Recombinant Mitochondrial Transcription Factor A with N-terminal Mitochondrial Transduction Domain Increases Respiration and Mitochondrial Gene Expression in G11778A Leber's Hereditary Optic Neuropathy Cybrid Cells

Diseases involving mitochondrial defects usually manifest themselves in high-energy, post-mitotic tissues such as brain, retina, skeletal and cardiac muscle and frequently cause deficiencies in mitochondrial bioenergetics. We have developed a scalable procedure to produce recombinant human mitochondrial transcription factor A (TFAM) modified with an N-terminal protein transduction domain (PTD) and mitochondrial localization signal (MLS) that allow it to cross membranes and enter mitochondria through its "mitochondrial transduction domain" (MTD,=PTD+MLS). _In vitro_ studies in a classic mitochondrial disease cell model demonstrated that Alexa488-labeled MTD-TFAM rapidly entered the mitochondrial compartment. MTD-TFAM treatment of these cell lines reversibly increased oxygen consumption (respiration) rates 3-fold, levels of respiratory proteins and mitochondrial gene expression. _In vivo_ results demonstrated that respiration increased to lesser degrees in mitochondria from tissues of mice injected with MTD-TFAM. MTD-TFAM can alter mitochondrial bioenergetics and holds promise for treatment of mitochondrial diseases involving deficiencies of energy production

Epigenetic Modifications of the PGC-1α Promoter during Exercise Induced Expression in Mice

The transcriptional coactivator, PGC-1α, is known for its role in mitochondrial biogenesis. Although originally thought to exist as a single protein isoform, recent studies have identified additional promoters which produce multiple mRNA transcripts. One of these promoters (promoter B), approximately 13.7kb upstream of the canonical PGC-1α promoter (promoter A), yields alternative transcripts present at levels much lower than the canonical PGC-1α mRNA transcript. In skeletal muscle, exercise resulted in a substantial, rapid increase of mRNA of these alternative PGC-1α transcripts. Although the β2-adrenergic receptor was identified as a signaling pathway that activates transcription from PGC-1α promoter B, it is not yet known what molecular changes occur to facilitate PGC-1α promoter B activation following exercise. We sought to determine whether epigenetic modifications were involved in this exercise response in mouse skeletal muscle. We found that DNA hydroxymethylation correlated to increased basal mRNA levels from PGC-1α promoter A, but that DNA methylation appeared to play no role in the exercise-induced activation of PGC-1α promoter B. The level of the activating histone mark H3K4me3 increased with exercise 2–4 fold across PGC- 1α promoter B, but remained unaltered past the canonical PGC-1α transcriptional start site. Together, these data show that epigenetic modifications partially explain exercise-induced changes in the skeletal muscle mRNA levels of PGC-1α isoforms

Mitochondrial DNA Copy Numbers in Pyramidal Neurons are Decreased and Mitochondrial Biogenesis Transcriptome Signaling is Disrupted in Alzheimer’s Disease Hippocampi

Alzheimer\u27s disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD

Anticardiolipin (aCL) in sera from periodontitis subjects activate Toll-like receptor 4 (TLR4).

Anticardiolipin antibodies (aCL) have been reported to be present in 15-20% of sera from subjects with periodontitis at concentrations exceeding those found in 95% of the healthy adult population. These antibodies, albeit at concentrations exceeding those generally found in periodontitis subjects, are typically present in patients with the antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. aCL from APS patients are proinflammatory and can activate trophoblasts, macrophages, and platelets via cell-surface interactions with their target antigen beta-2-glycoprotein-I (β2GPI). β2GPI is an anionic phospholipid-binding serum protein that can associate with toll-like receptors (TLR's) on the cell-surface, leading to cell activation following interaction with autoimmune aCL. We examined an expanded series of 629 sera from clinically characterized subjects for aCL content, and observed that 14-19% of these sera contained elevated (>95th %-tile) levels of aCL. We purified IgG from 16 subjects with elevated or normal levels of aCL and examined their ability to activate TLR2- or TLR4-transfected human embryonic kidney (HEK) cells, and observed that IgG from periodontitis patients with elevated aCL activated HEK-TLR4 cells, but not HEK-TLR2 cells. Prior removal of aCL by immunoabsorption significantly reduced the ability of IgG preparations from these sera to activate TLR4. Further experiments using a human first trimester trophoblastic cell line (HTR8 sv/neo) revealed that aCL from periodontitis patients stimulated IL-8 production, which was profoundly decreased if aCL was removed by immunoabsorption or if HTR8 sv/neo were pretreated with blocking anti-TLR4 antibodies. Thus, it appears that aCL from periodontitis patients can be proinflammatory, activating cells via TLR4. Since these antibodies are likely produced via molecular mimicry due to similarities between oral bacterial antigens and β2GPI, the data indicate that circulating serum aCL may induce or influence inflammatory responses at sites distant from the oral cavity

PGC-1α mRNA levels in skeletal muscle, by RT-qPCR, after rotarod exercise.

<p>(A) Schematic diagram of PGC-1α isoforms examined in this study. PGC-1α-a is expressed from one promoter, whereas PGC-1α-b and PGC-1α-c are both expressed from the same promoter. (B) Paired mice (n = 6 each group) were either exposed to rotarod exercise as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129647#pone.0129647.g001" target="_blank">Fig 1</a>, or kept sedentary. RT-qPCR is shown for PGC1α using isoform-specific primers against PGC-1α-a, PGC-1α-b, or PGC-1α-c. Known quantities from separate cloned plasmids containing each isoform were used to quantitate absolute copy numbers of PGC-1α isoforms or total PGC-1α, which were normalized to geometric means of GAPDH and beta actin mRNA levels. Bars are shown ±SEM. (** p<0.005 by paired t-test).</p

Chromatin Immunoprecipitation of histone modifications across Exon 1a and Exon 1b/c transcriptional start sites of PGC-1α.

<p>(A) Presence of an activating histone modification (H3K4me3) was examined via qPCR 1000bp in each direction from the transcriptional start site of Exon 1a (left) or Exon 1b/c (right). (B) Presence of a repressing histone modification (H3K27me3) was examined via qPCR 1000bp in each direction from the transcriptional start site of Exon 1a (left) or Exon 1b/c (right). 2-way ANOVA analysis found significant difference between the level of H3K4me3 only across the Exon 1b/c transcriptional start site (p<0.0001). Individual points were compared for statistical significance using student’s t-test (* p<0.05, ** p<0.005).</p

Measurement of 5mC and 5hmC across PGC-1α Exon1a and Exon1b/c promoter regions.

<p>(A) Percent protected cytosines at each CpG site within the promoter region of PGC-1α Exon1a (right) or Exon1b/c (left) after bisulfite treatment of skeletal muscle DNA. (B) Percent protected cytosines after TAB-Seq, showing the level of 5hmC at each CpG site. (C) The level of 5mC is shown at each CpG site across the PGC-1α promoters.</p

Mouse exercise session on a rotarod.

<p>After initial training, individual mice were run on an increasing rotarod speed of 35, 40, and 45 rpm. Graph shows time spent at each speed and cumulative time, in minutes, for each mouse.</p