Oxygen exchange and energy metabolism in erythrocytes of Rett syndrome and their relationships with respiratory alterations.

Rett syndrome (RTT) is a neurodevelopmental disorder, mainly affecting females, which is associated to a mutation on the methyl-CpG-binding protein 2 gene. In the pathogenesis and progression of classic RTT, red blood cell (RBC) morphology has been shown to be an important biosensor for redox imbalance and chronic hypoxemia. Here we have evaluated the impact of oxidation and redox imbalance on several functional properties of RTT erythrocytes. In particular, we report for the first time a stopped-flow measurement of the kinetics of oxygen release by RBCs and the analysis of the intrinsic affinity of the hemoglobin (Hb). According to our experimental approach, RBCs from RTT patients do not show any intrinsic difference with respect to those from healthy controls neither in Hb's oxygen-binding affinity nor in O2 exchange processes at 37 °C. Therefore, these factors do not contribute to the observed alteration of the respiratory function in RTT patients. Moreover, the energy metabolism of RBCs, from both RTT patients and controls, was evaluated by ion-pairing HPLC method and related to the level of malondialdehyde and to the oxidative radical scavenging capacity of red cells. Results have clearly confirmed significant alterations in antioxidant defense capability, adding important informations concerning the high-energy compound levels in RBCs of RTT subjects, underlying possible correlations with inflammatory tissue alterations.

Inflammatory protein response in CDKL5-Rett syndrome: evidence of a subclinical smouldering inflammation.

BACKGROUND: Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (CDKL5-RTT). A role for the acute-phase response (APR) is emerging in typical RTT caused by methyl-CpG-binding protein 2 gene mutations (MECP2-RTT). No information is, to date, available on the inflammatory protein response in CDKL5-RTT. We evaluated, for the first time, the APR protein response in CDKL5-RTT. METHODS: Protein patterns in albumin- and IgG-depleted plasma proteome from CDKL5-RTT patients were evaluated by two-dimensional gel electrophoresis/mass spectrometry. The resulting data were related to circulating cytokines and compared to healthy controls or MECP2-RTT patients. The effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) were evaluated. RESULTS: CDKL5-RTT mutations resulted in a subclinical attenuated inflammation, specifically characterized by an overexpression of the complement component C3 and CD5 antigen-like, both strictly related to the inflammatory response. Cytokine dysregulation featuring a bulk increase of anti-inflammatory cytokines, predominantly IL-10, could explain the unchanged erythrocyte sedimentation rate and atypical features of inflammation in CDKL5-RTT. Omega-3 PUFAs were able to counterbalance the pro-inflammatory status. CONCLUSION: For the first time, we revealed a subclinical smouldering inflammation pattern in CDKL5-RTT consisting in the coexistence of an atypical APR coupled with a dysregulated cytokine response.

Persistent Unresolved Inflammation in the Mecp2-308 Female Mutated Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is a rare neurodevelopmental disorder usually caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). Several Mecp2 mutant mouse lines have been developed recapitulating part of the clinical features. In particular, Mecp2-308 female heterozygous mice, bearing a truncating mutation, are a validated model of the disease. While recent data suggest a role for inflammation in RTT, little information on the inflammatory status in murine models of the disease is available. Here, we investigated the inflammatory status by proteomic 2-DE/MALDI-ToF/ToF analyses in symptomatic Mecp2-308 female mice. Ten differentially expressed proteins were evidenced in the Mecp2-308 mutated plasma proteome. In particular, 5 positive acute-phase response (APR) proteins increased (i.e., kininogen-1, alpha-fetoprotein, mannose-binding protein C, alpha-1-antitrypsin, and alpha-2-macroglobulin), and 3 negative APR reactants were decreased (i.e., serotransferrin, albumin, and apolipoprotein A1). CD5 antigen-like and vitamin D-binding protein, two proteins strictly related to inflammation, were also changed. These results indicate for the first time a persistent unresolved inflammation of unknown origin in the Mecp2-308 mouse model.

Red blood cells in Rett syndrome: oxidative stress, morphological changes and altered membrane organization.

In this review, we summarize the current evidence on the erythrocyte as a previously unrecognized target cell in Rett syndrome, a rare (1:10 000 females) and devastating neurodevelopmental disorder caused by loss-of-function mutations in a single gene (i.e. MeCP2, CDKL5, or rarely FOXG1). In particular, we focus on morphological changes, membrane oxidative damage, altered membrane fatty acid profile, and aberrant skeletal organization in erythrocytes from patients with typical Rett syndrome and MeCP2 gene mutations. The beneficial effects of ω-3 polyunsaturated fatty acids (PUFAs) are also summarized for this condition to be considered as a 'model' condition for autism spectrum disorders.

Subclinical inflammatory status in Rett syndrome.

