Efficient mitochondrial biogenesis drives incomplete penetrance in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies.

Drosophila nuclear factor DREF regulates the expression of the mitochondrial DNA helicase and mitochondrial transcription factor B2 but not the mitochondrial translation factor B1.

DREF [DRE (DNA replication-related element)-binding factor] controls the transcription of numerous genes in Drosophila, many involved in nuclear DNA (nDNA) replication and cell proliferation, three in mitochondrial DNA (mtDNA) replication and two in mtDNA transcription termination. In this work, we have analysed the involvement of DREF in the expression of the known remaining genes engaged in the minimal mtDNA replication (d-mtDNA helicase) and transcription (the activator d-mtTFB2) machineries and of a gene involved in mitochondrial mRNA translation (d-mtTFB1). We have identified their transcriptional initiation sites and DRE sequences in their promoter regions. Gel-shift and chromatin immunoprecipitation assays demonstrate that DREF interacts in vitro and in vivo with the d-mtDNA helicase and d-mtTFB2, but not with the d-mtTFB1 promoters. Transient transfection assays in Drosophila S2 cells with mutated DRE motifs and truncated promoter regions show that DREF controls the transcription of d-mtDNA helicase and d-mtTFB2, but not that of d-mtTFB1. RNA interference of DREF in S2 cells reinforces these results showing a decrease in the mRNA levels of d-mtDNA helicase and d-mtTFB2 and no changes in those of the d-mtTFB1. These results link the genetic regulation of nuclear DNA replication with the genetic control of mtDNA replication and transcriptional activation in Drosophila.

A modified method for the purification of active large enzymes using the glutathione S-transferase expression system.

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.

Nuclear respiratory factor 2 induces the expression of many but not all human proteins acting in mitochondrial DNA transcription and replication.

In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.

Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype.

The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

Accumulation of overoxidized Peroxiredoxin III in aged rat liver mitochondria.

Overoxidation and subsequent inactivation of Peroxiredoxin III (PrxIII), a mitochondrial H(2)O(2) scavenging enzyme, have been reported in oxidative stress conditions. No data are available in the literature about the presence of overoxidized forms of PrxIII in aged tissues. Liver mitochondria from 12-month-old rats and 28-month-old rats were here analyzed by two-dimensional gel electrophoresis. A spot corresponding to the native form of PrxIII was present in adult and old rats with the same volume, whereas an additional, more acidic spot, of the same molecular weight of the native form, accumulated only in old rats. The acidic spot was identified, by MALDI-MS analysis, as a form of PrxIII bearing the cysteine of the catalytic site overoxidized to sulphonic acid. This modified PrxIII form corresponds to the irreversibly inactivated enzyme, here reported, for the first time, in aging. Three groups of 28-month-old rats treated with acetyl-l-carnitine were also examined. Reduced accumulation of the overoxidized PrxIII form was found in all ALCAR-treated groups.

The MTERF family proteins: mitochondrial transcription regulators and beyond.

The MTERF family is a wide protein family, identified in Metazoa and plants, which consists of 4 subfamilies named MTERF1-4. Proteins belonging to this family are localized in mitochondria and show a modular architecture based on repetitions of a 30 amino acid module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes the characterized transcription termination factors human mTERF, sea urchin mtDBP and Drosophila DmTTF. In vitro and in vivo studies show that these factors play different roles which are not restricted to transcription termination, but concern also transcription initiation and the control of mtDNA replication. The multiplicity of functions could be related to the differences in the gene organization of the mitochondrial genomes. Studies on the function of human and Drosophila MTERF3 factor showed that the protein acts as negative regulator of mitochondrial transcription, possibly in cooperation with other still unknown factors. The complete elucidation of the role of the MTERF family members will contribute to the unraveling of the molecular mechanisms of mtDNA transcription and replication.

Variations at the H-strand replication origins of mitochondrial DNA and mitochondrial DNA content in the blood of type 2 diabetes patients.

