MicroRNA-210 regulates the metabolic and inflammatory status of primary human astrocytes

Abstract

Background: Astrocytes are the most numerous glial cell type with important roles in maintaining homeostasis and responding to diseases in the brain. Astrocyte function is subject to modulation by microRNAs (miRs), which are short nucleotide strands that regulate protein expression in a post-transcriptional manner. Understanding the miR expression profile of astrocytes in disease settings provides insight into the cellular stresses present in the microenvironment and may uncover pathways of therapeutic interest.

Methods: Laser-capture microdissection was used to isolate human astrocytes surrounding stroke lesions and those from neurological control tissue. Astrocytic miR expression profiles were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Primary human fetal astrocytes were cultured under in vitro stress conditions and transfection of a miR mimic was used to better understand how altered levels of miR-210 affect astrocyte function. The astrocytic response to stress was studied using qPCR, enzyme-linked immunosorbent assays (ELISAs), measurement of released lactate, and Seahorse.

Results: Here, we measured miR expression levels in astrocytes around human ischemic stroke lesions and observed differential expression of miR-210 in chronic stroke astrocytes compared to astrocytes from neurological control tissue. We also identified increased expression of miR-210 in mouse white matter tissue around middle cerebral artery occlusion (MCAO) brain lesions. We aimed to understand the role of miR-210 in primary human fetal astrocytes by developing an in vitro assay of hypoxic, metabolic, and inflammatory stresses. A combination of hypoxic and inflammatory stresses was observed to upregulate miR-210 expression. Transfection with miR-210-mimic (210M) increased glycolysis, enhanced lactate export, and promoted an anti-inflammatory transcriptional and translational signature in astrocytes. Additionally, 210M transfection resulted in decreased expression of complement 3 (C3) and semaphorin 5b (Sema5b).

Conclusions: We conclude that miR-210 expression in human astrocytes is modulated in response to ischemic stroke disease and under in vitro stress conditions, supporting a role for miR-210 in the astrocytic response to disease conditions. Further, the anti-inflammatory and pro-glycolytic impact of miR-210 on astrocytes makes it a potential candidate for further research as a neuroprotective agent.

Keywords: Astrocyte; Hypoxia; Inflammation; Ischemia; MicroRNA-210; Multiple sclerosis; Stroke.

Fig. 1

microRNAs are differentially expressed in astrocytes from neurological control tissue versus astrocytes surrounding stroke lesions. A Hematoxylin and eosin staining of early acute (upper panel) and late chronic (lower panel) infarcts. Subtle rarefaction/vacuolization of the parenchyma, loss of viable neurons, and acute ischemic, hypereosinophilic neurons (inset) are visible in the acute lesion. Cavitation of the parenchyma with a rim of well-developed astrocytosis (inset) is visible in chronic infarct lesions. B–D GFAP + astrocytes were captured from white (WM) and gray (GM) matter of unaffected (Neurological Control) brain tissue and around acute or chronic stroke lesions using laser-capture microdissection (LCM). Around 35 cells were pooled from each slide for RT-qPCR assessment of microRNA expression. B Bright-field images showing a brain section before and after LCM of astrocytes. C Heatmap of microRNA (miR) expression in GFAP + astrocytes. Color code represents the fold change of miR expression in lesioned WM and GM astrocytes compared to respective neurological control astrocytes. miRs are grouped according to their associated function. D Histograms of miR-210 and miR-21 expression in GFAP + astrocytes. Data are presented as mean ± SEM of n = 5 donors, except for WM C for which n = 4. One-way ANOVA was used for significance testing with Dunnett’s multiple comparison test. All miRs with statistically significant differences between neurological control and disease conditions are graphed in red in part D. *p < 0.05

