Dysregulation of mitochondrial function and biogenesis modulators in adipose tissue of obese children.

BACKGROUND/OBJECTIVES: We aimed to evaluate mitochondrial biogenesis (MB), structure, metabolism and dysfunction in abdominal adipose tissue from male pediatric patients with obesity. SUBJECTS/METHODS: Samples were collected from five children with obesity (percentile ⩾95) and five eutrophic boys (percentile ⩾5/⩽85) (8-12 years old) following parental informed consent. We analyzed the expression of key genes involved in MB (sirtuin-1 (SIRT1), peroxisome proliferator-activated receptor-γ (PPARγ), PPARγ coactivator-1α (PGC1α), nuclear respiratory factors 1 and 2 (NRF1, NRF2) and mitochondrial transcription factor A (TFAM) and surrogates for mitochondrial function/structure/metabolism (porin, TOMM20, complex I and V, UCP1, UCP2, SIRT3, SOD2) by western blot. Citrate synthase (CS), complex I (CI) activity, adenosine triphosphate (ATP) levels, mitochondrial DNA (mtDNA) content and oxidative stress end points were also determined. RESULTS: Most MB proteins were significantly decreased in samples from children with obesity except complex I, V and superoxide dismutase-2 (SOD2). Similarly, CS and CI activity showed a significant reduction, as well as ATP levels and mtDNA content. PPARγ, PGC1α, complex I and V and SOD2 were hyperacetylated compared with lean samples. Concurrently, in samples from children with obesity, we found decreased SOD2 activity and redox state imbalance highlighted by decreased reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and significant increases in protein carbonylation. CONCLUSIONS: Adipose tissue from children with obesity demonstrates a dysregulation of key modulators of MB and organelle structure, and displays hyperacetylation of key proteins and altered expression of upstream regulators of cell metabolism.

Cell cloning-based transcriptome analysis in Rett patients: relevance to the pathogenesis of Rett syndrome of new human MeCP2 target genes.

More than 90% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene that encodes the methyl-CpG-binding protein 2, a transcriptional modulator. Because MECP2 is subjected to X chromosome inactivation (XCI), girls with RTT either express the wild-type or mutant allele in each individual cell. To test the consequences of MECP2 mutations resulting from a genome-wide transcriptional dysregulation and to identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we carried out gene expression profiling of clonal populations derived from fibroblast primary cultures expressing exclusively either the wild-type or the mutant MECP2 allele. Clonal cultures were obtained from skin biopsy of three RTT patients carrying either a non-sense or a frameshift MECP2 mutation. For each patient, gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis. Firstly, clustering analysis classified the RTT patients according to their genetic background and MECP2 mutation. Secondly, expression profiling by microarray analysis and quantitative RT-PCR indicated four up-regulated genes and five down-regulated genes significantly dysregulated in all our statistical analysis, including excellent potential candidate genes for the understanding of the pathophysiology of this neurodevelopmental disease. Thirdly, chromatin immunoprecipitation analysis confirmed MeCP2 binding to respective CpG islands in three out of four up-regulated candidate genes and sequencing of bisulphite-converted DNA indicated that MeCP2 preferentially binds to methylated-DNA sequences. Most importantly, the finding that at least two of these genes (BMCC1 and RNF182) were shown to be involved in cell survival and/or apoptosis may suggest that impaired MeCP2 function could alter the survival of neurons thus compromising brain function without inducing cell death.

Impairment of CDKL5 nuclear localisation as a cause for severe infantile encephalopathy.

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause infantile spasms as well as Rett syndrome-like phenotype. To date, fewer than 20 different mutations have been reported. So far, no clear genotype-phenotype correlation has been established. We screened the entire coding region of CDKL5 in 151 affected girls with a clinically heterogeneous phenotype ranging from encephalopathy with epilepsy to atypical Rett syndrome by denaturing high liquid performance chromatography and direct sequencing, and we identified three novel missense mutations located in catalytic domain (p.Ala40Val, p.Arg65Gln, p.Leu220Pro). Segregation analysis showed that p.Arg65Gln was inherited from the healthy father, which rules out the involvement of CDKL5 in the aetiology of the phenotype in this patient. However, the de novo occurrence was shown for p.Ala40Val and p.Leu220Pro. The p.Ala40Val mutation was observed in two unrelated patients and represented the first recurrent mutation in the CDKL5 gene. For the two de novo mutations, we analysed the cellular localisation of the wild-type and CDKL5 mutants by transfection experiments. We showed that the two CDKL5 mutations cause mislocalisation of the mutant CDKL5 proteins in the cytoplasm. Interestingly these missense mutations that result in a mislocalisation of the CDKL5 protein are associated with severe developmental delay which was apparent within the first months of life characterised by early and generalised hypotonia, and autistic features, and as well as early infantile spasms.

