Mitochondria-associated microRNAs in rat hippocampus following traumatic brain injury

Highlights

• MiRNA machinery proteins Argonaute and Dicer are present in hippocampal mitochondria.

• Several miRNAs are enriched in hippocampal mitochondria under normal conditions.

• Numerous mitochondria-associated miRNAs are significantly altered following TBI.

• MiRNAs enriched in mitochondria decreased and cytosolic levels increased after TBI.

Abstract

Traumatic brain injury (TBI) is a major cause of death and disability. However, the molecular events contributing to the pathogenesis are not well understood. Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. There is clear evidence of compromised mitochondrial function following TBI; however, the underlying mechanisms and consequences are not clear. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally, and function as important mediators of neuronal development, synaptic plasticity, and neurodegeneration. Several miRNAs show altered expression following TBI; however, the relevance of mitochondria in these pathways is unknown. Here, we present evidence supporting the association of miRNA with hippocampal mitochondria, as well as changes in mitochondria-associated miRNA expression following a controlled cortical impact (CCI) injury in rats. Specifically, we found that the miRNA processing proteins Argonaute (AGO) and Dicer are present in mitochondria fractions from uninjured rat hippocampus, and immunoprecipitation of AGO associated miRNA from mitochondria suggests the presence of functional RNA-induced silencing complexes. Interestingly, RT-qPCR miRNA array studies revealed that a subset of miRNA is enriched in mitochondria relative to cytoplasm. At 12 h following CCI, several miRNAs are significantly altered in hippocampal mitochondria and cytoplasm. In addition, levels of miR-155 and miR-223, both of which play a role in inflammatory processes, are significantly elevated in both cytoplasm and mitochondria. We propose that mitochondria-associated miRNAs may play an important role in regulating the response to TBI.

Introduction

Traumatic brain injury (TBI) is a major cause of death and disability affecting an estimated 1.7 million people annually in the United States alone (Faul et al., 2010). The personal and health consequences associated with TBI are substantial with an estimated annual financial burden of over $75 billion in direct medical costs and other indirect costs (Finkelstein et al., 2006). At the present time, no effective treatment exists due, in part, to the widespread impact of numerous complex secondary biochemical and pathophysiological events occurring at different time points following the initial injury (McIntosh et al., 1998, Rosenfeld et al., 2012). These secondary injury events include, but are not limited to edema, excitotoxicity, inflammation, oxidative stress/damage, activation of necrotic and apoptotic cell death-signaling events, and impaired mitochondrial function (Rink et al., 1995, Clark et al., 2000, Sullivan et al., 2002, Lifshitz et al., 2004, Raghupathi, 2004, Singh et al., 2006, Ziebell and Morganti-Kossmann, 2010). Mitochondria play an essential role in maintaining cellular homeostasis by responding to cellular energy demands and participating directly in a wide range of cellular events including cell signaling, metabolism, and survival, in addition to cell death pathways (Saraste, 1999, McBride et al., 2006). Importantly, it has been well documented that the degree of mitochondrial dysfunction and recovery following TBI is a critical determinant of subsequent cell survival or death (Sullivan et al., 2002, Lifshitz et al., 2003, Lifshitz et al., 2004, Singh et al., 2006, Pandya et al., 2009, Patel et al., 2009). These vital roles for mitochondria in cellular function and survival have resulted in increased efforts to identify the molecular events associated with mitochondrial impairment in TBI.

Mitochondria respond rapidly to a wide range of stressors and cellular requirements. Several studies have documented the localization of microRNA (miRNA) processing machinery and various miRNA in mitochondrial fractions isolated from a variety of cells and tissues (Lung et al., 2006, Kren et al., 2009, Bian et al., 2010, Bandiera et al., 2011, Barrey et al., 2011, Huang et al., 2011, Mercer et al., 2011, Das et al., 2012, Hu et al., 2012, Sripada et al., 2012). MiRNAs are short, non-coding RNA molecules that bind to recognition elements in mRNA and regulate gene expression post-transcriptionally either by mRNA degradation or translational repression (Bartel, 2004, Chen and Rajewsky, 2007, Bartel, 2009, Huntzinger and Izaurralde, 2011). Disruption of miRNA function contributes to many disease states including cancer, cardiovascular disease, and neurodegeneration (Calin and Croce, 2006, Nelson et al., 2008a, Hata, 2013). The biological actions of miRNA are thought to require the cytoplasmic processing by Dicer, interaction with Argonaute (AGO) proteins and assembly into RNA-induced silencing complexes (RISC) (Liu et al., 2008, Bartel, 2009), which promotes and stabilizes miRNA base pair annealing with target mRNA. Recently, mitochondria have been shown to interact with AGO-miRNA containing processing bodies (P-bodies), and that mild uncoupling of mitochondria decreases cellular miRNA functional efficiency (Huang et al., 2011). The ability of a single miRNA or miRNA family to regulate the post-transcriptional expression of hundreds of genes makes them ideal candidates for coordinating complex gene expression programs, including modifying a cell's response to stressors or mounting a protective or pathological response following TBI. Recent experimental studies have reported altered expression levels of several miRNAs in both cortex and hippocampus following controlled cortical impact (CCI), fluid percussion injury, and contusive spinal cord injury (Lei et al., 2009, Liu et al., 2009, Redell et al., 2009, Redell et al., 2010, Strickland et al., 2011, Truettner et al., 2011, Hu et al., 2012, Yunta et al., 2012, Hu et al., 2013, Truettner et al., 2013, Di et al., 2014, Liu et al., 2014, Sabirzhanov et al., 2014, Sun et al., 2014). However, the signaling events regulating cellular miRNA activity and/or function in response to TBI are largely unknown.

In the present study, we demonstrate the presence of miRNA protein machinery in mitochondrial fractions of rat hippocampal tissue. In addition, miRNAs were found in AGO-containing complexes co-immunoprecipitated from purified mitochondria supporting a role for mitochondria in regulating miRNA activity. Importantly, a subset of miRNAs was preferentially enriched in hippocampal mitochondria preparations under normal conditions, and several miRNAs were found to decrease following a severe TBI. These studies point towards a novel role for mitochondrial regulation of miRNA expression in response to TBI.