Bcl-xL-mediated antioxidant function abrogates the disruption of mitochondrial dynamics induced by LRRK2 inhibition.

We have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-xL (SH-SY5Y/Bcl-xL) to clarify the effects of this mitochondrial protein on the control of mitochondrial dynamics and the autophagic processes which occur after the inhibition of leucine-rich repeat kinase 2 (LRRK2) with GSK2578215A. In wild type (SH-SY5Y/Neo) cells, GSK2578215A (1nM) caused a disruption of mitochondrial morphology and an imbalance in intracellular reactive oxygen species (ROS) as indicated by an increase in dichlorofluorescein fluorescence and 4-hydroxynonenal. However, SH-SY5Y/Bcl-xL cells under GSK2578215A treatment, unlike the wild type, preserved a high mitochondrial membrane potential and did not exhibit apoptotical chromatins. In contrast to wild type cells, in SH-SY5Y/Bcl-xL cells, GSK2578215A did not induce mitochondrial translocation of neither dynamin related protein-1 nor the proapoptotic protein, Bax. In SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, mitochondrial fragmentation elicited by GSK2578215A precedes an autophagic response. Furthermore, the overexpression of Bcl-xL protein restores the autophagic flux pathway disrupted by this inhibitor. SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, responded to LRRK2 inhibition by an increase in the levels of acetylated tubulin, indicating that this was abrogated by Bcl-xL overexpression. This hyperacetylation of tubulin took place earlier than any of the above-mentioned events suggesting that it is involved in the autophagic flux interruption. Pre-treatment with tempol prevented the GSK2578215A-induced mitochondrial fragmentation, autophagy and the rise in acetylated tubulin in SH-SY5Y/Neo cells. Thus, these data support the notion that ROS act as a second messenger connexion between LRRK2 inhibition and these deleterious responses, which are markedly alleviated by the Bcl-xL-mediated ROS generation blockade.

Effect of training status on the changes in platelet parameters induced by short-duration exhaustive exercise.

It is now well known that hemostasis is directly involved in the benefits induced by physical activity. It has recently been shown that the baseline mean platelet volume (MPV) may be a predictor of endurance performance. We aimed to explore whether platelet parameters are associated with VO2max as well as running duration and speed in a short-duration exhaustive exercise test. Thirty healthy male subjects (10 sedentary and 20 trained) performed an incremental running test until exhaustion. MPV, platelet distribution width (PDW), platelet (Plt) count, and plateletcrit (Pct) were determined before exercise, immediately after exercise and after 30' recovery. Training status did not produce any difference in the baseline levels or in the post-exercise increases found in all the parameters tested. VO2max, test duration, and running speed were not correlated with any baseline parameter. Although MPV was found to be a predictor of endurance performance in long-duration exercise, the results of the present study are consistent with the hypothesis that MPV may not be a significant marker of performance in short-duration exhaustive exercise. Likewise, more research is needed to ascertain whether platelet activation is a reliable performance predictor in other exercise settings.

In vitro methylation of nuclear respiratory factor-2 binding sites suppresses the promoter activity of the human TOMM70 gene.

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported that two binding sites for transcription factor NRF-2 in the promoter region of the human TOMM70 gene are essential in activating transcription (Blesa et al., Mitochondrion 2004; 3:251-59. Blesa et al., Biochem Cell Biol 2006; 84:813-22). This region contains thirteen CpG methylation sites, three of which occur in the sequence 5'-CCGG-3' that is specifically recognized by HpaII methylase which modifies the internal cytosine residue. Interestingly, each NRF-2 site contains one CCGG sequence, allowing specific methylation of the NRF-2 sites and, therefore, providing an ideal model to study how methylation of these sites affects promoter activity. In this paper we report that site-specific methylation of the NRF-2 binding sites in the TOMM70 promoter down-regulated expression of a luciferase reporter in HeLa S3 cells. Electrophoretic mobility shift assays confirmed abrogation of NRF-2 binding at the methylated sites. These results suggest that methylation of the TOMM70 promoter in mammalian cells may silence TOMM70 expression. However, studies of methylation degree on DNAs from different sources found no methylation in the promoter regions of TOMM70 and other TOMM/TIMM family genes. Thus, although in vitro methylation inactivates the expression of TOMM70, our results suggest that this is not the mechanism modulating its expression in vivo. Since a number of nuclear genes encoding mitochondrial translocases have NRF-2 binding sequences containing CpG methylation sites, a possible role of methylation as a regulatory mechanism of mitochondrial biogenesis can be ruled out.

NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene.

The human TOMM34 gene encodes a cytosolic protein with chaperone-like activity that helps import some preproteins to the mitochondria by keeping them in an unfolded, import-compatible state. TOMM34 was found to be upregulated frequently in colorectal tumors, suggesting that it also has a role in the growth of cancer cells. In this context, TOMM34 is a potential target for novel anticancer drugs, and it might also be used in the diagnosis of colorectal cancer. Nuclear respiratory factors (NRFs) play an important role in governing the nuclear-mitochondrial interactions implicated in mitochondrial biogenesis. Our previous studies revealed that NRFs promote the expression of the major members of the mitochondrial transport machinery, TOMM70 and TOMM20. Here we report the existence of binding sites for NRF-1, Sp1, and NRF-2 in the 5' region of the human TOMM34 gene. We determined the effects of mutations at these sites on promoter activity in HeLa S3 and A204 cells, in conjunction with chromatin immunoprecipitation experiments, electrophoretic mobility shift assays, and in vivo methylation analysis of the promoter region. We conclude that NRF-1 is the main transcription factor regulating the expression of TOMM34. Sp1 interacts with NRF-1 to stimulate the promoter's full activity.

Expression of the human TIMM23 and TIMM23B genes is regulated by the GABP transcription factor.

The TIM23 protein is a key component of the mitochondrial import machinery in yeast and mammals. TIM23 is the channel-forming subunit of the translocase of the inner mitochondrial membrane (TIM23) complex, which mediates preprotein translocation across the mitochondrial inner membrane. In this paper, we aimed to characterize the promoter region of the highly similar human TIM23 orthologs: TIMM23 and TIMM23B. Bioinformatic analysis revealed putative sites for the GA-binding protein (GABP) and the recombination signal binding protein for immunoglobulin kappa J (RBPJ) transcription factors in both promoters. Luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments showed three functional sites for GABP and one functional site for RBPJ in both promoters. Moreover, silencing of GABPA, the gene encoding the DNA-binding subunit of the GABP transcription factor, resulted in reduced expression of TIMM23 and TIMM23B. Our results show an essential role of GABP in activating TIMM23 expression. More broadly, they suggest that physiological signals involved in activating mitochondrial biogenesis and oxidative function also enhance the transcription but not the protein level of TIMM23, which is essential for maintaining mitochondrial function and homeostasis.

NRF-2 transcription factor is required for human TOMM20 gene expression.

TOMM20 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of precursor proteins with N-terminal cleavable presequences targeted to the mitochondria. Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) play an important role in governing nucleo-mitochondrial interactions implicated in mitochondrial biogenesis. It was recently reported that NRF-2 is critical for maintaining normal transcriptional levels of the TOMM20 gene, but the promoter of this gene is uncharacterized. We report the presence of a NRF-2 and two NRF-1 binding motifs in the 5'-flanking region of the human TOMM20 gene and provide insight into their roles for promoter activity by using chromatin immunoprecipitation experiments and reporter assays in HeLa S3 and A204 cells. We show that only NRF-2 and the proximal NRF-1 motifs are involved in the expression of the gene. The NRF-2 binding site is required to activate transcription. The proximal NRF-1 site cooperates with NRF-2 in regulating the expression of the gene. The distal NRF-1 binding site is not functional.

Inhibition of xanthine oxidase to prevent statin-induced myalgia and rhabdomiolysis.

