Downregulation of miR-23a and miR-27a following Experimental Traumatic Brain Injury Induces Neuronal Cell Death through Activation of Proapoptotic Bcl-2 Proteins

Abstract

MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level. To identify miRs that may regulate neuronal cell death after experimental traumatic brain injury (TBI), we profiled miR expression changes during the first several days after controlled cortical impact (CCI) in mice. miR-23a and miR-27a were rapidly downregulated in the injured cortex in the first hour after TBI. These changes coincided with increased expression of the proapoptotic Bcl-2 family members Noxa, Puma, and Bax. In an etoposide-induced in vitro model of apoptosis in primary cortical neurons, miR-23a and miR-27a were markedly downregulated as early as 1 h after exposure, before the upregulation of proapoptotic Bcl-2 family molecules. Administration of miR-23a and miR-27a mimics attenuated etoposide-induced changes in Noxa, Puma, and Bax, reduced downstream markers of caspase-dependent (cytochrome c release and caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways, and limited neuronal cell death. In contrast, miRs hairpin inhibitors enhanced etoposide-induced neuronal apoptosis and caspase activation. Importantly, administration of miR-23a and miR-27a mimics significantly reduced activation of Puma, Noxa, and Bax as well as attenuated markers of caspase-dependent and -independent apoptosis after TBI. Furthermore, miR-23a and miR-27a mimics significantly attenuated cortical lesion volume and neuronal cell loss in the hippocampus after TBI. These findings indicate that post-traumatic decreases in miR-23a and miR-27a contribute to neuronal cell death after TBI by upregulating proapoptotic Bcl-2 family members, thus providing a novel therapeutic target.

miR-711 upregulation induces neuronal cell death after traumatic brain injury

“…Neither TBI nor central administration of miR-711 hairpin inhibitor changed the levels of APAF1, cytochrome c and AIF-1 in whole cell lysates after TBI (data not shown), which is consistent with our previous results. 20 Western blot revealed that TBI induced the release of cytochrome c and AIF-1 from mitochondria to the cytoplasm, and AIF-1 translocation to nucleus (Figures 6a and b). Treatment with miR-711 hairpin inhibitor significantly reduced TBI-induced release of AIF-1 and cytochrome c into the cytosol and the level of AIF-1 in the nucleus (Figures 6a and b).…”

“…20 Although Bcl2 family molecules are not among miR-711-predicted targets, Akt is such a target. 26 Akt (protein kinase B) can attenuate neuronal apoptosis through phosphorylation and inhibition of caspases, pro-apoptotic Bcl2 family molecules and selected transcription factors (FoxO3a).…”

“…This concentration resulted in optimal transfection efficiency (~50%), was devoid of nonspecific changes in non-targeted miRs and had no neurotoxic effects. 20 Moreover, the chosen concentration was Figure 3 miR-711 hairpin inhibitor rescued Akt expression and attenuated translocation of AIF-1 and FoxO3a into nucleus. Controlled cortical impact in mice and etoposide treatment of cortical neurons decreased levels of Akt.…”

“…Verso cDNA Kit (Thermo Scientific) was used to synthesize cDNA from purified total RNA as described previously. 20 Quantitative real-time PCR amplification was performed by using cDNA TaqMan Universal Master Mix II (Applied Biosystems, Grand Island, NY, USA). TaqMan Gene Expression assays for following mouse genes were performed: GAPDH (Mm99999915_g1) and Trib3 (Mm00454879_m1) (Applied Biosystems).…”

“…We profiled miRs expression in the injured cortex of mice exposed to moderate-level CCI using miRNA array as described. 20 TBI significantly upregulated the level of miR-711 in brain (Figure 1a). We also performed a detailed expression profiling of miR-711 in the injured cortex using qPCR, and observed a rapid upregulation of miR-711 starting as early as 1 h post-injury, peaking at 6 h and persisting through 72 h (Figure 1a).…”