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microRNA‐132 is overexpressed in glia in temporal lobe epilepsy and reduces the expression of pro‐epileptogenic factors in human cultured astrocytes

Korotkov, Anatoly, Broekaart, Diede W. M., Banchaewa, Leyla, Pustjens, Ben, van Scheppingen, Jackelien, Anink, Jasper J., Baayen, Johannes C., Idema, Sander, Gorter, Jan A., van Vliet, Erwin A., Aronica, Eleonora·2020·Glia
DOI 10.1002/glia.23700PMC PMC6899748
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Abstract

Temporal lobe epilepsy (TLE) is a chronic neurological disease in humans, which is refractory to pharmacological treatment in about 30% of the patients. Reactive glial cells are thought to play a major role during the development of epilepsy (epileptogenesis) via regulation of brain inflammation and remodeling of the extracellular matrix (ECM). These processes can be regulated by microRNAs (miRs), a class of small non‐coding RNAs, which can control entire gene networks at a post‐transcriptional level. The expression of miRs is known to change dynamically during epileptogenesis. miR‐132 is one of the most commonly upregulated miRs in animal TLE models with important roles shown in neurons. However, the possible role of miR‐132 in glia remains largely unknown. The aim of this study was to characterize the cell‐type specific expression of miR‐132 in the hippocampus of patients with TLE and during epileptogenesis in a rat TLE model. Furthermore, the potential role of miR‐132 was investigated by transfection of human primary cultured astrocytes that were stimulated with the cytokines IL‐1β or TGF‐β1. We showed an increased expression of miR‐132 in the human and rat epileptogenic hippocampus, particularly in glial cells. Transfection of miR‐132 in human primary astrocytes reduced the expression of pro‐epileptogenic COX‐2, IL‐1β, TGF‐β2, CCL2, and MMP3. This suggests that miR‐132, particularly in astrocytes, represents a potential therapeutic target that warrants further in vivo investigation.