Although statins remain the cornerstone of lipid-lowering therapy for reducing the burden of atherosclerotic vascular disease, their administration has been associated with muscle-related adverse effects, including myalgia and rhabdomyolysis. Such adverse events are probably due to reduced antioxidant defenses associated with fewer intermediate metabolites in the cholesterol synthesis pathway. We hypothesize that the concomitant inhibition of xanthine oxidase via coadministration of allopurinol with statins could diminish reactive oxygen species (ROS)-related muscle damage, which would have in turn have positive effects on both the incidence of muscle-related adverse events and cardiovascular outcomes. Accordingly, inhibition of xanthine oxidase has been previously shown to be effective for reducing biomarkers of muscle damage following exercise in professional athletes. Because of the widespread statin utilization and increasing trends in their therapeutic use in atherosclerotic vascular diseases, the proposed strategy could have important clinical implications for reducing statin-induced myalgia and rhabdomyolysis.

NRF-2 transcription factor is essential in promoting human Tomm70 gene expression.

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic pre-proteins targeted to the mitochondria. We report the presence of two nuclear respiratory factor 2 (NRF-2) binding motifs in the 5'-flanking region of the human Tomm70 gene and establish their essential role for promoter activity by using reporter assays in HeLa cells. We show that both NRF-2 binding sites are functional and present evidence that interactions between these sites and a CpG island contribute to expression. Mobility shift assays show that these NRF-2 sites are specifically recognized by NRF-2 present in HeLa nuclear extracts.

Tau-protein expression in human blood lymphocytes: a promising marker and suitable sample for life-time diagnosis of Alzheimer's disease.

OBJECTIVES: Taking into account the hypothesis that Alzheimer's disease (AD) might be a systemic disease that affects several tissues in the body, the aim of this study was to try to detect the expression of tau-protein in human peripheral blood lymphocytes (PBL) in patients with AD. MATERIAL AND METHODS: Blood samples were obtained from patients with AD (n=16, age 67-98) and from volunteers without psychoneurological pathology (n=10, age 65-78). PBL were isolated on Ficoll-Paque gradient centrifugation. For cell fixation and permeabilization we used a fixative solution (4% formaldehyde and 0.1% glutaraldehyde) and 0.03% Triton X-100. Immunocytochemical detection of tau-protein was carried out by biotin-streptavidin complex method with tau monoclonal antibody (1:100, clone TAU-2, ICN) and universal immunostaining kit IMMU-MARK (ICN). RESULTS: The expression of tau-protein was shown in PBL in absolute majority of AD patients studied. Only in two healthy volunteers a single lymphocyte from many cells (i.e. a smear) demonstrated a very weak-positive immunostaining to tau-protein CONCLUSION: This first demonstration of clear difference in localization of tau-protein in blood lymphocytes between healthy and sick people testifies to the fact that tau-protein could be considered as a promising marker and blood lymphocytes as a suitable sample for life-time diagnosis of AD.

Distinct functional contributions of 2 GABP-NRF-2 recognition sites within the context of the human TOMM70 promoter.

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP-NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP-NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP-NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

Report of hereditary persistence of alpha-fetoprotein in a Spanish family: molecular basis and clinical concerns.

The serum level of alpha-fetoprotein in normal adults is lower than 10 ng/ml. High levels of alpha-fetoprotein in adults are linked to cirrhosis, acute or chronic hepatitis, hepatocellular carcinomas and other pathologies, as well as to foetal malformation, and this protein is therefore used as a regular clinical marker for these diseases. We report a Spanish family in which very high levels of alpha-fetoprotein have been detected in nine members from the screening of a total of 17 relatives. These levels of alpha-fetoprotein are not accompanied by a causing pathology, are inherited as an autosomal dominant genetic trait, and are associated to a G-->A substitution at position -116 of the 5'-flanking region of the alpha-fetoprotein gene. This is an unusual benign trait of hereditary persistence of alpha-fetoprotein. This paper provides a detailed clinical report of the family including a study of the molecular basis of this trait. The desirability of a test to detect and/or rule out this benign trait in adults with abnormal levels of alpha-fetoprotein is considered.