Inflammation has been advocated as a possible common central mechanism for developmental cognitive impairment. Rett syndrome (RTT) is a devastating neurodevelopmental disorder, mainly caused by de novo loss-of-function mutations in the gene encoding MeCP2. Here, we investigated plasma acute phase response (APR) in stage II (i.e., "pseudo-autistic") RTT patients by routine haematology/clinical chemistry and proteomic 2-DE/MALDI-TOF analyses as a function of four major MECP2 gene mutation types (R306C, T158M, R168X, and large deletions). Elevated erythrocyte sedimentation rate values (median 33.0 mm/h versus 8.0 mm/h, P < 0.0001) were detectable in RTT, whereas C-reactive protein levels were unchanged (P = 0.63). The 2-DE analysis identified significant changes for a total of 17 proteins, the majority of which were categorized as APR proteins, either positive (n = 6 spots) or negative (n = 9 spots), and to a lesser extent as proteins involved in the immune system (n = 2 spots), with some proteins having overlapping functions on metabolism (n = 7 spots). The number of protein changes was proportional to the severity of the mutation. Our findings reveal for the first time the presence of a subclinical chronic inflammatory status related to the "pseudo-autistic" phase of RTT, which is related to the severity carried by the MECP2 gene mutation.

Inflammatory lung disease in Rett syndrome.

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly linked to mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Respiratory dysfunction, historically credited to brainstem immaturity, represents a major challenge in RTT. Our aim was to characterize the relationships between pulmonary gas exchange abnormality (GEA), upper airway obstruction, and redox status in patients with typical RTT (n = 228) and to examine lung histology in a Mecp2-null mouse model of the disease. GEA was detectable in ~80% (184/228) of patients versus ~18% of healthy controls, with "high" (39.8%) and "low" (34.8%) patterns dominating over "mixed" (19.6%) and "simple mismatch" (5.9%) types. Increased plasma levels of non-protein-bound iron (NPBI), F2-isoprostanes (F2-IsoPs), intraerythrocyte NPBI (IE-NPBI), and reduced and oxidized glutathione (i.e., GSH and GSSG) were evidenced in RTT with consequently decreased GSH/GSSG ratios. Apnea frequency/severity was positively correlated with IE-NPBI, F2-IsoPs, and GSSG and negatively with GSH/GSSG ratio. A diffuse inflammatory infiltrate of the terminal bronchioles and alveoli was evidenced in half of the examined Mecp2-mutant mice, well fitting with the radiological findings previously observed in RTT patients. Our findings indicate that GEA is a key feature of RTT and that terminal bronchioles are a likely major target of the disease.

Genes related to mitochondrial functions, protein degradation, and chromatin folding are differentially expressed in lymphomonocytes of Rett syndrome patients.

Rett syndrome (RTT) is mainly caused by mutations in the X-linked methyl-CpG binding protein (MeCP2) gene. By binding to methylated promoters on CpG islands, MeCP2 protein is able to modulate several genes and important cellular pathways. Therefore, mutations in MeCP2 can seriously affect the cellular phenotype. Today, the pathways that MeCP2 mutations are able to affect in RTT are not clear yet. The aim of our study was to investigate the gene expression profiles in peripheral blood lymphomonocytes (PBMC) isolated from RTT patients to try to evidence new genes and new pathways that are involved in RTT pathophysiology. LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses on microarray data from 12 RTT patients and 7 control subjects identified 482 genes modulated in RTT, of which 430 were upregulated and 52 were downregulated. Functional clustering of a total of 146 genes in RTT identified key biological pathways related to mitochondrial function and organization, cellular ubiquitination and proteosome degradation, RNA processing, and chromatin folding. Our microarray data reveal an overexpression of genes involved in ATP synthesis suggesting altered energy requirement that parallels with increased activities of protein degradation. In conclusion, these findings suggest that mitochondrial-ATP-proteasome functions are likely to be involved in RTT clinical features.

Erythrocyte shape abnormalities, membrane oxidative damage, and ß-actin alterations: an unrecognized triad in classical autism.

Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. Although red blood cells (RBCs) membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, RBCs shape in autism. Here, we investigated RBC morphology by scanning electron microscopy in patients with classical autism, that is, the predominant ASDs phenotype (age range: 6-26 years), nonautistic neurodevelopmental disorders (i.e., "positive controls"), and healthy controls (i.e., "negative controls"). A high percentage of altered RBCs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. The RBCs altered morphology in autistic subjects was related to increased erythrocyte membrane F2-isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membrane ß-actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and ß-actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.

