A novel <i>HSD17B10</i> mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression

<p>We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene <i>HSD17B10</i> for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in <i>HSD17B10</i> cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5′-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.</p

Distinctive examples of genes showing significant sub-gene changes in RC disease.

<p>Each vertical bar represents a microarray probeset, while narrower bars on gene ends indicate UTRs. Arrowheads indicate the direction of transcription. (<b>A</b>) <b><i>MRPL27</i></b>, a mitochondrial RP gene, had significant changes at both its 5' and 3'-UTRs toward opposite directions in RC disease both cell types. Consequently, it was not identified as a DEG by whole transcript level analysis. (<b>B</b>) <b><i>mTOR</i></b> was consistently and significantly changed across the entire gene in RC disease. (<b>C</b>) <b><i>ENC1</i></b> had modified antisense transcription in RC disease. The opposite direction of expression changes within its 5'-UTR suggested that the ENC1 antisense transcript might be functional. See also <b>Fig. S7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069282#pone.0069282.s003" target="_blank">File S3</a></b>.</p

Primary Respiratory Chain Disease Causes Tissue-Specific Dysregulation of the Global Transcriptome and Nutrient-Sensing Signaling Network

<div><p>Primary mitochondrial respiratory chain (RC) diseases are heterogeneous in etiology and manifestations but collectively impair cellular energy metabolism. Mechanism(s) by which RC dysfunction causes global cellular sequelae are poorly understood. To identify a common cellular response to RC disease, integrated gene, pathway, and systems biology analyses were performed in human primary RC disease skeletal muscle and fibroblast transcriptomes. Significant changes were evident in muscle across diverse RC complex and genetic etiologies that were consistent with prior reports in other primary RC disease models and involved dysregulation of genes involved in RNA processing, protein translation, transport, and degradation, and muscle structure. Global transcriptional and post-transcriptional dysregulation was also found to occur in a highly tissue-specific fashion. In particular, RC disease muscle had decreased transcription of cytosolic ribosomal proteins suggestive of reduced anabolic processes, increased transcription of mitochondrial ribosomal proteins, shorter 5′-UTRs that likely improve translational efficiency, and stabilization of 3′-UTRs containing AU-rich elements. RC disease fibroblasts showed a strikingly similar pattern of global transcriptome dysregulation in a reverse direction. In parallel with these transcriptional effects, RC disease dysregulated the integrated nutrient-sensing signaling network involving FOXO, PPAR, sirtuins, AMPK, and mTORC1, which collectively sense nutrient availability and regulate cellular growth. Altered activities of central nodes in the nutrient-sensing signaling network were validated by phosphokinase immunoblot analysis in RC inhibited cells. Remarkably, treating RC mutant fibroblasts with nicotinic acid to enhance sirtuin and PPAR activity also normalized mTORC1 and AMPK signaling, restored NADH/NAD⁺ redox balance, and improved cellular respiratory capacity. These data specifically highlight a common pathogenesis extending across different molecular and biochemical etiologies of individual RC disorders that involves global transcriptome modifications. We further identify the integrated nutrient-sensing signaling network as a common cellular response that mediates, and may be amenable to targeted therapies for, tissue-specific sequelae of primary mitochondrial RC disease.</p></div

Nicotinic acid treatment in a complex I mutant FCL reverses mTORC1 and AMPK activation and rescues cellular redox poise and respiratory capacity.

