Representative proteome map of differentially expressed proteins from the mitochondrial fractions of fibroblasts from (A) LHON cases (n = 7) and (U) unaffected LHON individuals (n = 3).

<p>Equal amounts of proteins (100 µg) from each sample were resolved by 2-DE. The numbers indicate the spot IDs of proteins whose expression levels differ significantly between the LHON cases and their unaffected relatives.</p

Functional categories and sub-cellular localizations of proteins identified in the study based on the MitoMiner Database and Nextprot.

<p>M = Mitochondria, MI = Mitochondrial intermembrane space, ER = Endoplasmic Reticulum, C = Cytoplasm, N = Nucleus, CSK = cytoskeleton.</p><p>Functional categories and sub-cellular localizations of proteins identified in the study based on the MitoMiner Database and Nextprot.</p

Characteristics of LHON cases and unaffected relatives recruited in the study.

<p>*MtDNA/nuclear DNA from fibroblast of each individual was measured according to Pejznochova M, 2008 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106779#pone.0106779-Pejznochova1" target="_blank">[62]</a> by amplification of <i>ND5</i> gene in mitochondrial genome from position 13466–13650 (GenBank sequence NC_012920 gi:251831106), and <i>PARL</i> gene from nuclear genome from 16912–17165 (GenBank sequence: NC_000003.12 gi:224589815). Real-time PCR amplification was performed on Bio-Rad CFX 96 thermo cycler and the threshold cycle (Ct) was obtained. MtDNA/nuclear DNA ratio was calculated from the Ct (mtDNA)/Ct (nDNA) ratio. The increasing ratio shows decrease in amount of mtDNA per cell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106779#pone.0106779-Pejznochova1" target="_blank">[62]</a>.</p><p>Characteristics of LHON cases and unaffected relatives recruited in the study.</p

Differentially expressed proteins which show similar trends of down-regulation in the mitochondrial proteomes of both LHON cases and unaffected relatives when compared with controls.

<p>Differentially expressed proteins which show similar trends of down-regulation in the mitochondrial proteomes of both LHON cases and unaffected relatives when compared with controls.</p

Assessment of the purity of fibroblasts from a cultured skin biopsy.

<p>Fibroblast surface protein (FSP) was used as a marker in immunofluorescence of the cultured fibroblasts obtained directly obtained from the skin biopsy. The green represents fibroblasts and the nucleus was stained with Hoechst-dye 33342 which shows blue.</p

Summary of significantly altered proteins between the LHON cases and the control group.

<p>*Gene name according to HUGO Gene Nomenclature Committee.</p>#<p>indicates <i>P</i> value based on Post Hoc Tukey Test after one way ANOVA and the value less than 0.05 is considered significant in both tests.</p><p>Summary of significantly altered proteins between the LHON cases and the control group.</p

Summary of significantly altered proteins between the unaffected LHON individuals and the control group.

<p>*Gene name according to HUGO Gene Nomenclature Committee.</p>#<p>indicates <i>P</i> value based on Post Hoc Tukey Test after one way ANOVA and the value less than 0.05 is considered significant in both tests.</p><p>Summary of significantly altered proteins between the unaffected LHON individuals and the control group.</p

Summary of significantly altered proteins between the LHON cases and the unaffected relatives.

<p>*Gene name according to HUGO Gene Nomenclature Committee.</p>#<p>indicates <i>P</i> value based on Post Hoc Tukey Test after one way ANOVA and the value less than 0.05 is considered significant in both tests.</p><p>Summary of significantly altered proteins between the LHON cases and the unaffected relatives.</p

Western blot analyses for assessment of mitochondrial enrichment and purity.

<p>20 µg of mitochondrial lysate and whole cell lysate from fibroblasts were separated by 12% SDS-PAGE gel and checked with specific antibodies against various sub-cellular organelles. (W = whole cell lysate; M = mitochondrial enriched fraction) (The same membrane for each cell type was stripped and probed with subsequent antibodies.).</p

Profiling the Mitochondrial Proteome of Leber’s Hereditary Optic Neuropathy (LHON) in Thailand: Down-Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the 11778G>A Mutation

<div><p>Leber’s Hereditary Optic Neuropathy (LHON) is one of the commonest mitochondrial diseases. It causes total blindness, and predominantly affects young males. For the disease to develop, it is necessary for an individual to carry one of the primary mtDNA mutations 11778G>A, 14484T>C or 3460G>A. However these mutations are not sufficient to cause disease, and they do not explain the characteristic features of LHON such as the higher prevalence in males, incomplete penetrance, and relatively later age of onset. In order to explore the roles of nuclear encoded mitochondrial proteins in development of LHON, we applied a proteomic approach to samples from affected and unaffected individuals from 3 pedigrees and from 5 unrelated controls. Two-dimensional electrophoresis followed by MS/MS analysis in the mitochondrial lysate identified 17 proteins which were differentially expressed between LHON cases and unrelated controls, and 24 proteins which were differentially expressed between unaffected relatives and unrelated controls. The proteomic data were successfully validated by western blot analysis of 3 selected proteins. All of the proteins identified in the study were mitochondrial proteins and most of them were down regulated in 11778G>A mutant fibroblasts. These proteins included: subunits of OXPHOS enzyme complexes, proteins involved in intermediary metabolic processes, nucleoid related proteins, chaperones, cristae remodelling proteins and an anti-oxidant enzyme. The protein profiles of both the affected and unaffected 11778G>A carriers shared many features which differed from those of unrelated control group, revealing similar proteomic responses to 11778G>A mutation in both affected and unaffected individuals. Differentially expressed proteins revealed two broad groups: a cluster of bioenergetic pathway proteins and a cluster involved in protein quality control system. Defects in these systems are likely to impede the function of retinal ganglion cells, and may lead to the development of LHON in synergy with the primary mtDNA mutation.</p></div