Up-regulation of key microRNAs, and inverse down-regulation of their predicted oxidative phosphorylation target genes, during aging in mouse brain

https://doi.org/10.1016/j.neurobiolaging.2009.04.020Get rights and content

Abstract

Although significant advances have been made in the study of the molecular mechanisms controlling brain aging, post-transcriptional gene regulation in normal brain aging has yet to be explored. Our lab recently reported that predominant microRNA up-regulation is observed in liver during aging, with key microRNAs predicted to target detoxification genes. Here we examine the role of microRNA regulation in brain during the normal aging process. MicroRNA microarrays and global proteomic profiling were used to compare the brain tissues of 10-, 18-, 24-, and 33-month-old mice. Our results suggest that: (1) like liver, during aging the brain exhibits predominant microRNA up-regulation, and this trend starts in mid-life; (2) of the 70 up-regulated microRNAs, 27 are predicted to target 10 genes of mitochondrial complexes III, IV, and F0F1-ATPase, which exhibit inversely correlated expression; (3) mice of extreme longevity (33-month old) exhibit fewer microRNA expression changes from 10-month-old levels than do old adult mice (24-month old). We found unique de-regulated microRNAs shared between aging brain and aging liver, as well as brain- vs. liver-specific microRNAs during normal aging.

Introduction

Mitochondrial dysfunction and oxidative damage are two pivotal causes among many factors contributing to age-related brain deterioration (Boveris and Navarro, 2008, Reeve et al., 2008, Slemmer et al., 2008). In the study of the correlation between mitochondrial complexes and aging brain, aging rat brains exhibit mitochondrial complex I dysfunction (Petrosillo et al., 2008), and mitochondrial complexes I and IV display a marked decrease in expression in the hippocampus (Navarro et al., 2008). Reduced F0F1-ATP synthase subunits are also reported in aging rat brains (Dencher et al., 2007). Despite the significance of these recent findings, and their impact on the overall understanding of normal aging processes, there is a significant lack of knowledge about the post-transcriptional regulation of molecular events controlling the decreased oxidative phosphorylation in the aging brain.

Glycolysis and the citric acid cycle (CAC) are the two main pathways of ATP production in brain cells (Hertz and Kala, 2007). Unlike substrate level phosphorylation, the higher ATP-yielding CAC is coupled to oxidative phosphorylation via the electron transport chain (ETC), and thus requires oxygen for efficient energy production. The CAC harnesses energy by gathering protons and high-energy electrons from substrates interacting with dehydrogenases. The mitochondrial ETC consists of a series of enzymatic complexes and a quinone pool. In order of decreasing electropotential, these complexes are NADH dehydrogenase (complex I); succinate dehydrogenase (complex II); cytochrome bc1 complex (complex III); cytochrome c oxidase (COX) (complex IV). The ETC functions to pump protons across the inner membrane (IM) to establish proton motive force, which in turn affords the electrochemical gradient needed for ATPase-catalyzed phosphorylation to occur. A deficiency in any of these key components may result in a decline of efficient ATP production. While oxidative phosphorylation is known to decline and suffer loss of efficiency in aging tissues (Sen et al., 2007), the regulatory mechanisms and causative factors related to this decline have not been identified.

MicroRNAs (miRNAs) are short (19–22 nucleotides), non-coding RNAs that play critical roles in post-transcriptional regulation of cellular gene expression (Ambros, 2004). Regulation via miRNA is involved in physiological processes ranging from development to apoptosis and aging (Wang, 2007). Mature miRNAs associate with the RNA-induced silencing complex and either: (1) inhibit protein translation, via incomplete base pairing to the 3′ untranslated region (3′-UTR) of target gene messenger RNA (mRNA); or (2) signal for degradation of target mRNA, via complete homologous binding to the mRNA coding region. In either case, post-transcriptional regulation is achieved by silencing the mRNA transcripts, preventing them from producing their final protein products. The versatility of these two mechanisms enables the targeting both of single genes by multiple miRNAs, achieving an additive or fine-tuning effect, and multiple genes by single miRNAs, contributing to the dynamic versatility observed in intracellular signal transduction.

