Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

Physical training and hypertension have opposite effects on endothelial brain-derived neurotrophic factor expression.

AIMS: Changes in circulating brain-derived neurotrophic factor (BDNF) levels were reported in patients with or at risk for cardiovascular diseases associated with endothelial dysfunction, suggesting a link between BDNF and endothelial functionality. However, little is known on cardiovascular BDNF. Our aim was to investigate levels/localization, function, and relevance of cardiovascular BDNF. METHODS AND RESULTS: BDNF levels (western blotting) and localization (immunostaining) were assessed in the heart and aorta from rats with impaired (spontaneously hypertensive rats [SHR]), normal (Wistar Kyoto rats [WKY]), and improved (SHR and WKY subjected to physical training) endothelial function. BDNF levels were also measured in cultured endothelial cells (CECs) subjected to low and high shear stress. The cardiovascular effects of BDNF were investigated in isolated aortic rings and hearts. The results showed high BDNF levels in the heart and aorta, the expression being prominent in endothelial cells as compared with other cell types. Exogenous BDNF vasodilated aortic rings but changed neither coronary flow nor cardiac contractility. Hypertension was associated with decreased expression of BDNF in the endothelium, whereas physical training led to endothelial BDNF up-regulation not only in WKY but also in SHR. Exposure of CECs to high shear stress stimulated BDNF production and secretion. CONCLUSION: Cardiovascular BDNF is mainly localized within endothelial cells in which its expression is dependent on endothelial function. These results open new perspectives on the role of endothelial BDNF in cardiovascular health.

Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant variant of human glial tumors. A prominent feature of this tumor is the occurrence of necrosis and vascular proliferation. The regulation of glial neovascularization is still poorly understood and the characterization of factors involved in this process is of major clinical interest. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine released by leukocytes and by a variety of cells outside of the immune system. Recent work has shown that MIF may function to regulate cellular differentiation and proliferation in normal and tumor-derived cell lines, and may also contribute to the neovascularization of tumors. Our immunohistological analysis of MIF distribution in GBM tissues revealed the strong MIF protein accumulation in close association with necrotic areas and in tumor cells surrounding blood vessels. In addition, MIF expression was frequently associated with the presence of the tumor-suppressor gene p53. To substantiate the concept that MIF might be involved in the regulation of angiogenesis in GBM, we analyzed the MIF gene and protein expression under hypoxic and hypoglycemic stress conditions in vitro. Northern blot analysis showed a clear increase of MIF mRNA after hypoxia and hypoglycemia. We could also demonstrate that the increase of MIF transcripts on hypoxic stress can be explained by a profound transcriptional activation of the MIF gene. In parallel to the increase of MIF transcripts, we observed a significant rise in extracellular MIF protein on angiogenic stimulation. The data of our preliminary study suggest that the up-regulation of MIF expression during hypoxic and hypoglycemic stress might play a critical role for the neovascularization of glial tumors.

aIr-1, a newly found locus on mouse chromosome 16 encoding a trans-acting activator factor for MHC class II gene expression.

RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.

Interleukin-1 receptor antagonist gene (IL-1RN) polymorphism is a predictive factor of clinical pregnancy after IVF.

BACKGROUND: Only 25% of IVF transfer cycles lead to a clinical pregnancy, calling for continued technical progress but also more in depth analysis of patients' individual characteristics. The interleukin-1 (IL-1) system and matrix metalloproteinases (MMPs) are strongly implicated in embryo implantation. The genes coding for IL-1Ra (gene symbol IL-1RN), IL-1beta, MMP2 and MMP9 bear functional polymorphisms. We analysed the maternal genetic profile at these polymorphic sites in IVF patients, to determine possible correlations with IVF outcome. METHODS: One hundred and sixty women undergoing an IVF cycle were enrolled and a buccal smear was obtained. The presence of IL-1RN variable number of tandem repeats and IL-1B + 3953, MMP2-1306 and MMP9-1562 single nucleotide substitutions were determined. Patients were divided into pregnancy failures (119), biochemical pregnancies (8) and clinical pregnancies (33). RESULTS: There was a 40% decrease in IL-1RN*2 allele frequency (P = 0.024) and a 45% decrease in IL-1RN*2 carrier status in the clinical pregnancy group as compared to the pregnancy failure group (P = 0.017). This decrease was still statistically significant after a multivariate logistic regression analysis. The likelihood of a clinical pregnancy was decreased accordingly in IL-1RN*2 carriers: odds ratio = 0.349, 95% confidence interval = 0.2-0.8, P = 0.017. The IL-1B, MMP2 and MMP9 polymorphisms showed no correlation with IVF outcome. CONCLUSIONS: IL-1RN*2 allele carriage is associated with a poor prognosis of achieving a pregnancy after IVF.

Non-viral gene therapy for GDNF production in RCS rat: the crucial role of the plasmid dose.