<p>(<b>A</b>) Upregulated P-S6 and P-AMPK expression in the Q1039 CI mutant FCL was mitigated in a dose-dependent fashion with 24 hour nicotinic acid treatment (lanes 5–6). Cells were grown in the same low glucose concentration (5 mMol) as was used for microarray studies. The proband has Leigh syndrome caused by known pathogenic mtDNA mutations in two complex I subunits, ND4 and ND6 (<b>Fig. 1</b>). (<b>B</b>) Total cellular deficiency in both NADH and NAD⁺ (as measured by HPLC as pMol/mg protein), along with an increased NADH/NAD⁺ ratio, was evident in the same CI mutant FCL relative to control FCL (gray). NADH and NAD⁺ cellular levels, as well as NADH/NAD⁺ redox balance, were all reversed by 24 hour nicotinic acid (10 mMol) treatment in CI mutant FCLs relative to untreated CI mutant FCLs (black). Percent change in mean [NAD⁺], [NADH], and calculated NADH/NAD⁺ ratios are shown, as determined in 3 biologic replicates per condition. (<b>C</b>) Intact FCLs from the same Q1039 CI-mutant proband (black) showed no significant change in total cellular routine (basal) respiratory capacity relative to unaffected control (white) oxygen flux as measured by high-resolution respirometry (Oxygraph-2K, Oroboros). However, nicotinic acid (10 mmol) treatment for 24 hours significantly increased total cellular respiratory capacity both in control (slash) and affected (dotted) cells. *, p<0.05 by ANOVA followed by paired t-test. Maximal (electron transport system, ETS) respiration was significantly greater than routine respiration for three of the conditions studied (CI mutant cells with no treatment, p = 0.0033; CI mutant cells with nicotinic acid treatment, p = 0.007, and control cells with no treatment, p = 0.002 by paired t-tests. No significance was reached when comparing basal and maximal respiratory for the nicotinic acid treated control cells, possibly due to small sample size (n = 3, p = 0.1126). Leak (background or non-mitochondrial) respiration showed no difference in any condition. Bars indicate mean±SEM. (<b>D</b>) Digitonin-permeabilized fibroblast analyses (right panel) reliably discriminated the specific complex site of RC dysfunction in affected CI mutant (Q1039) cells (black), as complex I-dependent (“GMADP, glutamate+malate+ADP) cellular respiratory capacity was significantly reduced in CI mutant FCLs relative to unaffected control FCLs (white). The genetic-based deficiency in CI-dependent respiratory capacity was not improved by 24 hour treatment with nicotinic acid (10 mMol, gray). Bars indicate mean±SEM. *, p<0.05.</p

Primary RC dysfunction transcriptionally and post-transcriptionally dysregulates the integrated nutrient-sensing signaling network.

<p>(<b>A</b>) <b>Integrated overview modeling general interactions between central nutrient-sensing signaling pathways.</b> Arrows and bars convey activating and inhibiting effects, respectively. TFs, physiologic signals, and drugs known to modulate this pathway are indicated in green, blue, and purple font, respectively. “P” indicates pathway components whose activity is modulated by phosphorylation. Red boxes detail physiologic effects. (<b>B</b>) <b>Oligomycin-based pharmacologic RC inhibition in human FCLs alters mTORC1 and AMPK pathway activities.</b> To confirm primary mitochondrial RC dysfunction was sufficient to alter mTORC1 signaling, FCLs from a healthy individual were treated in DMEM medium containing 20% fetal bovine serum for 24 hours and either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose, with either the complex V inhibitor, Oligomycin, (“O”, 5 uMol), an AMPK activator, AICAR (“A”, 2 mMol), or the mTORC1 inhibitor, rapamycin (“R”, 100 nMol). Regardless of glucose concentration, expression of a standard readout of mTORC1 pathway activity, phospho-S6 protein level, was reduced by oligomycin treatment, while increased phospho-AMPK expression was evident in high glucose media. (<b>C</b>) <b>Oligomycin-based alteration of mTORC1 and AMPK pathway activities is dose- and nutrient-dependent.</b> The same control FCLs as in (B) were treated for 24 hours in either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose media with oligomycin in doses ranging from 0.25 uMol to 5 uMol. A general trend is evident of overall reduced phospho-S6 and phospho-AMPK at increasing doses of oligomycin. See also <b>Fig. S13 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069282#pone.0069282.s003" target="_blank">File S3</a></b>. (<b>D</b>) <b>Pharmacologic RC inhibition increases </b><b><i>FOXO1</i></b><b> expression in high-glucose conditions.</b> FCLs were treated for 24 hours in either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose media with a RC inhibitor targeting either complex I (rotenone, 0.1 uMol), III (antimycin A, 0.5 uMol), or V (oligomycin, 1 uMol). As expected, <i>FOXO1</i> expression was inhibited by high-glucose media. All three RC inhibitors increased <i>FOXO1</i> relative expression in high-glucose conditions, whereas <i>FOXO1</i> expression was decreased by oligomycin in low-glucose media. (<b>E–F</b>) <b>Integrated view of detailed gene and sub-gene level transcriptional alterations in RC disease among central nutrient-sensing signaling network modulators.</b> Their major downstream targets and pathways were presented in separated plots. An overall inverse expression pattern is evident in RC disease between (<b>E</b>) skeletal muscle and (<b>F</b>) FCL.</p

Differential gene expression in RC disease FCL.