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Figure 1 Expression of miR‐132 in the rat hippocampus. (a)—Taqman RT‐qPCR showed a 2.1‐fold higher (p < .05) miR‐132 expression in the DG during the acute stage; (b–g)—in situ hybridization for miR‐132 in the control (b, c), during the acute (d, e) and the chronic (f, g) stages. Mostly neuronal expression was observed in the control DG (b) and CA1 (c). Higher expression was observed in cells with glial morphology in the DG ML (d, black arrowheads), CA1 SO and CA1 SR (e) during the acute stage as well as during the chronic stage (f, g); (h) OD analysis in different hippocampal layers relative to control; (i–k)—double labeling of miR‐132 with cell‐type specific markers: miR‐132 was co‐localized with NeuN (i), GFAP (j) and Iba1 (k), the insets show higher magnification of cells indicated by black arrows; scale bar in (b–g) 100 μm; scale bar in (i–k) 50 μm; scale bar in (i) inset 10 μm and applies to (j, k) insets; DG, dentate gyrus; GCL, granule cell layer; Hil, hilus; ML, molecular layer of the DG; PCL, pyramidal cell layer; SR, stratum radiatum; SO, stratum oriens; Mann–Whitney U test *p < .05, **p < .01 [Color figure can be viewed at https://wileyonlinelibrary.com]
Figure 1 Expression of miR‐132 in the rat hippocampus. (a)—Taqman RT‐qPCR showed a 2.1‐fold higher (p < .05) miR‐132 expression in the DG during the acute stage; (b–g)—in situ hybridization for miR‐132 in the control (b, c), during the acute (d, e) and the chronic (f, g) stages. Mostly neuronal expression was observed in the control DG (b) and CA1 (c). Higher expression was observed in cells with glial morphology in the DG ML (d, black arrowheads), CA1 SO and CA1 SR (e) during the acute stage as well as during the chronic stage (f, g); (h) OD analysis in different hippocampal layers relative to control; (i–k)—double labeling of miR‐132 with cell‐type specific markers: miR‐132 was co‐localized with NeuN (i), GFAP (j) and Iba1 (k), the insets show higher magnification of cells indicated by black arrows; scale bar in (b–g) 100 μm; scale bar in (i–k) 50 μm; scale bar in (i) inset 10 μm and applies to (j, k) insets; DG, dentate gyrus; GCL, granule cell layer; Hil, hilus; ML, molecular layer of the DG; PCL, pyramidal cell layer; SR, stratum radiatum; SO, stratum oriens; Mann–Whitney U test *p < .05, **p < .01 [Color figure can be viewed at https://wileyonlinelibrary.com]CC BY · PubMed Central
Figure 2 Expression of miR‐132 in the human hippocampus. (a)—Taqman RT‐qPCR showed a 3.4‐fold higher (p < .001) miR‐132 expression in the hippocampus of patients with TLE‐HS; (b–j)—in situ hybridization for miR‐132 in the hippocampus of TLE without HS (e–g) and TLE with HS (h–j) patients compared to autoptic control (b–d); mostly neuronal expression of miR‐132 was observed in the DG (b), hilus (c), and CA1 regions of control hippocampus. Higher expression was observed in cells with glial morphology in the DG ML (e, here and further indicated by black arrowheads), hilus (f) and CA1 (g) which was most prominent in the DG ML of TLE‐HS (h); hilus (i) and CA1 (j) and was associated with GCL dispersion and severe neuronal loss in the hilus and CA1 PCL; (k)—OD analysis showed a higher expression in CA1 SR (p < .05) and a lower expression in the GCL and PCL (both p < .05) of the TLE‐HS hippocampus relative to control; DG, dentate gyrus; GCL, granule cell layer; Hil, hilus; ML, molecular layer of the dentate gyrus; PCL, pyramidal cell layer; SO, stratum oriens; SR, stratum radiatum; scale bar 100 μm; Mann–Whitney U test *p < .05, ***p < .001 [Color figure can be viewed at https://wileyonlinelibrary.com]
Figure 2 Expression of miR‐132 in the human hippocampus. (a)—Taqman RT‐qPCR showed a 3.4‐fold higher (p < .001) miR‐132 expression in the hippocampus of patients with TLE‐HS; (b–j)—in situ hybridization for miR‐132 in the hippocampus of TLE without HS (e–g) and TLE with HS (h–j) patients compared to autoptic control (b–d); mostly neuronal expression of miR‐132 was observed in the DG (b), hilus (c), and CA1 regions of control hippocampus. Higher expression was observed in cells with glial morphology in the DG ML (e, here and further indicated by black arrowheads), hilus (f) and CA1 (g) which was most prominent in the DG ML of TLE‐HS (h); hilus (i) and CA1 (j) and was associated with GCL dispersion and severe neuronal loss in the hilus and CA1 PCL; (k)—OD analysis showed a higher expression in CA1 SR (p < .05) and a lower expression in the GCL and PCL (both p < .05) of the TLE‐HS hippocampus relative to control; DG, dentate gyrus; GCL, granule cell layer; Hil, hilus; ML, molecular layer of the dentate gyrus; PCL, pyramidal cell layer; SO, stratum oriens; SR, stratum radiatum; scale bar 100 μm; Mann–Whitney U test *p < .05, ***p < .001 [Color figure can be viewed at https://wileyonlinelibrary.com]CC BY · PubMed Central
Figure 3 Double labeling of miR‐132 with cell type‐specific markers in human TLE‐HS. (a)—miR‐132 was expressed in neurons throughout hippocampus, including granule cells, pyramidal cells and hilar neurons; co‐localization of miR‐132 was also found with markers of astrocytes GFAP (b), EAAT1 (c) and GS (d); miR‐132 was also co‐localized with Iba1+ microglial cells (e) in the areas of glial scar in CA1 and with CD34 (f) associated with blood vessels; the co‐localization with reactivity markers showed miR‐132 expression in vimentin+ cells with astrocytic morphology (g), HLA‐DR/DP/DQ‐expressing cells with microglial morphology (h) and STAT3+ cells in the CA1; black arrows indicate cells shown in higher magnification in insets; scale bar 50 μm; scale bar in inset a =10 μm and applies for insets a‐i [Color figure can be viewed at https://wileyonlinelibrary.com]