Glial cell line-derived neurotrophic factor (GDNF) is one of the candidate molecules among neurotrophic factors proposed for a potential treatment of retinitis pigmentosa (RP). It must be administered repeatedly or through sustained releasing systems to exert prolonged neuroprotective effects. In the dystrophic Royal College of Surgeon's (RCS) rat model of RP, we found that endogenous GDNF levels dropped during retinal degeneration time course, opening a therapeutic window for GDNF supplementation. We showed that after a single electrotransfer of 30 μg of GDNF-encoding plasmid in the rat ciliary muscle, GDNF was produced for at least 7 months. Morphometric, electroretinographic and optokinetic analyses highlighted that this continuous release of GDNF delayed photoreceptors (PRs) as well as retinal functions loss until at least 70 days of age in RCS rats. Unexpectedly, increasing the GDNF secretion level accelerated PR degeneration and the loss of electrophysiological responses. This is the first report: (i) demonstrating the efficacy of GDNF delivery through non-viral gene therapy in RP; (ii) establishing the efficacy of intravitreal administration of GDNF in RP associated with a mutation in the retinal pigment epithelium; and (iii) warning against potential toxic effects of GDNF within the eye/retina.

Regulation of neurotrophic factors and energy metabolism by antidepressants in astrocytes.

There is growing evidence that astrocytes are involved in the neuropathology of major depression. In particular, decreases in glial cell density observed in the cerebral cortex of individuals with major depressive disorder are accompanied by a reduction of several astrocytic markers suggesting that astrocyte dysfunction may contribute to the pathophysiology of major depression. In rodents, glial loss in the prefrontal cortex is sufficient to induce depressive-like behaviors and antidepressant treatment prevents the stress-induced reduction of astrocyte number in the hippocampus. Collectively, these data support the existence of a link between astrocyte loss or dysfunction, depressive-like behavior and antidepressant treatment. Astrocytes are increasingly recognized to play important roles in neuronal development, neurotransmission, synaptic plasticity and maintenance of brain homeostasis. It is also well established that astrocytes provide trophic, structural, and metabolic support to neurons. In this article, we review evidence that antidepressants regulate energy metabolism and neurotrophic factor expression with particular emphasis on studies in astrocytes. These observations support a role for astrocytes as new targets for antidepressants. The contribution of changes in astrocyte glucose metabolism and neurotrophic factor expression to the therapeutic effects of antidepressants remains to be established.

The T309G Murine Double Minute 2 Gene Polymorphism Is an Independent Prognostic Factor for Patients with Renal Cell Carcinoma.

The purpose of this study was to evaluate the association of the T309G MDM2 gene polymorphism with renal cell carcinoma (RCC) risk, pathology, and cancer-specific survival (CSS). T309G MDM2 was genotyped in 449 Caucasians, including 240 with RCC and 209 cancer-free controls. The T309G MDM2 genotype was TT in 174 (38.8%), GT in 214 (47.7%), and GG in 61 (13.6%) subjects, without any significant differences between cases and controls on both univariable (p=0.58) and multivariable logistic regression (each p>0.25). Furthermore, T309G MDM2 was not linked with T stage (p=0.75), N stage (p=0.37), M stage (p=0.94), grade (p=0.21), and subtype (p=0.55). There was, however, a statistically significant association of T309G MDM2 with CSS (p=0.022): patients with TT had significantly worse survival than GG/GT (p=0.009), while those with GT and GG had similar outcomes (p=0.92). The 5-year survival rate for patients with TT, GT, and GG was 69.5%, 84.5%, and 89.7%, respectively. On the multivariable analysis, T309G was identified as an independent prognostic factor. The T309G MDM2 polymorphism is an independent prognostic factor for patients with RCC, with the TT genotype being associated with worse prognosis. In this study, there were no significant associations with RCC risk and pathology.

Insulin-Like growth-factor 1 myocardial expression decreases in chronic alcohol consumption