A plasma proteomic approach in Rett syndrome: classical versus preserved speech variant.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Although over 200 mutations types have been identified so far, nine of which the most frequent ones. A wide phenotypical heterogeneity is a well-known feature of the disease, with different clinical presentations, including the classical form and the preserved speech variant (PSV). Aim of the study was to unveil possible relationships between plasma proteome and phenotypic expression in two cases of familial RTT represented by two pairs of sisters, harbor the same MECP2 gene mutation while being dramatically discrepant in phenotype, that is, classical RTT versus PSV. Plasma proteome was analysed by 2-DE/MALDI-TOF MS. A significant overexpression of six proteins in the classical sisters was detected as compared to the PSV siblings. A total of five out of six (i.e., 83.3%) of the overexpressed proteins were well-known acute phase response (APR) proteins, including alpha-1-microglobulin, haptoglobin, fibrinogen beta chain, alpha-1-antitrypsin, and complement C3. Therefore, the examined RTT siblings pairs proved to be an important benchmark model to test the molecular basis of phenotypical expression variability and to identify potential therapeutic targets of the disease.

Effects of ω-3 polyunsaturated fatty acids on plasma proteome in Rett syndrome.

The mechanism of action of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) is only partially known. Prior reports suggest a partial rescue of clinical symptoms and oxidative stress (OS) alterations following ω -3 PUFAs supplementation in patients with Rett syndrome (RTT), a devastating neurodevelopmental disorder with transient autistic features, affecting almost exclusively females and mainly caused by sporadic mutations in the gene encoding the methyl CpG binding protein 2 (MeCP2) protein. Here, we tested the hypothesis that ω-3 PUFAs may modify the plasma proteome profile in typical RTT patients with MECP2 mutations and classic phenotype. A total of 24 RTT girls at different clinical stages were supplemented with ω-3 PUFAs as fish oil for 12 months and compared to matched healthy controls. The expression of 16 proteins, mainly related to acute phase response (APR), was changed at the baseline in the untreated patients. Following ω-3 PUFAs supplementation, the detected APR was partially rescued, with the expression of 10 out of 16 (62%) proteins being normalized. ω-3 PUFAs have a major impact on the modulation of the APR in RTT, thus providing new insights into the role of inflammation in autistic disorders and paving the way for novel therapeutic strategies.

Analysis of aqueous humour proteins in patients with retinoblastoma.

BACKGROUND: To investigate aqueous humour protein composition from retinoblastoma patients. DESIGN: Prospective, hospital-based study. PARTICIPANTS: Eighteen retinoblastoma patients (Reese-Ellsworth stage V or ABC classification group E RB) undergoing ocular enucleation, and 10 normal subjects undergoing cataract surgery. Five of 18 patients presented with associated secondary glaucoma whereas 13 had no secondary glaucoma; 5 of 13 patients with no secondary glaucoma received chemotherapeutical treatment with melphalan. METHODS: Aqueous humour samples were collected by limbal paracentesis of the anterior chamber after ocular enucleation in patients and after the stab peripheral corneal incision in controls. Total protein concentration according to Bradford method and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the samples were performed. MAIN OUTCOME MEASURE: Aqueous humour protein concentration. RESULTS: Aqueous humour protein concentration was significantly higher in retinoblastoma patients than controls (P < 0.01); patients with secondary glaucoma presented the highest values (P < 0.05 vs. controls); patients treated with melphalan presented a significant decrease (P < 0.01) versus non-treated; controls did not significantly differ from treated patients. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis pattern in retinoblastoma patients who did not receive any treatment was very different either from treated or from controls. CONCLUSION: This study represents a preliminary step towards a more accurate two dimensional electrophoresis (2DE) pattern, which will be combined with mass spectrometry analysis to clarify the potential role of specific proteins in tumour development and progression; although these results suggest that aqueous humour protein pattern in retinoblastoma is characteristic, several aspects of the study are still under investigation.

Effects of Mucuna pruriens protease inhibitors on Echis carinatus venom.

The medicinal plant Mucuna pruriens, with reputed anti-snake venom properties has been reported to contain a kunitz-type trypsin inhibitor. This study was undertaken to further evaluate the protease inhibitory potential of gpMuc, a multiform glycoprotein, and other protein fractions from M. pruriens seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease-recognizing epitope on one of its isoforms. The whole protein extract of M. pruriens seeds inhibited prothrombin activation by ecarin and whole E. carinatus venom, and also thrombin-like activity using chromogenic assay.

Erythrocyte sedimentation rate measurement by VES Matic Cube 80 in relation to inflammation plasma proteins.

