Elsevier

Methods

Volume 46, Issue 4, December 2008, Pages 288-294
Methods

Review Article
Assessing mitochondria biogenesis

https://doi.org/10.1016/j.ymeth.2008.09.026Get rights and content

Abstract

Mitochondria have their own DNA (mtDNA) and hence biogenesis of mitochondria requires a coordination of nuclear and mtDNA, both of which encode for mitochondria proteins. Our understanding of the molecular control of mitochondria biogenesis has increased in recent years, providing key signatures of the process. To determine whether or not a tissue or an organ of human or animal origin is undergoing mitochondria biogenesis, multiple parameters should be analyzed. First and foremost is visualization and measurement of mitochondria mass/volume in histological sections using fluorescent mitochondria dyes and light microscopy or transmission electron microscopy to yield quantitative results. To confirm or extend these types of analysis, biochemical markers of mitochondria biogenesis should also be included, including assessment of mtDNA copy number, steady-state levels of biogenesis-related transcription factors (e.g. mitochondria transcription factor A, mitochondrial transcription specificity factors, nuclear respiratory factors 1 and 2, and peroxisome proliferator activated receptor gamma coactivator-1-α), mtDNA-encoded transcripts and proteins, and rates of mitochondria translation. These techniques are described in isolation and in the context of transgenic and dietary animal models that have been used as tools to study the regulation of mitochondria biogenesis and its role in disease pathology.

Introduction

There are genetic, metabolic, and dietary events that result in mitochondria biogenesis and thereby may impact health and disease. Mitochondria diseases may be due to base pair substitutions in the mitochondria genome and/or may involve defects in the nuclear encoded mitochondria proteins. Also, the mechanisms or proteins responsible for ferrying some mitochondria proteins (chaperone proteins) synthesized in the cytoplasm to the mitochondria could be defective and the import of such proteins into the mitochondria could be impaired. All of these factors collectively can lead to mitochondria dysfunction, pathology, and to mitochondria biogenesis.

A number of groups have studied several diseases in humans that affect skeletal and cardiac muscle and peripheral and central nervous system tissue, particularly the brain, the liver, bone marrow, the endocrine and exocrine pancreas, the kidneys and the intestines. [1], [2], [3], [4], [5]. Fibroblasts isolated from a child afflicted with Leigh’s syndrome revealed a disorder involving a nuclear mutation in cytochrome c oxidase, but all subunits were present to lesser degrees [6]. Mita et al. [7] reported that a quadriceps muscle biopsy of a young patient afflicted with Kearns-Sayre syndrome revealed mitochondria deletion of all of subunit III, parts of NADH-coenzyme Q reductase (subunits III and IV), all of ATP synthase subunit VI and part of ATP synthase subunit VIII. The DNA responsible for encoding cytochrome c oxidase subunit IV was present but not the DNA of mitochondria encoded cytochrome c oxidase subunit II. Another disorder, myoclonus epilepsy with ragged red fibers (MERRF), affects both brain and muscle tissue. Western blot analysis revealed a decrease in cytochrome c oxidase subunit II relative to the other subunits, but Northern analysis failed to show any change in subunits I–III [1]. Schwartzkopff et al. [8] reported a case of a 30 year old female exhibiting tachycardia in which there was no overt signs of cardiac failure. Subsequent biopsy of the right septal endocardium revealed enlarged and vacuolated mitochondria and increased mitochondria: myofibril values, the appearance of glycogen granules and lipid droplets and a marked decrease in cytochrome c oxidase activity. Mullen-Hocker et al. [9] and Zeviani et al. [10] reported similar observations, particularly lower cardiac cytochrome c oxidase activity in patients suffering from cardiomyopathy. Horvath et al. [11] discovered that the copper chaperone protein, SCO2, is mutated in several forms of fatal infantile cardiomyopathy leading to cytochrome c oxidase deficiency. Salviati et al. [12] demonstrated that copper supplementation of cultured cells (myoblasts, myotubes and fibroblasts) from patients with SCO2 mutations and decreased cytochrome c oxidase activity, could restore the cytochrome c oxidase activity to control levels. Furthermore, a patient with SCO2 mutations and severe hypertrophic cardiomyopathy was reversed with copper-histidine supplementation [13]. Mutations in SCO1 or SCO2 results in cell copper deficiency [14].