Figure 3 Double labeling of miR‐132 with cell type‐specific markers in human TLE‐HS. (a)—miR‐132 was expressed in neurons throughout hippocampus, including granule cells, pyramidal cells and hilar neurons; co‐localization of miR‐132 was also found with markers of astrocytes GFAP (b), EAAT1 (c) and GS (d); miR‐132 was also co‐localized with Iba1+ microglial cells (e) in the areas of glial scar in CA1 and with CD34 (f) associated with blood vessels; the co‐localization with reactivity markers showed miR‐132 expression in vimentin+ cells with astrocytic morphology (g), HLA‐DR/DP/DQ‐expressing cells with microglial morphology (h) and STAT3+ cells in the CA1; black arrows indicate cells shown in higher magnification in insets; scale bar 50 μm; scale bar in inset a =10 μm and applies for insets a‐i [Color figure can be viewed at https://wileyonlinelibrary.com]CC BY · PubMed Central
Figure 4 miR‐132 overexpression modulates TGF‐β pathway. The stimulation of primary human fetal astrocytes with TGF‐β1 resulted in higher expression of TGFB1 (p < .01), TGFB2 (p < .001), TGFBR1 (p < .001), THBS1 (p < .01), MMP2 (p < .001), MMP14 (p < .001), and miR‐132 (p < .001) as compared to non‐stimulated control (a); the higher expression of TGFB2 was attenuated by miR‐132 mimic transfection (p < .05) after TGF‐β1 stimulation (b); no change was found in expression of SMAD2 (c) and THBS1 (d); the expression of TGFBR2 was higher after miR‐132 transfection (p < .001) (e); western blot analysis showed that the expression of TGF‐β2 protein was lower (p < .01) after miR‐132 transfection (f, g); the data was normalized to the stimulated control condition and the dotted line indicates non‐stimulated control; veh, vehicle (Lipofectamine 2000); α‐132 indicates antagomir; n = 5 for RT‐qPCR, n = 3 for western blot, Mann–Whitney U test, *p < .05, **p < .01, ***p < .001
Figure 4 miR‐132 overexpression modulates TGF‐β pathway. The stimulation of primary human fetal astrocytes with TGF‐β1 resulted in higher expression of TGFB1 (p < .01), TGFB2 (p < .001), TGFBR1 (p < .001), THBS1 (p < .01), MMP2 (p < .001), MMP14 (p < .001), and miR‐132 (p < .001) as compared to non‐stimulated control (a); the higher expression of TGFB2 was attenuated by miR‐132 mimic transfection (p < .05) after TGF‐β1 stimulation (b); no change was found in expression of SMAD2 (c) and THBS1 (d); the expression of TGFBR2 was higher after miR‐132 transfection (p < .001) (e); western blot analysis showed that the expression of TGF‐β2 protein was lower (p < .01) after miR‐132 transfection (f, g); the data was normalized to the stimulated control condition and the dotted line indicates non‐stimulated control; veh, vehicle (Lipofectamine 2000); α‐132 indicates antagomir; n = 5 for RT‐qPCR, n = 3 for western blot, Mann–Whitney U test, *p < .05, **p < .01, ***p < .001CC BY · PubMed Central
Figure 5 miR‐132 overexpression downregulates IL‐1β‐induced factors. The stimulation of primary human fetal astrocytes with IL‐1β resulted in increased expression of PTGS2, IL1B, CCL2, IL6, MMP3 (all p < .001) and MMP9 (p < .01) (a); miR‐132 transfection attenuated the increased expression of PTGS2 (b, p < .01), IL1B (c, p < .001), CCL2 (d, p < .01) and MMP3 (f, p < .001), whereas inhibition of miR‐132 further increased the expression of PTGS2 (b, p < .001), IL1B (c, p < .001), MMP3 (f, p < .001) and MMP9 (g, p < .001); no modulation of expression was found for IL6 (e); semi‐quantitative analysis of the western blot showed that the expression of COX‐2 protein was also decreased (p < .01) after miR‐132 transfection (h, i); the data were normalized to the stimulated control condition and the dotted line indicates non‐stimulated control; veh, vehicle (Lipofectamine 2000); α‐132 indicates antagomir; n = 5 for RT‐qPCR, n = 3 for western blot, Mann–Whitney U test, *p < .05, **p < .01, ***p < .001
Figure 5 miR‐132 overexpression downregulates IL‐1β‐induced factors. The stimulation of primary human fetal astrocytes with IL‐1β resulted in increased expression of PTGS2, IL1B, CCL2, IL6, MMP3 (all p < .001) and MMP9 (p < .01) (a); miR‐132 transfection attenuated the increased expression of PTGS2 (b, p < .01), IL1B (c, p < .001), CCL2 (d, p < .01) and MMP3 (f, p < .001), whereas inhibition of miR‐132 further increased the expression of PTGS2 (b, p < .001), IL1B (c, p < .001), MMP3 (f, p < .001) and MMP9 (g, p < .001); no modulation of expression was found for IL6 (e); semi‐quantitative analysis of the western blot showed that the expression of COX‐2 protein was also decreased (p < .01) after miR‐132 transfection (h, i); the data were normalized to the stimulated control condition and the dotted line indicates non‐stimulated control; veh, vehicle (Lipofectamine 2000); α‐132 indicates antagomir; n = 5 for RT‐qPCR, n = 3 for western blot, Mann–Whitney U test, *p < .05, **p < .01, ***p < .001CC BY · PubMed Central

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Metadata

Authors
Korotkov, A., Broekaart, D. W. M., Banchaewa, L., Pustjens, B., van Scheppingen, J., Anink, J. J., Baayen, J. C., Idema, S., Gorter, J. A., van Vliet, E. A., & Aronica, E.
Affiliations
  • Amsterdam UMC, University of Amsterdam Department of (Neuro)Pathology, Amsterdam Neuroscience, Meibergdreef 9 Amsterdam the Netherlands
  • Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Neurosurgery, Amsterdam Neuroscience, De Boelelaan 1117 Amsterdam the Netherlands
  • Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam Amsterdam the Netherlands
ORCID
Venue
Glia
Publisher
Wiley
Published
2019-08-13
Volume
68
Issue
1
Pages
60-75
ISSN
  • 0894-1491
  • 1098-1136
DOI
10.1002/glia.23700
PMC
PMC6899748
Cited by
70
References
11
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Epilepsiefonds (16‐05)
  • FP7 Health (602102)
  • H2020 Marie Skłodowska-Curie Actions (642881)

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