Stressed-Induced TMEM135 Protein Is Part of a Conserved Genetic Network Involved in Fat Storage and Longevity Regulation in Caenorhabditis elegans

Disorders of mitochondrial fat metabolism lead to sudden death in infants and children. Although survival is possible, the underlying molecular mechanisms which enable this outcome have not yet been clearly identified. Here we describe a conserved genetic network linking disorders of mitochondrial fat metabolism in mice to mechanisms of fat storage and survival in Caenorhabditis elegans (C. elegans). We have previously documented a mouse model of mitochondrial very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency.[1] We originally reported that the mice survived birth, but, upon exposure to cold and fasting stresses, these mice developed cardiac dysfunction, which greatly reduced survival. We used cDNA microarrays[2], [3], [4] to outline the induction of several markers of lipid metabolism in the heart at birth in surviving mice. We hypothesized that the induction of fat metabolism genes in the heart at birth is part of a regulatory feedback circuit that plays a critical role in survival.[1] The present study uses a dual approach employing both C57BL/6 mice and the nematode, C. elegans, to focus on TMEM135, a conserved protein which we have found to be upregulated 4.3 (±0.14)-fold in VLCAD-deficient mice at birth. Our studies have demonstrated that TMEM135 is highly expressed in mitochondria and in fat-loaded tissues in the mouse. Further, when fasting and cold stresses were introduced to mice, we observed 3.25 (±0.03)- and 8.2 (±0.31)- fold increases in TMEM135 expression in the heart, respectively. Additionally, we found that deletion of the tmem135 orthologue in C. elegans caused a 41.8% (±2.8%) reduction in fat stores, a reduction in mitochondrial action potential and decreased longevity of the worm. In stark contrast, C. elegans transgenic animals overexpressing TMEM-135 exhibited increased longevity upon exposure to cold stress. Based on these results, we propose that TMEM135 integrates biological processes involving fat metabolism and energy expenditure in both the worm (invertebrates) and in mammalian organisms. The data obtained from our experiments suggest that TMEM135 is part of a regulatory circuit that plays a critical role in the survival of VLCAD-deficient mice and perhaps in other mitochondrial genetic defects of fat metabolism as well

Genetic determinants of heel bone properties: genome-wide association meta-analysis and replication in the GEFOS/GENOMOS consortium

Quantitative ultrasound of the heel captures heel bone properties that independently predict fracture risk and, with bone mineral density (BMD) assessed by X-ray (DXA), may be convenient alternatives for evaluating osteoporosis and fracture risk. We performed a meta-analysis of genome-wide association (GWA) studies to assess the genetic determinants of heel broadband ultrasound attenuation (BUA; n = 14 260), velocity of sound (VOS; n = 15 514) and BMD (n = 4566) in 13 discovery cohorts. Independent replication involved seven cohorts with GWA data (in silico n = 11 452) and new genotyping in 15 cohorts (de novo n = 24 902). In combined random effects, meta-analysis of the discovery and replication cohorts, nine single nucleotide polymorphisms (SNPs) had genome-wide significant (P < 5 × 10(-8)) associations with heel bone properties. Alongside SNPs within or near previously identified osteoporosis susceptibility genes including ESR1 (6q25.1: rs4869739, rs3020331, rs2982552), SPTBN1 (2p16.2: rs11898505), RSPO3 (6q22.33: rs7741021), WNT16 (7q31.31: rs2908007), DKK1 (10q21.1: rs7902708) and GPATCH1 (19q13.11: rs10416265), we identified a new locus on chromosome 11q14.2 (rs597319 close to TMEM135, a gene recently linked to osteoblastogenesis and longevity) significantly associated with both BUA and VOS (P < 8.23 × 10(-14)). In meta-analyses involving 25 cohorts with up to 14 985 fracture cases, six of 10 SNPs associated with heel bone properties at P < 5 × 10(-6) also had the expected direction of association with any fracture (P < 0.05), including three SNPs with P < 0.005: 6q22.33 (rs7741021), 7q31.31 (rs2908007) and 10q21.1 (rs7902708). In conclusion, this GWA study reveals the effect of several genes common to central DXA-derived BMD and heel ultrasound/DXA measures and points to a new genetic locus with potential implications for better understanding of osteoporosis pathophysiology

TMEM135 is elevated with the stresses of cold and fasting in the worm and in C57BL/6 mice.

