Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

Background: Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods: We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area - BA22p) identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results: Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6) and glial fibrillary acidic protein (GFAP) were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion: Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

Dysregulation of renal transient receptor potential melastatin 6/7 but not paracellin-1 in aldosterone-induced hypertension and kidney damage in a model of hereditary hypomagnesemia

Rationale Hyperaldosteronism, important in hypertension, is associated with electrolyte alterations, including hypomagnesemia, through unknown mechanisms. Objective To test whether aldosterone influences renal Mg(2+) transporters, (transient receptor potential melastatin (TRPM) 6, TRPM7, paracellin-1) leading to hypomagnesemia, hypertension and target organ damage and whether in a background of magnesium deficiency, this is exaggerated. Methods and results Aldosterone effects in mice selectively bred for high-normal (MgH) or low (MgL) intracellular Mg(2+) were studied. Male MgH and MgL mice received aldosterone (350 mu g/kg per day, 3 weeks). SBP was elevated in MgL. Aldosterone increased blood pressure and albuminuria and increased urinary Mg(2+) concentration in MgH and MgL, with greater effects in MgL. Activity of renal TRPM6 and TRPM7 was lower in vehicle-treated MgL than MgH. Aldosterone increased activity of TRPM6 in MgH and inhibited activity in MgL. TRPM7 and paracellin-1 were unaffected by aldosterone. Aldosterone-induced albuminuria in MgL was associated with increased renal fibrosis, increased oxidative stress, activation of mitogen-activated protein kinases and nuclear factor-NF-kappa B and podocyte injury. Mg(2+) supplementation (0.75% Mg(2+)) in aldosterone-treated MgL normalized plasma Mg(2+), increased TRPM6 activity and ameliorated hypertension and renal injury. Hence, in a model of inherited hypomagnesemia, TRPM6 and TRPM7, but not paracellin-1, are downregulated. Aldosterone further decreased TRPM6 activity in hypomagnesemic mice, a phenomenon associated with hypertension and kidney damage. Such effects were prevented by Mg(2+) supplementation. Conclusion Amplified target organ damage in aldosterone-induced hypertension in hypomagnesemic conditions is associated with dysfunctional Mg(2+)-sensitive renal TRPM6 channels. Novel mechanisms for renal effects of aldosterone and insights into putative beneficial actions of Mg(2+), particularly in hyperaldosteronism, are identified. J Hypertens 29: 1400-1410 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Cytokines and dysregulation of the immune response in human malaria

The dysregulation of the immune response by malaria parasite has been considered as a possible constraint to the effectiveness of malaria vaccination. In spite of the important role interleukin-I (IL-1) in malaria are lacking. We found that only 2 out of 35 subjectswith acute malaria showed increased levels of serum IL-1 alpha by enzyme immunoassay. To assess whether IL-1 could interfere with T- lymphocyte responses, blood mononuclear cells from patients infected with Plasmodium falciparum, P. vivax, or healthy subjects were cultured with phytohemagglutinin, and lymphocyte proliferation measured 72h later by 3H-thymidine incorporation. Our data showed that T-lymphocyte responses are depressed both in P. falciparum (10,500 ñ 2,900) and P. vivax malaria (13,000 ñ 3,300), as compared to that of healthy individuals (27,000 ñ 3,000). Addition of IL-1 partially reserved depression of malaria lymphocytes, but had no effect on normal cells. On the other hand, T-lymphocytes from malaria infected-subjects presented a minimal decrease in proliferation, when cultured in the presence of exogenous PGE2. These data indicate the occurrence of two defects of immunoregulation in malaria: a deficiency of IL-1 production by monocytes/macrophages, and an increased resistance of lymphocytes to the antiproliferative effect of PGE2.

Developmental critical period in gentic redox dysregulation: animal and human studies in schizophrenia