Background Chronic alcohol ingestion may cause severe biochemical and pathophysiological derangements to skeletal muscle. Unfortunately, these alcohol-induced events may also prime skeletal muscle for worsened, delayed, or possibly incomplete repair following acute injury. As alcoholics may be at increased risk for skeletal muscle injury, our goals were to identify the effects of chronic alcohol ingestion on components of skeletal muscle regeneration. To accomplish this, age- and gender-matched C57Bl/6 mice were provided normal drinking water or water that contained 20% alcohol (v/v) for 1820 wk. Subgroups of mice were injected with a 1.2% barium chloride (BaCl2) solution into the tibialis anterior (TA) muscle to initiate degeneration and regeneration processes. Body weights and voluntary wheel running distances were recorded during the course of recovery. Muscles were harvested at 2, 7 or 14 days post-injection and assessed for markers of inflammation and oxidant stress, fiber cross-sectional areas, levels of growth and fibrotic factors, and fibrosis. Results Body weights of injured, alcohol-fed mice were reduced during the first week of recovery. These mice also ran significantly shorter distances over the two weeks following injury compared to uninjured, alcoholics. Injured TA muscles from alcohol-fed mice had increased TNFα and IL6 gene levels compared to controls 2 days after injury. Total protein oxidant stress and alterations to glutathione homeostasis were also evident at 7 and 14 days after injury. Ciliary neurotrophic factor (CNTF) induction was delayed in injured muscles from alcohol-fed mice which may explain, in part, why fiber cross-sectional area failed to normalize 14 days following injury. Gene levels of TGFβ1 were induced early following injury before normalizing in muscle from alcohol-fed mice compared to controls. However, TGFβ1 protein content was consistently elevated in injured muscle regardless of diet. Fibrosis was increased in injured, muscle from alcohol-fed mice at 7 and 14 days of recovery compared to injured controls. Conclusions Chronic alcohol ingestion appears to delay the normal regenerative response following significant skeletal muscle injury. This is evidenced by reduced cross-sectional areas of regenerated fibers, increased fibrosis, and altered temporal expression of well-described growth and fibrotic factors.

A functional microsatellite of the macrophage migration inhibitory factor gene associated with meningococcal disease.

Macrophage migration inhibitory factor (MIF) is an abundantly expressed proinflammatory cytokine playing a critical role in innate immunity and sepsis and other inflammatory diseases. We examined whether functional MIF gene polymorphisms (-794 CATT(5-8) microsatellite and -173 G/C SNP) were associated with the occurrence and outcome of meningococcal disease in children. The CATT(5) allele was associated with the probability of death predicted by the Pediatric Index of Mortality 2 (P=0.001), which increased in correlation with the CATT(5) copy number (P=0.04). The CATT(5) allele, but not the -173 G/C alleles, was also associated with the actual mortality from meningoccal sepsis [OR 2.72 (1.2-6.4), P=0.02]. A family-based association test (i.e., transmission disequilibrium test) performed in 240 trios with 1 afflicted offspring indicated that CATT(5) was a protective allele (P=0.02) for the occurrence of meningococcal disease. At baseline and after stimulation with Neisseria meningitidis in THP-1 monocytic cells or in a whole-blood assay, CATT(5) was found to be a low-expression MIF allele (P=0.005 and P=0.04 for transcriptional activity; P=0.09 and P=0.09 for MIF production). Taken together, these data suggest that polymorphisms of the MIF gene affecting MIF expression are associated with the occurrence, severity, and outcome of meningococcal disease in children.

Early adversity and 5-HTT-BDNF genes: new evidences of gene-environment interactions on depressive symptoms in a general population

Adverse childhood experiences have been described as one of the major environmental risk factors for depressive disorder. Likewise, the deleterious impact of early traumatic experiences on depression seems to be moderated by individual genetic variability. Serotonin transporter (5-HTT) and the Brain-Derived Neurotrophic Factor (BDNF) seem to modulate the effect of childhood adversity on adult depression, although inconsistencies across studies have been found. Moreover, the GxE interaction concerning the different types of childhood adversity remains poorly understood. The aim of this study is to analyse the putative interaction between the 5-HTT gene (5-HTTLPR polymorphism), BDNF gene (Val66Met polymorphism) and childhood adversity in accounting for adult depressive symptoms.

Signalling by neurotrophins and hepatocyte growth factor regulates axon morphogenesis by differential β-catenin phosphorylation

Tyrosine phosphorylation of ß-catenin, a component of adhesion complexes and the Wnt pathway, affects cell adhesion, migration and gene transcription. By reducing ßcatenin availability using shRNA-mediated gene silencing or expression of intracellular N-cadherin, we show that ß-catenin is required for axon growth downstream of Brain Derived Neurotrophic Factor (BDNF) and Hepatocyte Growth Factor (HGF) signalling. We demonstrate that receptor tyrosine kinases (RTK) Trk and Met interact with and phosphorylate ß-catenin. Neurotrophins (NT) stimulation of Trk receptors results in phosphorylation of ß-catenin at residue Y654 and increased axon growth and branching. Conversely, pharmacological inhibition of Trk or a Y654F mutant blocks these effects. ß-catenin phospho(P)-Y654 colocalizes with the cytoskeleton at growth cones. However, HGF that also increases axon growth and branching, induces ß-catenin phosphorylation at Y142 and a nuclear localization. Interestingly, dominant negative ΔN-TCF4 abolishes the effects of HGF in axon growth and branching, but not of NT. We conclude that NT and HGF signalling differentially phosphorylate ß-catenin, targeting ß-catenin to distinct compartments to regulate axon morphogenesis by TCF4-transcription-dependent and independent mechanisms. These results place ß-catenin downstream of growth factor/RTK signalling in axon differentiation.

Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.