<p>(<b>A–D</b>) Differential expression of the same GSEA-defined pathways are shown for RC disease versus control FCLs as identified in skeletal muscle in <b>Fig. 2</b> (See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069282#pone.0069282.s002" target="_blank">File S2</a></b> for complete results). Positive and negative enrichment scores, respectively, indicate up- and down-regulation in RC disease. Numbers indicate counts of genes in each gene set that were measured by our experiment. * p<0.05; ** p<0.01; *** p<0.001. (<b>B</b>) The “Cytokine-cytokine receptor interaction” gene-set was identified in GSEA as among the most up-regulated gene sets in RC disease FCLs. (<b>C</b>) RNA processing pathway genes were generally down-regulated in RC disease FCLs. (<b>D</b>) Unsupervised clustering via principal component analysis (PCA) identified distinctive transcriptome groupings among FCLs from subjects categorized as color-detailed and defined in <b>Fig. 1</b>, where numbered circles indicate subjects also studied in the muscle data set (<b>Fig. 2D</b>).</p

Differential UTR expression in RC disease.

<p>(<b>A</b>) <b>Association between AU-rich elements and 3′-UTR changes in RC disease.</b> All genes were split into five exclusive groups based on the presence and type of AU-rich elements (ARE) in their 3′-UTRs. Genes in muscle without ARE had little change of their 3′-UTR, while those with long ARE motifs showed the most significant average increase. The reverse trend of lesser magnitude occurred in RC disease FCLs. Only effective probesets having expression significantly higher than background were analyzed. (<b>B</b>) <b><i>PAPD4</i></b> encodes the GDL-2 protein that acts as a cytoplasmic poly(A) polymerase, and was dysregulated by RC disease in a fashion consistent with its overall 3′-UTR changes in both cell types. (<b>C</b>) <b><i>DHX36</i></b>, also known as <i>RHAU</i>, enhances RNA decay by binding to AU-rich elements (AREs) in 3′-UTRs. Interestingly, DHX36 dysregulation in RC disease positively correlated to changes in 3′-UTRs containing AREs, which likely reflects its novel role in the regulation of RNA structure and synthesis. (<b>D</b>) <b>Position-specific differential expression of 3′-UTRs in RC disease.</b> Probes mapped to 3′-UTRs were assigned to 1% intervals from the 5′ to 3′ ends. Since 3′-UTR degradation starts from the 3′-end, the increasing difference from the 3′ to 5′ indicates a gradually decreased pace of 3′-UTR degradation occurs in RC disease. (<b>E</b>) <b>Relative 5</b>′<b>-UTR change in RC disease is dependent on the baseline absolute abundance of 5</b>′<b>-UTR in muscle.</b> Significantly changed 5′-UTRs (p<0.05) are highlighted in red, with the green line generated by Lowess smoothing. This plot demonstrates the nearly unanimous downregulation in RC disease of 5′-UTRs with the highest baseline transcription levels. See also <b>Fig. S12 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069282#pone.0069282.s003" target="_blank">File S3</a></b>.</p

Co-regulation of gene groups by upstream regulators.