<p>Figures <b>A</b> and <b>B</b> are representative experiments in the worm. Fig. <b>A</b> shows the induction of TMEM135 with cold stress (4°C) in the worm, and Fig. <b>B</b> is the quantification of average fluorescence levels exemplified in Fig. <b>A</b>. Values in these experiments are expressed as relative fluorescence intensity in mean ± SEM. Figures <b>C</b> and <b>D</b> were experiments done using C57BL/6 mice. The animals were subjected to overnight fasting and were exposed to the cold for 2 hours, as previously published<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014228#pone.0014228-Exil2" target="_blank">[10]</a>. (<b>C</b>) Western blots of TMEM135 expression with fasting and cold stresses. (<b>D</b>) Quantification by densitometry of the western blots shown in C, N = 3 per group.</p

Nile Red staining and MitoTracker Red staining, survival differences and DAF-16 levels among the three <i>C. elegans</i> strains.

<p>Fig. <b>6A</b> is representative staining with Nile Red in the worm. (<b>B</b>) Represents semi-quantitative assessment of Nile Red staining intensity, N = 4 per group. (<b>C</b>) Represents survival analysis among the three <i>C. elegans</i> strains at 20°C. (<b>D</b>) Represents survival analysis among the three <i>C. elegans</i> strains at 15°C. Wild-type  =  controls shown in red, <i>tmem 135</i>(−<i>/</i>−) <i> =  tmem135</i>-deleted animals shown in black, TMEM135::GFP =  <i>C. elegans</i> animal overexpressing TMEM135 shown in green. (<b>E</b>) Quantitative assessment of MitoTracker Red fluorescence. (<b>F</b>) Western blot analysis and quantification of DAF-16 levels in the three <i>C. elegans</i> strains, N = 6 per group, values are mean ± SEM.</p

RNA and protein expression of TMEM135 in mouse tissues.

<p>(<b>A</b>) Northern blot analysis of TMEM135 transcripts in newborn hearts of mice deficient in the very long chain ACAD gene. (<b>B</b>) <b>B</b> is representative of quantification by densitometry of Northern blot in <b>A</b>. (<b>C</b>) Protein expression of TMEM135 in different mouse tissues. (<b>D</b>) Quantification by densitometry of western blots in <b>C</b>. N =  3 per group for wild-type control mice labeled (+/+) for VLCAD +/+, heterozygous mice (+/−) for VLCAD+/−, and null mutant mice (−/−) for VLCAD−/−.</p

TMEM135 localizes to fat droplets in the mouse and in <i>C. elegans</i> and to mitochondria close to fat droplets in the mouse.

<p>Experiments in A, B and C were done in mouse tissue. (<b>A</b>) Western blot to assess protein expression of TMEM135 in sub-cellular fractions of the mouse heart. TMEM135 specific antibody, 1∶1000. Lane1: total protein, lane 2: nuclear fraction, lane 3: mitochondrial fraction, lane 4: microsomal fraction, lane 5: a low speed sediment containing intercalated discs sediment and microsomes enriched in heart sarcoplasmic reticulum, lane 6: cytosol. (<b>B</b>) Quantification by densitometry of western blot in <b>A</b>. <b>C1</b>, <b>C2</b> and <b>C3</b> are representative of Transmitted Immuno-Electron microscopy in mouse heart tissue. (<b>C1</b>) Immunogold staining showing TMEM135 mostly in the mitochondria and along the Z-line in the sarcomere. M =  mitochondria, S =  sarcomere. <b>C2 and C3</b> show accumulation of the TMEM135 epitope in the surrounding of lipid droplets in mouse hearts. M =  mitochondria, Arrows =  fat droplets, Arrowheads =  TMEM135 gold stain. (<b>D</b>) Is representative of TMEM135::GFP staining with Mitotracker Red, showing no co-localization of TMEM135 and the mitochondria in the worm. (<b>E</b>) Represents Nile red staining in the TMEM135 over-expressing animals. This figure shows co-localization of the GFP and fat droplets in the worm, as indicated by the arrows. Experiments in this section were performed 3 or 4 times.</p

Comparison of sequence homology analysis of TMEM135 in different species.

<p>TMEM135 is well conserved across species.</p

Western blot analysis of possible downstream targets of TMEM135.

<p><b>7A</b> is representative western blot for NRF2/SKN1 targets. <b>7B</b> is representative western blot analysis of fatty acid metabolism target genes. N = 3 in each group. <i>tmem135</i>(<i>−/−</i>) for the tmem135-deletion strain, and TMEM135::GFP for the overexpressing strain. N = 3 in each group.</p