Association of PPARG2 Pro12Ala variant with larger body mass index in Mestizo and Amerindian populations of Mexico.

Previous studies have sought to associate the Pro12Ala variant of the peroxisome proliferator-activated receptor gamma2 (PPARG2) gene with type 2 diabetes, insulin resistance, and obesity, with controversial results. We have determined the Pro12Ala variant frequency in 370 nondiabetic Mexican Mestizo subjects and in five Mexican Amerindian groups and have investigated its possible association with lipid metabolism, insulin serum levels, and obesity in three of these populations. Two independent case-control studies were conducted in 239 nondiabetic individuals: 135 case subjects (BMI > or = 25 kg/m2) and 104 control subjects (BMI < 25 kg/m2). The PPARG2 Ala12 allele frequency was higher in most Amerindian populations (0.17 in Yaquis, 0.16 in Mazahuas, 0.16 in Mayans, and 0.20 in Triquis) than in Asians, African Americans, and Caucasians. The Pro12Ala and Ala12Ala (X12Ala) genotypes were significantly associated with greater BMI in Mexican Mestizos and in two Amerindian groups. X12Ala individuals had a higher risk of overweight or obesity than noncarriers in Mestizos (OR = 3.67; 95% CI, 1.42-9.48; p = 0.007) and in Yaquis plus Mazahuas (OR = 3.21; 95% CI, 1.27-8.11; p = 0.013). Our results provide further support of the association between the PPARG2 Ala12 allele and risk of overweight or obesity in Mestizos and two Amerindian populations from Mexico.

[Case report. Patient with varicocele & oligozoospermia with Y chromosome microdeletion: AZFb+c]. / Aportación de un caso. Paciente con varicocele y oligozoospermia, con microdeleción en cromosoma Y: AZFb+c.

The 13-18% of the couples in reproductive age gets to present some kind of fertility problem, the male factor participates in near 50% of the cases. The microdeletions in the Yq chromosome occupy single the 7,6%; severe oligozoospermia and azoospermia are related with these alterations. The structure of region AZF of Yq is divided in AZFa, AZFb, AZFc and AZFd. The microdeletions can be simple or combined, the microdeletion AZFc (59-65%) is the commonest. In cases with severe oligozoospermia (or azoospermia), study of microdeletions in Y chromosome is suggested. ICSI with selection of sex, would be the treatment adapted in these cases, is in phase of study.

Aportación de un caso. Paciente con varicocele y oligozoospermia, con microdeleción en cromosoma Y: AZFb+c / Case report. Patient with varicocele & oligozoospermia with Y chromosome microdeletion: AZFb+c

El 13-18% de las parejas en edad reproductiva llegan a presentar algún tipo de problema de fertilidad, el factor masculino participa en cerca del 50% de los casos. Las microdeleciones en el cromosoma Yq ocupan sólo el 7,6% y se relacionan en forma importante con oligozoospermia severa (<5millones x mililitro OMS 1999) (2-5%) y azoospermia (5-10%). La estructura de la región AZF de Yq se divide en AZFa, AZFb, AZFc y AZFd. Las microdeleciones pueden ser simples o combinadas, siendo la microdelección AZFc (59-65%), la más común. En casos con oligozoospermia severa (o azoospermia) se sugiere estudio de microdeleciones en cromosoma Y, independientemente de las enfermedades concomitantes (ejemplo varicocele), como parte del pronóstico de fertilidad. ICSI con selección de sexo, sería el tratamiento adecuado en estos casos, se encuentra en fase de estudio

The 13-18% of the couples in reproductive age gets to present some kind of fertility problem, the male factor participates in near 50% of the cases. The microdeletions in the Yq chromosome occupy single the 7,6%; severe oligozoospermia and azoospermia are related with these alterations. The structure of region AZF of Yq is divided in AZFa, AZFb, AZFc and AZFd. The microdeletions can be simple or combined, the microdeletion AZFc (59-65%) is the commonest. In cases with severe oligozoospermia (or azoospermia), study of microdeletions in Y chromosome is suggested. ICSI with selection of sex, would be the treatment adapted in these cases, is in phase of study

Dysferlin homozygous mutation G1418D causes limb-girdle type 2B in a Mexican family.

Dysferlin protein (DYSF) is a ferlin family member found in sarcolemma and is involved in membrane repair, muscle differentiation, membrane fusion, etc. The deficiency of DYSF due to mutations is associated with different pathologic phenotypes including the autosomal recessive limb-girdle type 2B phenotype (LGMD2B), a distal anterior compartment myopathy (DMAT), and the Miyoshi myopathy (MM). In this study, we determined a missense mutation c.4253G>A on the DYSF gene in a Mexican family from an endogamic population. This mutation was assumed to be the cause of dystrophy because only homozygous individuals of the family manifest a clinical phenotype. Structural implications caused by G/D substitution at amino acid position 1418 are discussed in terms of potential importance of the dysferlin neighboring sequence.