Recently our lab reported that miRNAs are involved in the aging process in liver. MicroRNAs exhibiting variation with age – including microRNA- (miR-) 669c, 709, 214, and 93 – are predominantly up-regulated, and most of them target the mRNAs of genes related to detoxification and regeneration, presumably contributing to their observed functional decline (Maes et al., 2008). A recent report by Maes et al. provides a comprehensive review of many changes in lead miRNA expression related to several neurodegenerative diseases, including Alzheimer's disease (Maes et al., 2009). While this report describes a plethora of miRNA changes in neuronal diseases, normal age-dependent changes in miRNA expression patterns remain to be explored. The present study sets out to identify baseline changes in miRNA expression during normal brain aging, using a systematic investigation from mid to late and even very late stages of normal mouse life span, focusing initially on the well-studied mouse strain, C57BL/6J.

This study combines miRNA array and mass spectrometry proteomic analysis to detect variations in expression levels of miRNAs, and corresponding affects on their predicted target proteins, in 10-, 18-, 24- and 33-month-old mouse brains. As in liver, the majority of miRNAs deregulated during brain aging are up-regulated. Most of the up-regulated miRNAs target oxidative phosphorylation; key genes of this family are found to be down-regulated by our Tandem Mass proteomic profiling, suggesting the expected inverse expression pattern between miRNA up-regulation and their candidate targets’ down-regulation. Surprisingly, compared with 10-month old, the extremely old mouse brain (33 months of age) demonstrates less distinct variation than old mice (24 months of age) in both mitochondrial complex gene and miRNA expression levels, and exhibits expression levels resembling that of 18-month-old mice. The miRNAs that demonstrate differential expression between 24- and 33-month-old mice may offer insight as to some of the contributing factors that lead to extended longevity in a very small percentage of the population. Moreover, combined with our previous study, this work demonstrates that in aging brain and liver, mmu-miR-30d, -34a, -468, -669b and -709 are similarly up-regulated. In contrast, aging brain-specific up-regulated miRNAs include mmu-miR-22, -101a, -720, and -721, with predicted target genes involved in oxidative phosphorylation, while aging liver-specific up-regulation is seen with miR-669c, -712, -214, and -93, with predicted target genes involved in detoxification- and regeneration-related gene families.

Section snippets

Mouse strain and samples

Normal wild-type male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) at 2 months of age. Subsequently, mice were housed, fed, and maintained in a barrier facility according to National Institutes of Health animal care guidelines, to reach the designated age groups. Euthanasia was carried out by cervical dislocation, in order to avoid confounding factors associated with the isoflurane sacrificing procedure, which may induce gene expression changes. Brain regions

Proteomic results

Proteomic analysis revealed numerous proteins exhibiting variation between 10-, 18-, 24- and 33-month-old mouse brains. Among them, 190 proteins were identified at 95% confidence level; 13 subunits of the mitochondrial ETC complexes and F0F1-ATPase were identified, of which 10 were down-regulated (mitochondrial complexes III: subunits 2, 7 and Rieske; IV: 5A, 5B and 7A; F0F1-ATPase: beta, b, d and O) (Table 1). The sharpest decreases in protein expression levels were exhibited between 10- and

Discussion

In order to investigate post-transcriptional regulation of gene expression by miRNA changes during normal brain aging, we examined four different age groups: 10-, 18-, 24- and 33-month-old mice, representing adult, middle-aged, old, and extremely old age groups, respectively. With the consideration that the brain aging process underlies a comprehensive functional decline, we employed whole brain analyses in this study. Ten proteins related to mitochondrial ETC complexes III, IV and F0F1-ATP

Conflict of interest

No conflicts of interest are reported for this study.

Acknowledgements

This work is supported by grant NNX08AP23G from the U.S. National Aeronautics and Space Administration (NASA) to EW. We would like to thank Mrs. Vickie Chen and Mr. Alan N. Bloch for proofreading the manuscript, as well as Patrick R. Lyninger for computational and technical services.

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