Fig. 2

microRNAs are differentially expressed in stressed astrocytes compared to unstressed astrocytes. A Primary human fetal astrocytes were stained for expression of Glial Fibrillary Acidic Protein (GFAP) and DAPI to assess purity of human cultures. Scale bar = 500 µm. B–D Primary human astrocytes were untreated (“U”) or subjected to inflammatory (“I”), metabolic (“M”), and/or hypoxic (“H”) in vitro stress conditions using Interleukin-1 beta (IL1b), glucose-free media, or 1% oxygen, respectively. C Heatmap presenting the RT-qPCR assessment of microRNA expression in stress conditions relative to untreated control. Color code represents log₂(fold change) of miR expression in disease conditions compared to the expression levels in untreated cells. D Plot of miR-210 expression levels measured by RT-qPCR. Each dot represents a separate human donor, with 5–6 donors assayed per condition. One-way ANOVA with Sidak’s multiple comparison correction was used for significance testing in image D. *p < 0.05

Fig. 3

Primary human astrocytes downregulate known targets of miR-210 following transfection with miR-210-Mimic. A Primary human astrocytes were transfected with AlexaFluor BLOCK-iT as a positive control of transfection. Representative images of non-transfected (left) and transfected (right) cells immunostained for GFAP and overlayed with brightfield. Scale bar = 100 µm. B Primary human astrocytes were transfected with miR-210-Mimic (210M) or miR-210-Seed-mutant (210S) as a control. The expression of miR-210 in 210M-transfected relative to 210S-transfected cells was assessed by RT-qPCR, n = 6. C MiRabel analysis of miR-210 shows the five most likely gene-targets of miR-210 based on sequence-specific predicted targeting, with lower MiRabel scores signifying a higher likelihood of being targeted by miR-210. D RT-qPCR assessment of microRNA-210 targets Iron Sulfur Cluster Protein (ISCU, n = 9) and E Cytoglobin (CYGB, n = 8) in 210M-transfected relative to 210S-transfected cells. All data is graphed as mean ± SEM. Each dot represents a separate human sample. Paired t tests were performed for tests of significance. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

210M induces glycolysis and lactate export of primary human astrocytes. A miRPathDB was used to predict pathways targeted by miR-210. The pathways with the most hits are listed in descending order, and the p-value of each pathway is included inside the respective bar. B Oxygen Consumption Rate (OCR) was measured in primary human astrocytes 48 h after transfection with 210S or 210M. The basal oxygen consumption (before oligomycin) and maximal respiratory capacity (between FCCP and Rotenone/Antimycin A) are presented in the histogram. n = 3 with 10 technical replicates for OCR experiments. C Extracellular Acidification Rate (ECAR) was measured in primary human astrocytes 48 h after transfection with 210S or 210M. The basal (before glucose), normal glycolysis (between glucose and oligomycin) and glycolytic capacity (between oligomycin and 2-DG addition) are presented in the histogram. n = 4 with 20 technical replicates for ECAR experiments. D RT-qPCR assessment of MCT4 in 210M-transfected cells relative to 210S-transfected cells 48 h after transfection. Mean ± SEM of 8 donors. E RT-qPCR assessment of GPD1L in 210M-transfected cells relative to 210S-transfected cells 48 h after transfection. Mean ± SEM of 8 donors. F Immunoblotted bands of GPD1L and Beta-tubulin (Beta-Tub) proteins and their quantification in 210M-transfected relative to 210S-transfected cells 48 h after transfection. Mean ± SEM of 6 donors. G Primary human astrocytes were transfected with 210S or 210M. After 48 h, cells were washed, and the concentration of lactate was measured in media that was incubated with astrocytes for 2 h. Mean ± SEM of 8 donors. Statistical comparisons were made by two-way ANOVA with Sidak’s correction (B and C) or by a paired t-test (D–H). *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

210M transfected astrocytes exhibit an anti-inflammatory and neuroprotective phenotype. Primary human astrocytes were transfected with 210S or 210M, with or without prior exposure to hypoxic and inflammatory stress (“H-I”) using 1% oxygen and interleukin 1-beta, respectively. A, C RT-qPCR assessment of CXCL10, IL6, IGF1 gene expression. B, D CXCL10, IL6, and IGF1 protein levels in cell supernatant measured by ELISA. E, F RT-qPCR assessment of C3 and Sema5b gene expression. All data are presented as mean ± SEM of n = 9 (A, C and E), 5 (B and D) or 7 (F) donors. Statistical comparisons between groups were made by a one-way ANOVA (A and E) or by a paired t-test (B–D, F). *p < 0.05; **p < 0.01