<p>(<b>A</b>) <b>TF activity analysis of genes with promoter binding sites.</b> Average change of genes whose [−1 kb, 1 kb] promoter regions containing sites matching the position weight matrix (PWM) of 258 transcription factors (TFs) indicated that many TFs had modified activity in primary RC disease. Each dot represents a known TF PWM and its size corresponds to the combined p value in muscle and FCLs. (<b>B</b>) <b>TF activity analysis of genes with intronic binding sites.</b> The same analysis as in (A) was performed on genes whose introns contained sites matching the PWMs. (<b>C</b>) <b>Inverse expression of cytosolic and mitochondrial ribosomal genes in RC disease.</b> Overall differential expression of cytosolic and mitochondrial RP genes in RC disease had opposite patterns in muscle and FCLs. This analysis included genes encoding ribosome subunits, but not RP pseudogenes and genes regulating ribosome biogenesis, such as <i>RRS1</i> and <i>RPS6KA1</i>. (<b>D</b>) <b>Differential correlation of FOXO1 expression with cytosolic and mitochondrial ribosomal proteins. </b><i>FOXO1</i> was differentially correlated to cytosolic (green) and mitochondrial (blue) RP genes. Each green or blue bar represents the correlation coefficient between <i>FOXO1</i> and one RP gene summarized from four sample groups of all tissue-disease combinations using the DerSimonian-Laird meta-analysis method. The red bars on both ends indicate the overall average, with p values computed by Student’s t-test. (<b>E</b>) <b><i>FOXO1</i></b><b> differential expression at gene and sub-gene levels.</b> Differential <i>FOXO1</i> expression in both cell types is symbolized as two icons. Colors convey direction (green = down, red = up) and statistical significance of differential expression in RC disease. Triangles on the left are 5'-UTRs whose differential expression was more significant than the respective whole transcript average (see detailed keys in <b>Fig. 6</b>). See also <b>Fig. S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069282#pone.0069282.s003" target="_blank">File S3</a></b>.</p

Differential gene expression in RC disease muscle.

<p>(<b>A</b>) Significantly differentially expressed gene sets identified by GSEA (See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069282#pone.0069282.s002" target="_blank">File S2</a></b> for complete results). Positive and negative enrichment scores, respectively, indicate up- and down-regulation in RC disease. Numbers indicate counts of genes in each gene set that were measured by our experiment. * p<0.05; ** p<0.01; *** p<0.001. (<b>B–C</b>) Running enrichment scores of two significantly differentially expressed gene sets identified by GSEA. In each plot, the green and black lines, respectively, convey the running enrichment score and the genes categorized into the gene set as defined by KEGG pathway or GO gene ontology. The colored panel at the bottom indicates the level of differential expression from the most up-regulated (red) to most down-regulated (blue) in RC disease. (<b>B</b>) Proteasome pathway genes were generally up-regulated in RC disease muscle. (<b>C</b>) Structural muscle proteins were generally down-regulated in RC disease. (<b>D</b>) Unsupervised clustering via principal component analysis (PCA) identified distinctive transcriptome groupings among muscle from subjects categorized as color-detailed and defined in <b>Fig. 1</b> based on having “definite” RC disease (red), “suspected” RC disease with abnormal muscle RC biochemistry (dark pink), “suspected” RC disease with either no biopsy performed or no definitive biochemical abnormalities identified (light pink), pyruvate metabolism defect controls (light gray), ‘other disease” controls (dark gray), and healthy controls (brown). Numbered circles indicate subjects also studied in the fibroblast data set (<b>Fig. 3D</b>).</p

The global pattern of transcriptome changes in RC disease was reversed in muscle and FCL.

<p>(<b>A</b>) The global pattern of differential gene expression showed a highly significant negative correlation between RC disease muscle and FCLs (r = −0.49, p<10⁻¹⁶). The x- and y-axis units are signed p values, where dot size of individual genes is proportional to the combined fold-change of the two tissue types in RC disease relative to controls. (<b>B</b>) Genes were categorized based on their relative differential expression patterns in both muscle and FCL RC disease. Each subset was submitted to DAVID for over-representation analysis, where the most significant categories identified in DAVID are detailed for each of 8 unique gene subsets. (<b>C–D</b>) One of the spontaneously constructed IPA gene networks centered on MTOR. Red and green convey up- and down-regulation in RC disease, respectively. Twelve of the genes in this network have previously been associated to PI3K/AKT pathway signaling (blue lines). (<b>C</b>) Most genes in the MTOR IPA gene network showed significant up-regulation in RC disease muscle, with the exception of only a few downstream network members, such as <i>mir-132</i>. (<b>D</b>) Almost all genes identified in the IPA-centered MTOR network showed reversed expression change in RC disease FCL relative to controls as had been seen in muscle, including <i>mir-132</i> (<b>Fig. 4C</b>).</p