Mitochondrial DNA (mtDNA) sequence variation in the segment of the D-loop region encompassing the initiation sites for replication and transcription was analyzed in the blood of 277 Italian type 2 diabetes patients and 277 Italian healthy subjects. Compared with the Cambridge Reference Sequence, diabetic patients show a slightly higher propensity to accumulate base changes in this region, with respect to controls, although no significant association can be established between any of the detected changes and the diabetic condition. Subjects, patients and controls, harbouring base changes at the replication origins (positions 57 and 151) and at position 58 were analyzed for mtDNA content. The mtDNA content increased three-four times only in the diabetic patients bearing the m.151C>T transition, whereas in those bearing the m.58T>C change the mtDNA content doubled, independently of the affiliation haplogroup. This result suggests that the m.151C>T transition and, to a lower extent, the m.58T>C might confer to the blood cells of diabetic patients the capability of increasing their mtDNA content, whereas the same transitions have no effect on control subjects.

The Drosophila nuclear factor DREF positively regulates the expression of the mitochondrial transcription termination factor DmTTF.

The DREF [DRE (DNA replication-related element)-binding factor], which regulates the transcription of a group of cell proliferation-related genes in Drosophila, also controls the expression of three genes involved in mtDNA (mitochondrial DNA) replication and maintenance. In the present study, by in silico analysis, we have identified DREs in the promoter region of a gene participating in mtDNA transcription, the DmTTF (Drosophila mitochondrial transcription termination factor). Transient transfection assays in Drosophila S2 cells, with mutated versions of DmTTF promoter region, showed that DREs control DmTTF transcription; moreover, gel-shift and ChIP (chromatin immunoprecipitation) assays demonstrated that the analysed DRE sites interact with DREF in vitro and in vivo. Accordingly, DREF knock-down in S2 cells by RNAi (RNA interference) induced a considerable decrease in DmTTF mRNA level. These results clearly demonstrate that DREF positively controls DmTTF expression. On the other hand, mtRNApol (mitochondrial RNA polymerase) lacks DREs in its promoter and is not regulated in vivo by DREF. In situ RNA hybridization studies showed that DmTTF was transcribed almost ubiquitously throughout all stages of Drosophila embryogenesis, whereas mtRNApol was efficiently transcribed from stages 11-12. Territories where transcription occurred mostly were the gut and Malpighi tubes for DmTTF, and the gut, mesoderm, pharyngeal muscle and Malpighi tubes for mtRNApol. The partial overlapping in the temporal and spatial mRNA expression patterns confirms that transcription of the two genes is differentially regulated during embryogenesis and suggests that DmTTF might play multiple roles in the mtDNA transcription process, for which different levels of the protein with respect to mtRNApol are required.

The PGC-1alpha-dependent pathway of mitochondrial biogenesis is upregulated in type I endometrial cancer.

PGC-1alpha-dependent pathway of mitochondrial biogenesis was investigated for the first time in type I endometrial cancer and in normal endometrium. In cancer endometrial tissue the citrate synthase activity, the mitochondrial DNA content and the TFAM level were found doubled compared to control endometrial tissue. Moreover, a 1.6- and 1.8-fold increase, respectively, of NRF-1 and PGG-1alpha expression was found. This study demonstrates, for the first time, that the increased mitochondrial biogenesis in type I endometrial cancer is associated to the upregulation of PGC-1alpha signalling pathway.

Methods for studying mitochondrial transcription termination with isolated components.

Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3'-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP-DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.

Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system.

Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms.

The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription.