These disease conditions have lead to a focus on the gene program that drives mitochondria biogenesis. Mitochondria transcription factor A (mtTFA) is a major transcription factor governing mitochondria DNA replication and transcription during mitochondria biogenesis [15]. Low levels of mtTFA transcript and protein are associated with overall decreased mitochondria gene transcription in HeLa cells. On-the-other-hand, expression of human mtTFA in Saccharomyces cerevisiae devoid of mtTFA, restores mitochondria DNA transcription and function [16]. Functional human mtTFA is a 25 kD protein, [17], [18] whereby its transcriptional activation initiates the synthesis of mitochondria RNAs by mitochondria RNA polymerase [17]. On-the-other-hand mitochondrial specificity factors, TFB1M and TFB2M, may have even more significant roles than mtTFA [19], [20].

The investigation of nuclear control of mitochondria gene expression has lead to the discovery of several other important transcription factors. Nuclear Respiratory Factor-1 (NRF-1) coordinates nuclear encoded respiratory chain expression with mitochondria gene transcription and replication [21]. NRF-1 recognition sites have been found in many genes encoding respiratory functional subunits, such as rat cytochrome c oxidase subunit VIc and the bovine ATP synthase γ-subunit. Therefore, NRF-1 activates mitochondria gene expression by upregulating mtTFA [22].

Another nuclear gene product, NRF-2, has also been implicated in the coordination between nuclear and mitochondria gene expression. Although the majority of genes encoding proteins in respiratory functions have a NRF-1 recognition site, some genes such as cytochrome c oxidase subunit IV and ATP synthase β-subunit, lack a NRF- mitochondria recognition site but contain a NRF-2 recognition site indicating that these respiratory chain genes may be differentially regulated [23]. In some genes, both NRF-1 and NRF-2 recognition sites have been identified [15], [24]. It is apparent that NRF-1 and NRF-2 may convey nuclear regulatory events to the mitochondria via mtTFA, and coordinate the gene expression between the nuclear and mitochondria genomes.

Peroxisomal proliferating activating receptor-γ coactivator (PGC-1), is thought to be a master regulator of mitochondria biogenesis and its interaction with mtTFA, NRF-1 and NRF-2 is the subject of investigation. This transcription factor has the ability to induce the production of mitochondria in brown adipose tissue [25]. There are various isoforms of PGC-1 which constitutes a family: PGC-1α, PGC-1β, and PGC-1-related coactivators. Both PGC-1α and PGC-1β have high expression in tissues rich in mitochondria. Unlike some other transcription factors, PGC-1α does not have any response elements, meaning it does not bind to a DNA promoter directly. Rather it acts via a protein–protein interaction but it does not have enzymatic activity [26]. Transfection of PGC-1α into C2C12 cells by Wu et al. [27] and into myocytes by Lehman et al. [28] all resulted in indices of mitochondria biogenesis in terms of mitochondria protein, transcripts and mitochondria volume densities of the cells. PGC-1α may act as a coactivator of NRF-1 [27], which then is thought to bind to the promoter of mtTFA to initiate the concomitant upregulation of both mitochondria and nuclear encoded proteins in a coordinated fashion. Another set of transcription factors needed to initiate mitochondria biogenesis are termed transcription specificity factors (TFB1M and TFB2M). There are recognition sites within the promoters for NRF-1 and NRF-2 for these two transcription factors. It is also reported that PGC-1α will upregulate these two transcription factors. Upregulation of mtTFA augments mitochondria biogenesis with these other transcription factors [20].

Section snippets

Description of methods to study mitochondria biogenesis

Because of the complex nature of mitochondria, multiple parameters need to be analyzed in order to ascertain whether a mitochondria biogenesis program is occurring in cells, tissues, and/or organs (Table 1). Below, I will describe methods for assessment of mitochondria volume/number by microscopy, mtDNA copy number by PCR, typical molecular markers of biogenesis by western blot, and mitochondria translation rates by in vivo labeling.

Inducing mitochondria biogenesis: over-expression models to nutritional models

While there are many models that have been and are being produced whereby there is an over-expression of PGC-1α to study mitochondria biogenesis, we will focus on those produced by the Kelly lab at Washington University School of Medicine. Trial studies using an over-expression model of PGC-1α resulted in 100% mortality in offspring due to over-production of mitochondria. Knowing the lethality of simple over-expression, an inducible system allows the animals to mature and then induce

Concluding remarks

Determination of whether a model system demonstrates the presence of mitochondria biogenesis cannot be relied upon by only one method. Multiple assessments should be done to muster peer review. Clearly histological methods are needed with transmission electron microscopy being the gold standard. However, in addition, some biochemical markers should be presented. In particular the preferred measures are mtDNA and mtTFA using Northerns or Westerns of mitochondria transcripts or proteins,

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