Conditional neuronal deletion of microRNA-141/200c cluster, but not microRNA-181a/b-1 cluster, is protective against experimental stroke in male mice

Abstract

MicroRNAs (miRs) regulate the translation of target genes often in a cell-type specific manner. We previously demonstrated that downregulation of either miR-181a or miR-200c with intracranial injection of an inhibitor is protective against experimental stroke in mice. Here, we generated genetic lines of inducible Ca²⁺-calmodulin kinase IIα (CKIIα) neuronal miR-181a/b-1 and miR-141/200c cluster deletion to investigate whether the protective effect of their inhibition could be neuron-specific. Jackson Lab strains Mirc14tm1.1Czc/J and Mirc13tm1Mtm/Mmjax were each crossed with the tamoxifen-inducible Cre-recombinase strain B6;129S6-Tg, CKIIα-cre/ERT2. Adult double transgenic male mice were randomized and treated with 3 mg tamoxifen or vehicle via oral gavage for 7 days prior to 1 h middle cerebral artery occlusion (MCAO) or sham surgery. Mice were assessed for gross motor function at 24 h and then sacrificed for quantification of infarct volume. Separate animals were assessed for cell-type specific brain expression of miR-181a and miR-200c via combined fluorescent immunohistochemistry and in situ hybridization. Brains from tamoxifen treated mice exhibited selective miR deletion in CKIIα neurons. Infarct volumes were significantly lower, and neurological scores significantly improved in CKIIα/miR-200c mice pretreated with tamoxifen versus vehicle alone. In contrast, no difference was observed in infarct volume or neurological score in CKIIα/miR-181a mice pretreated with tamoxifen versus vehicle.

Keywords: CKIIα; MCAO; cell type; miR; miRNA.

FIGURE 1

Representative images of fluorescent in situ hybridization (FISH) of miR‐181a (green, left panel) and miR‐200c (green, right panel), complexed with fluorescent immunohistochemistry indicating neurons (NeuN, red), astrocytes (glial fibrillary acidic protein, GFAP, violet), and all cell nuclei (DAPI, blue). Hippocampal cornu ammonis 1 (CA1) is identified as a representative region with high neuronal miR‐181a and miR‐200c expression. Scale bar represents 50 μm.

FIGURE 2

(a) Representative images of in situ hybridization for CKIIα mRNA expression in whole brain (left) and in hippocampus (right) from the Allen Brain Atlas project (Miller et al., 2017). Hippocampal CA1 is identified as a region with high CKIIα mRNA expression. (b) Representative FISH images of hippocampal CA1 in double transgenic CKIIα‐miR‐141/200c and CKIIα‐miR‐181a/b‐1 mice treated with either oil (left) or tamoxifen (right) for 7 days.

FIGURE 3

Quantification of infarct volume (a) and a representative TTC‐stained brain (b, white areas represent infarct) 24 h after MCAO in CKIIα‐miR‐141/200c mice pretreated with either oil or tamoxifen for 7 days. (c) Neurological deficit scores 24 h after MCAO. N = 10, mean ± SD.

FIGURE 4

Quantification of infarct volume (a) and a representative TTC‐stained brain (b) 24 h after MCAO in CKIIα‐miR‐181a/b‐1 mice treated with either oil or tamoxifen for 7 days. (c) Neurological deficit scores 24 h after MCAO. N = 7, mean ± SD.