Expression analysis of the SG-SSPN complex in smooth muscle and endothelial cells of human umbilical cord vessels.

Recently, participation of the sarcoglycan (SG)-sarcospan (SSPN) complex in the development of cardiomyopathy in patients with limb-girdle muscular dystrophy has been shown, and presence of the complex in smooth muscle may be important for the contraction/dilation process of vessels. However, there are few studies determining the SG-SSPN complex in vascular smooth muscle and endothelial cells of vessels. In this study, we analyzed by reverse transcriptase-polymerase chain reaction and immunofluorescence the expression of different components of the complex in vein/artery smooth muscle and endothelial cells of the human umbilical cord. By RNA analysis, we observed expression of alpha-, beta-, gamma-, delta-, epsilon-SG, and SSPN in smooth muscle cells. In endothelial cells, RNA expression was restricted to beta-, delta-, epsilon-SG, and SSPN. At protein level, we observed in smooth muscle the presence of beta-, delta-, epsilon-SG, and SSPN. In endothelial cells, immunostaining only evidenced the presence of epsilon-SG and SSPN. However, colocalization of SGs and SSPN with dystrophin and utrophin was noted. These results, interestingly, suggest that the SG-SSPN complex may either form with dystrophin or utrophin in smooth muscle cells, and with utrophin in endothelial cells. Additionally, we also observed in some smooth muscle regions the colocalization of the SG-SSPN complex with caveolin, with colocalization being more pronounced between epsilon-SG-SSPN and caveolin in endothelial cells.

Differential expression and subcellular distribution of dystrophin Dp71 isoforms during differentiation process.

Dp71 is the major product of the Duchenne muscular dystrophy gene in the brain. In order to study the function of Dp71 in the nervous system we examined the expression of Dp71 isoforms in PC12 rat pheochromocytoma cell line, a well-established system to study neuronal differentiation. We show by reverse transcriptase-polymerase chain reaction and Western blot assays that PC12 cells express two Dp71 isoforms. One isoform lacks exon 71 and the other isoform lacks exons 71 and 78 (Dp71d and Dp71f isoforms respectively). Nerve growth factor-induced neuronal differentiation of PC12 cells results in differential regulation of the expression and subcellular localization of Dp71 isoforms: a) the amount of Dp71f protein increases nine-fold in total extracts while Dp71d increases up to seven-fold in nuclear extracts; b) Dp71f relocates from the cytoplasm to neuritic processes, being prominent at varicosities and the growth cone; c) Dp71d relocates almost entirely to the nucleus and is detected to a lower extent in the cytoplasm and neuritic processes. Dp71f co-localizes with beta-dystroglycan and synaptophysin while Dp71d co-localizes with beta-dystroglycan in the nucleus. Dp71d accumulates at cell-cell contacts where Dp71f is absent. These results suggest that Dp71d and Dp71f associate with different subcellular complexes and therefore may have distinct functions in PC12 cells.

Expression and localization of utrophin in differentiating PC12 cells.

To ascertain the role of utrophin in cultured neuronal cells, we investigated its expression and distribution along the NGF-induced differentiation of PC12 cells grown on different substrata. Utrophin mRNA was measured by RT-PCR assay and utrophin protein was quantified by immunoblot analysis. The distribution of utrophin and beta-dystroglycan was analyzed by confocal microscopy. We demonstrate that utrophin protein was increased 4-fold during differentiation of cells grown laminin. Concomitant with this up-regulation, utrophin was enriched at the growth cones in differentiating cells, where it co-localizes with beta-dystroglycan. These data suggest the presence of a utrophin-beta-dystroglycan complex in PC12 cells that participates in the formation and/or stabilization of the growth cone-extracellular matrix adhesion.

Dystrophins in developing retina: Dp260 expression correlates with synaptic maturation.

Dystrophin, the protein altered in Duchenne muscular dystrophy (DMD), is necessary for normal retinal function and exists in several isoforms. We examined the expression of dystrophin and utrophin proteins and transcripts in the rat retina at different developmental stages using Western blots and semi-quantitative RT-PCR. Our results revealed the presence of utrophin (DRP1), G-utrophin and/or DRP2 and four dystrophin isoforms (Dp427, Dp260, Dp140, Dp71) in the normal adult rat retina. Only Dp260 showed a marked progressive increase with age at both protein and mRNA levels. This variation is consistent with the establishment of synaptic functions in the developing retina and suggests a key role for this apo-dystrophin in synaptogenesis.