DmTTF is a Drosophila mitochondrial DNA-binding protein, which recognizes two sequences placed at the boundary of clusters of genes transcribed in opposite directions. To obtain in vivo evidences on the role of DmTTF, we characterized a DmTTF knock-down phenotype obtained by means of RNA interference in D.Mel-2 cells. By a combination of RNase protection and real-time RT-PCR experiments we found that knock-down determines remarkable changes in mitochondrial transcription. In particular, protein depletion increases not only the level of (+) and (-)strand RNAs mapping immediately after of the two protein-binding site, but also that of transcripts located further downstream. Unexpectedly, depletion of the protein also causes the decrease in the content of those transcripts mapping upstream of the protein target sites, including the two rRNAs. The changes in transcript level do not depend on a variation in mitochondrial DNA (mtDNA) content, since mtDNA copy number is unaffected by DmTTF depletion. This work shows conclusively that DmTTF arrests in vivo the progression of the mitochondrial RNA polymerase; this is the first ever-obtained evidence for an in vivo role of an animal mitochondrial transcription termination factor. In addition, the reported data provide interesting insights into the involvement of DmTTF in transcription initiation in Drosophila mitochondria.

MTERF3, the most conserved member of the mTERF-family, is a modular factor involved in mitochondrial protein synthesis.

The MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved "mTERF-motifs", three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein-protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus.

Contrahelicase activity of the mitochondrial transcription termination factor mtDBP.

The sea urchin mitochondrial D-loop binding protein (mtDBP) is a transcription termination factor that is able to arrest bidirectionally mitochondrial RNA chain elongation. The observation that the mtDBP binding site in the main non-coding region is located in correspondence of the 3' end of the triplex structure, where the synthesis of heavy strand mitochondrial (mt) DNA is either prematurely terminated or allowed to continue, raised the question whether mtDBP could also regulate mtDNA replication. By using a helicase assay in the presence of the replicative helicase of SV40, we show that mtDBP is able to inhibit the enzyme thus acting as a contrahelicase. The impairing activity of mtDBP is bidirectional as it is independent of the orientation of the protein binding site. The inhibition is increased by the presence of the guanosine-rich sequence that flanks mtDBP binding site. Finally, a mechanism of abrogation of mtDBP contrahelicase activity is suggested that is based on the dissociation of mtDBP from DNA caused by the passage of the RNA polymerase through the protein-DNA complex. All these findings favour the view that mtDBP, besides serving as transcription termination factor, could also act as a negative regulator of mtDNA synthesis at the level of D-loop expansion.

Dietary supplementation with acetyl-l-carnitine counteracts age-related alterations of mitochondrial biogenesis, dynamics and antioxidant defenses in brain of old rats.

We previously reported the ability of dietary supplementation with acetyl-l-carnitine (ALCAR) to prevent age-related decreases of mitochondrial biogenesis in skeletal muscle and liver of old rats. Here, we investigate the effects of ALCAR supplementation in cerebral hemispheres and cerebellum of old rats by analyzing several parameters linked to mitochondrial biogenesis, mitochondrial dynamics and antioxidant defenses. We measured the level of the coactivators PGC-1α and PGC-1ß and of the factors regulating mitochondrial biogenesis, finding an age-related decrease of PGC-1ß, whereas PGC-1α level was unvaried. Twenty eight-month old rats supplemented with ALCAR for one and two months showed increased levels of both factors. Accordingly, the expression of the two transcription factors NRF-1 and TFAM followed the same trend of PGC-1ß. The level of mtDNA, ND1 and the activity of citrate synthase, were decreased with aging and increased following ALCAR treatment. Furthermore, ALCAR counteracted the age-related increase of deleted mtDNA. We also analyzed the content of proteins involved in mitochondrial dynamics (Drp1, Fis1, OPA1 and MNF2) and found an age-dependent increase of MFN2 and of the long form of OPA1. ALCAR treatment restored the content of the two proteins to the level of the young rats. No changes with aging and ALCAR were observed for Drp1 and Fis1. ALCAR reduced total cellular levels of oxidized PRXs and counteracted the age-related decrease of PRX3 and SOD2. Overall, our findings indicate a systemic positive effect of ALCAR dietary treatment and a tissue specific regulation of mitochondrial homeostasis in brain of old rats. Moreover, it appears that ALCAR acts as a nutrient since in most cases its effects were almost completely abolished one month after treatment suspension. Dietary supplementation of old rats with this compound seems a valuable approach to prevent age-related mitochondrial dysfunction and might ultimately represent a strategy to delay age-associated negative consequences in mitochondrial homeostasis.

