP green (a–d), Best3 is stained green, nestin and GFAP red (e,f), nuclear staining with DAPI blue, magnification 10× (a), 40× (b,c), 60× (d). Best3 was also expressed in astrocyte-like cells in cortical areas of the brain of a term infant with white matter gliosis (g, enlarged Best3-positive structure in h). Peroxidase-based immunostaining captured at magnification 20×.

FIGURE 2

PCR analysis of Best3 mRNA alternative splicing in mouse brain with and without HI injury. Alternative splicing of Best3 mRNA was studied by a set of primers (A) spanning the areas of expected splicing of exons 2, 3, and 6 in different combinations as described in detail previously (Golubinskaya et al., 2015). The results of these PCR experiments are presented in (B–D). Total Best3 mRNA can be detected by primer pair C localized in exons 9 and 10 in both healthy (C) and injured brain (B). Splice variants of Best3 with exons 2 and 3 present were not detected, neither in healthy brain nor in injury (pairs of primers: A, localized in exons 1 and 5; D in exons 2 and 7; E in exons 3 and 5; F in exons 2 and 3). Exon 6 can be either present or spliced out in Best3 mRNA. Primer pair B spanning exons 4–7 gives two bands, the heavier band corresponds to “+6” and the lighter one to “–6” splice variants. The pair of primers G localized in exons 6 and 8 shows a band corresponding to “+6” splice variant. Primer pair I detects the “–6” variant (D). Primer pairs G and I can potentially detect more than one transcript corresponding to “+6” and “–6” variants depending on possible splicing of exon 10. Positive controls for these primers have been published previously by our group (Golubinskaya et al., 2015). MWL, molecular weight ladder.

FIGURE 3

The expression of Best3 mRNA in mouse brain tissue at different time points after HI. Figures show relative expression of the studied mRNAs in uninjured (R, blue) and injured (L, red) hemispheres in relation to the average expression in corresponding hemispheres of sham animals at the respective time points (sham group mean set to zero in the figure). Statistical analysis was performed by mixed-model three-way ANOVA on Cp values; main effects were based on subsequently estimated marginal means. Symbols above graphs indicate significant difference between uninjured and sham (R vs. sham group at zero, ), between injured and sham (L vs. sham group at zero, ), and between uninjured and injured (R vs. L, ×), all symbols indicate P < 0.05. The total mRNA for Best3 increased in both hemispheres at early time points after HI, but returned to control values already 24 h after HI, and even showed a transient decrease at 72 h (A). The long “+6” splice variant increased only in the uninjured hemisphere, while in the injured hemisphere it decreased and stayed decreased through all time point except 24 h (B). The short “–6” splice variant transiently increased at 6 and 12 h, and in the injured hemisphere also again at 72 h (C). Expression of mRNA for nestin (D) decreased at early time points in non-injured hemisphere, but returned to control values at 72 h after HI, while it increased in the injured hemisphere during this period to return to control values at 7 days after injury. The ER-stress marker CHOP slightly decreased during the early period after HI in the non-injured hemisphere, but was elevated throughout the studied period in the injured hemisphere (E). A total of 41 pups (26 HI, 15 sham) were used; for details see section “Materials and Methods.” Detailed statistical results are given in the (Supplementary Table S1).

FIGURE 4

Best3 mRNA is expressed in the primary culture of mouse astrocytes. TG caused very small changes in total Best3 (A) and in its short splice variant expression (C), but induced a noticeable increase in expression of the long “+6” splice variant of Best3 (B). TG-treated cells showed a pronounced ER-stress (E) and increase in nestin expression (D). ^∗∗P < 0.01, ^∗∗∗P < 0.001 t-test control vs. treatment.

FIGURE 5

Best3 protein is downregulated in mouse brain tissue and cultured mouse astrocytes after injury. Western blot analysis of brain tissue (A,B) and cultured astrocytes (C). Brain specimens from right uninjured (R) and left injured (L) hemispheres were alternately placed on the gels in (A,B); the same was done for sham-operated animals (A). Two neighboring R and L lanes belong to the same animal. In total protein samples from brain (A) at approximately 75–80 kDa two bands with close molecular weights could be detected; their density was summarized in the analysis. These bands were reduced in injury. Additional lighter pairs of bands could be observed at approximately 65 kDa. The result of subcellular fractionation of brain tissue is shown in (B), where two bands of approximately 75–80 kDa were detected in the cytoplasmic fraction as well as in the total protein fraction. The content of Best3 in the cytoplasm did not change significantly in injury, while Best3 in the total protein fraction was downregulated. In cultured astrocytes (C) treatment with thapsigargin for 48 h (TG 48 h) also caused downregulation of Best3. ^∗P < 0.05 by two-way ANOVA and estimated marginal means; ^∗∗∗P < 0.001 t-test R vs. L or treated vs. untreated cells. A total of 18 pups (14 HI, 4 sham) were used; for details see section “Materials and Methods.”