Converging evidence favors an abnormal susceptibility to oxidative stress in schizophrenia. Decreased levels of glutathione (GSH), the major cellular antioxidant and redox regulator, was observed in cerebrospinal-fluid and prefrontal cortex of patients. Importantly, abnormal GSH synthesis of genetic origin was observed: Two case-control studies showed an association with a GAG trinucleotide repeat (TNR) polymorphism in the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) catalytic subunit (GCLC) gene. The most common TNR genotype 7/7 was more frequent in controls, whereas the rarest TNR genotype 8/8 was three times more frequent in patients. The disease associated genotypes (35% of patients) correlated with decreased GCLC protein, GCL activity and GSH content. Similar GSH system anomalies were observed in early psychosis patients. Such redox dysregulation combined with environmental stressors at specific developmental stages could underlie structural and functional connectivity anomalies. In pharmacological and knock-out (KO) models, GSH deficit induces anomalies analogous to those reported in patients. (a) morphology: spine density and GABA-parvalbumine immunoreactivity (PV-I) were decreased in anterior cingulate cortex. KO mice showed delayed cortical PV-I at PD10. This effect is exacerbated in mice with increased DA from PD5-10. KO mice exhibit cortical impairment in myelin and perineuronal net known to modulate PV connectivity. (b) physiology: In cultured neurons, NMDA response are depressed by D2 activation. In hippocampus, NMDA-dependent synaptic plasticity is impaired and kainate induced g-oscillations are reduced in parallel to PV-I. (c) cognition: low GSH models show increased sensitivity to stress, hyperactivity, abnormal object recognition, olfactory integration and social behavior. In a clinical study, GSH precursor N-acetyl cysteine (NAC) as add on therapy, improves the negative symptoms and decreases the side effects of antipsychotics. In an auditory oddball paradigm, NAC improves the mismatched negativity, an evoked potential related to pre-attention and to NMDA receptors function. In summary, clinical and experimental evidence converge to demonstrate that a genetically induced dysregulation of GSH synthesis combined with environmental insults in early development represent a major risk factor contributing to the development of schizophrenia Conclusion Based on these data, we proposed a model for PSIP1 promoter activity involving a complex interplay between yet undefined regulatory elements to modulate gene expression.

Genetic and epigenetic analysis of SSAT gene dysregulation in suicidal behavior.

It has recently been proposed that the SSAT gene plays a role in the predisposition to suicidal behavior. SSAT expression was found to be down-regulated in the brain of suicide completers. In addition, a single nucleotide polymorphism (SNP) rs6526342 was associated both with variation in SSAT expression and with suicidal behavior. In this study, we aimed to characterize the relationship between SSAT dysregulation and suicide behavior. To this end, we measured SSAT expression levels in the ventral prefrontal cortex (VPFC) of suicide completers (n = 20) and controls (n = 20) and found them to be significantly down-regulated in suicide victims (P = 0.007). To identify the basis of the regulation of SSAT expression, we performed an association analysis of 309 SNPs with SSAT transcript levels in 53 lymphoblastoid cell lines from the CEPH collection. We then examined the methylation status of the SSAT promoter region in males and females suicide completers and control subjects whose SSAT brain expression had been measured. We found no evidence to support a role for SNPs in controlling the level of SSAT expression. SSAT promoter methylation levels were not different between suicide completers and controls and did not correlate with SSAT expression levels. In addition, we found no indication of a genetic association between suicidal behavior and SNPs located within the SSAT gene. Our study provides new results which show that dysregulation of SSAT expression does play a role in suicide behavior. However, our data do not support any association between rs6526342 and variation in SSAT expression or suicidal behavior.

Redox dysregulation in schizophrenia : implication for oligodendrocyte maturation and structural connectivity