Growth hormone secretagogues hexarelin and JMV2894 protect skeletal muscle from mitochondrial damages in a rat model of cisplatin-induced cachexia.

Chemotherapy can cause cachexia, which consists of weight loss associated with muscle atrophy. The exact mechanisms underlying this skeletal muscle toxicity are largely unknown and co-therapies to attenuate chemotherapy-induced side effects are lacking. By using a rat model of cisplatin-induced cachexia, we here characterized the mitochondrial homeostasis in tibialis anterior cachectic muscle and evaluated the potential beneficial effects of the growth hormone secretagogues (GHS) hexarelin and JMV2894 in this setting. We found that cisplatin treatment caused a decrease in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM, mtDNA, ND1), mitochondrial mass (Porin and Citrate synthase activity) and fusion index (MFN2, Drp1), together with changes in the expression of autophagy-related genes (AKT/FoxO pathway, Atg1, Beclin1, LC3AII, p62) and enhanced ROS production (PRX III, MnSOD). Importantly, JMV2894 and hexarelin are capable to antagonize this chemotherapy-induced mitochondrial dysfunction. Thus, our findings reveal a key-role played by mitochondria in the mechanism responsible for GHS beneficial effects in skeletal muscle, strongly indicating that targeting mitochondrial dysfunction might be a promising area of research in developing therapeutic strategies to prevent or limit muscle wasting in cachexia.

DmTTF, a novel mitochondrial transcription termination factor that recognises two sequences of Drosophila melanogaster mitochondrial DNA.

Using a combination of bioinformatic and molecular biology approaches a Drosophila melanogaster protein, DmTTF, has been identified, which exhibits sequence and structural similarity with two mitochondrial transcription termination factors, mTERF (human) and mtDBP (sea urchin). Import/processing assays indicate that DmTTF is synthesised as a precursor of 410 amino acids and is imported into mitochondria, giving rise to a mature product of 366 residues. Band-shift and DNase I protection experiments show that DmTTF binds two homologous, short, non-coding sequences of Drosophila mitochondrial DNA, located at the 3' end of blocks of genes transcribed on opposite strands. The location of the target sequences coincides with that of two of the putative transcription termination sites previously hypothesised. These results indicate that DmTTF is the termination factor of mitochondrial transcription in Drosophila. The existence of two DmTTF binding sites might serve not only to stop transcription but also to control the overlapping of a large number of transcripts generated by the peculiar transcription mechanism operating in this organism.

The sea urchin mitochondrial transcription factor A binds and bends DNA efficiently despite its unusually short C-terminal tail.

Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM.

Mitochondrial DNA mutations in RRF of healthy subjects of different age.

To obtain information on the mechanisms responsible of the generation of ragged red fibers (RRF) during aging, we analyzed the mitochondrial genotype of single skeletal muscle fibers of healthy individuals having an age comprised between 45 and 92 years. The sequencing of the D-loop region showed many sequence changes with respect to the Cambridge reference sequence (CRS), in both RRF and normal fibers. These changes were more abundant in RRF and their number increased between 50 and 60, and 61 and 70 years and then remained approximately constant. The analysis of the sequence changes showed that each subject contained one or more changes associated to RRF in positions of D-loop region that either do not change or that change very rarely. In general the same type of RRF-associated change was not found in more than one individual; exceptions were changes in positions 189, 295, 374 and 514, detected in 20-50% of analyzed subjects. In particular the A189G age-associated mutation was found only in old individuals and prevalently in RRF. Sequencing of other two mtDNA regions showed no relevant changes in the 16S/ND1 region and two RRF-associated original mutations, G5847A and A5884C, in two very conserved positions of tRNATyr. These results indicate that each subject has its own pattern of RRF-associated mutations in both coding and non-coding region of human mtDNA.