Schizophrenia, which results from an interaction between gene and environmental factors, is a psychiatric disorder characterized by reality distortion. The clinical symptoms, which are generally diagnosed in late adolescence or early adulthood, partly derive from altered brain connectivity especially in prefrontal cortex. Disruption of neuronal networks implies oligodendrocyte and myelin abnormalities in schizophrenia pathophysiology. The mechanisms of these impairments are still unclear. Converging evidences indicate a role of redox dysregulation, generated by an imbalance between pro-oxidants and antioxidant defense mechanisms, in the development of schizophrenia pathophysiology. In particular, genetic and biochemical data indicate impaired synthesis of glutathione, the main cellular antioxidant and redox regulator. As oligodendrocyte maturation is dependent on redox state, we evaluated whether abnormal redox control could contribute to oligodendrocyte and myelin impairments in schizophrenia. We found that glutathione in prefrontal cortex of early psychosis patients and control subjects positively correlated with white matter integrity. We then further explored the interplay between glutathione and myelin using a translational approach. Our data showed that in mice with genetically impaired glutathione synthesis, oligodendrocyte late maturation as well as myelination was delayed in the anterior cingulate cortex. Specifically, oligodendrocyte number and myelin levels were lowered at peripubertal age, coincident in time with the peak of myelin- related gene expression during normal brain development. These data suggest that early adolescence is a vulnerable developmental period during which an adequate redox control is required for oligodendrocyte maturation and active myelination process. Consistently, oxidative stress mediated by psychosocial stress also delayed myelination in peripubertal mice. At cellular levels, impaired glutathione synthesis altered oligodendrocyte development at several levels. Using oligodendrocyte progenitor cells cultures, our data showed that glutathione deficiency was associated with (i) cell cycle arrest and a reduction in oligodendrocyte proliferation, and (ii) an impairment in oligodendrocyte maturation. Abnormal oligodendrocyte proliferation was mediated by upregulation of Fyn kinase activity. Consistently, under oxidative stress conditions, we observed abnormal regulation of Fyn kinase in fibroblasts of patients deficient in glutathione synthesis. Together, our data support that a redox dysregulation due to glutathione deficit could underlie myelination impairment in schizophrenia, possibly mediated by dysregulated Fyn pathway. Better characterization of Fyn mechanisms would pave the way towards new drug targets. -- La schizophrénie est une maladie psychiatrique qui se définit par une distorsion de la perception de la réalité. Les symptômes cliniques sont généralement diagnostiqués durant l'adolescence ou au début de l'âge adulte et proviennent de troubles de la connectivité, principalement au niveau du cortex préfrontal. Les dysfonctionnements des réseaux neuronaux impliquent des anomalies au niveau des oligodendrocytes et de la myéline dans la pathophysiologie de la schizophrénie. Les mécanismes responsables des ces altérations restent encore mal compris. Dans le développement de la schizophrénie, des évidences mettent en avant un rôle de la dérégulation rédox, traduit par un déséquilibre entre facteurs pro-oxydants et défenses antioxydantes. Des données génétiques et biochimiques indiquent notamment un défaut de la synthèse du glutathion, le principal antioxydant et rédox régulateur des cellules. Etant donné que la maturation des oligodendrocytes est dépendante de l'état rédox, nous avons regardé si une dérégulation rédox contribue aux anomalies de la myéline dans le cadre de la schizophrénie. Dans le cortex préfrontal des sujets contrôles et des patients en phase précoce de psychose, nous avons montré que le glutathion était positivement associé à l'intégrité de matière blanche. Afin d'explorer plus en détail la relation entre le glutathion et la myéline, nous avons mené une étude translationnelle. Nos résultats ont montré que des souris ayant un déficit de la synthèse du glutathion présentaient un retard dans les processus de maturation des oligodendrocytes et de la myélinisation dans le cortex cingulaire antérieure. Plus précisément, le nombre d'oligodendrocytes et le taux de myéline étaient uniquement diminués durant la période péripubertaire. Cette même période correspond au pic de l'expression des gènes en lien avec la myéline. Ces données soulignent le fait que l'adolescence est une période du développement particulièrement sensible durant laquelle un contrôle adéquat de l'état rédox est nécessaire aux processus de maturation des oligodendrocytes et de myélinisation. Ceci est en accord avec la diminution de myéline observée suite à un stress oxydatif généré par un stress psychosocial. Au niveau cellulaire, un déficit du glutathion affecte le développement des oligodendrocytes à différents stades. En effet, dans des cultures de progéniteurs d'oligodendrocytes, nos résultats montrent qu'une réduction du taux de glutathion était associée à (i) un arrêt du cycle cellulaire ainsi qu'une diminution de la prolifération des oligodendrocytes, et à (ii) des dysfonctionnements de la maturation des oligodendrocytes. Par ailleurs, au niveau moléculaire, les perturbations de la prolifération étaient générées par une augmentation de l'activité de la kinase Fyn. Ceci est en accord avec la dérégulation de Fyn observée dans les fibroblastes de patients ayant une déficience en synthèse du glutathion en condition de stress oxydatif. Les résultats de cette thèse soulignent qu'une dérégulation rédox induite par un déficit en glutathion peut contribuer aux anomalies des oligodendrocytes et de la myéline via le dysfonctionnement des voies de signalisation Fyn. Une recherche plus avancée de l'implication de Fyn dans la maladie pourrait ouvrir la voie à de nouvelles cibles thérapeutiques.

Genetic dysregulation of glutathione synthesis predicts alteration of plasma thiol redox status in schizophrenia.

Abstract Genetic studies have shown an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL), the key enzyme for glutathione (GSH) synthesis. The present study was aimed at analyzing the influence of a GSH dysregulation of genetic origin on plasma thiols (total cysteine, homocysteine, and cysteine-glycine) and other free amino acid levels as well as fibroblast cultures GSH levels. Plasma thiols levels were also compared between patients and controls. As compared with patients with a low-risk GCLC GAG TNR genotype, patients with a high-risk genotype, having an impaired GSH synthesis, displayed a decrease of fibroblast GSH and plasma total cysteine levels, and an increase of the oxidized form of cysteine (cystine) content. Increased levels of plasma free serine, glutamine, citrulline, and arginine were also observed in the high-risk genotype. Taken together, the high-risk genotypes were associated with a subgroup of schizophrenia characterized by altered plasma thiols and free amino acid levels that reflect a dysregulation of redox control and an increased susceptibility to oxidative stress. This altered pattern potentially contributes to the development of a biomarker profile useful for early diagnosis and monitoring the effectiveness of novel drugs targeting redox dysregulation in schizophrenia. Antioxid. Redox Signal. 15, 2003-2010.