Westergren method is considered as the reference procedure to measure Erythrocyte Sedimentation Rate (ESR) by the International Council for Standardization in Haematology. However, a closed automated method, VES Matic Cube 80 (DIESSE S.p.A., Siena, Italy), has been introduced as a new ESR measurement instrument. In this article, we report two different studies: first, we compared the two methods (Westergren and VES Matic Cube 80) and second, we correlated the inflammatory state of 248 patients with their ESR values. Total protein, albumin, C-reactive protein, and other inflammatory proteins were detected in each sample. The results obtained using VES Matic Cube 80 demonstrated a good correlation with those obtained using the Westergren method (Ordinary linear regression: y=0.955x-0.205, r(2) =0.816, P<0.05; Passing-Bablock regression equation: y=0.9153x-0.5763; Bland-Altman analysis: bias 1.2; limits of agreement -17.4-19.9) and with the inflammatory protein levels (CRP: r=0.554 and r=0.498 and Fibrinogen: r=0.699 and r=0.663 for Ves Matic Cube 80 and Westergren, respectively), supporting the hypothesis that VES Matic Cube 80 offers a fast and safe ESR determination, ensuring precision and a very good correlation with the reference method.

In vitro effects of Echis carinatus venom on the human plasma proteome.

Echis carinatus venom (EV) is a complex mixture of toxins that contribute to its lethality. EV proteolytic activity was analyzed by zymography, chromogenic assays, and SDS-PAGE. To understand the molecular mechanism of the envenomation, we investigated the in vitro effect of EV on human plasma proteins. We looked for EV protein substrates and their proteolytic fragments. We analyzed EV proteolytic activity on standard proteins such as prothrombin or fibrinogen. To set up the optimal EV:plasma protein ratio conditions, plasma was incubated with EV (treated plasma), depleted of abundant proteins, and subjected to SDS-PAGE. Samples from control and treated plasma were also analyzed by 2-DE/MALDI-TOF MS, leading to the identification of four classes of plasma proteins cleaved by EV: proteases, protease inhibitors, binding proteins, and transporters. EV mainly proteolyzes entire proteins but can also act on physiological fragments. In summary, the physiological effects of EV proteases involve other important processes in addition to blood coagulation; complement activation and hemoglobin metabolism are also affected. In particular, the cleavage of protease inhibitors appears to be the mechanism through which the venom neutralizes the body's defenses.

Brain protein changes in Mecp2 mouse mutant models: Effects on disease progression of Mecp2 brain specific gene reactivation.

Rett syndrome (RTT) is a leading cause of severe intellectual disability in females, caused by de novo loss-of function mutations in the X-linked methyl-CpG binding protein 2 (MECP2). To better investigate RTT disease progression/pathogenesis animal models of Mecp2 deficiency have been developed. Here, Mecp2 mouse models are employed to investigate the role of protein patterns in RTT. A proteome analysis was carried out in brain tissue from i) Mecp2 deficient mice at the pre-symptomatic and symptomatic stages and, ii) mice in which the disease phenotype was reversed by Mecp2 reactivation. Several proteins were shown to be differentially expressed in the pre-symptomatic (n = 18) and symptomatic (n = 20) mice. Mecp2 brain reactivated mice showed wild-type comparable levels of expression for twelve proteins, mainly related to proteostasis (n = 4) and energy metabolic pathways (n = 4). The remaining ones were found to be involved in redox homeostasis (n = 2), nitric oxide regulation (n = 1), neurodevelopment (n = 1). Ten out of twelve proteins were newly linked to Mecp2 deficiency. Our study sheds light on the relevance of the protein-regulation of main physiological process in the complex mechanisms leading from Mecp2 mutation to the RTT clinical phenotype. SIGNIFICANCE: We performed a proteomic study of a Mecp2stop/y mouse model for Rett syndrome (RTT) at the pre-symptomatic and symptomatic Mecp2 deficient mice stage and for the brain specific reactivated Mecp2 model. Our results reveal major protein expression changes pointing out to defects in proteostasis or energy metabolic pathways other than, to a lesser extent, in redox homeostasis, nitric oxide regulation or neurodevelopment. The Mecp2 mouse rescued model provides the possibility to select target proteins more susceptible to the Mecp2 gene mutation, potential and promising therapeutical targets.

Increased isoprostanoid levels in brain from murine model of Krabbe disease - Relevance of isoprostanes, dihomo-isoprostanes and neuroprostanes to disease severity.

Krabbe disease (KD) is a rare and devastating pediatric leukodystrophy caused by mutations in the galactocerebrosidase (GALC) gene. The disease leads to impaired myelin formation and extensive myelin damage in the brain. Oxidative stress is implicated in the pathogenesis of KD but insofar few information is available. The gray and white matter of the brain are rich in docosahexaenoic acid and adrenic acid respectively and under non-enzymatic oxidative stress, release isoprostanoids, i.e. F4-neuroprostanes (F4-NeuroPs) and F2-dihomo-isoprostanes (F2-dihomo-IsoPs). In this study, the formation of isoprostanoids in brain tissue was investigated in a well-established KD mouse model (twitcher) that recapitulates the human pathology. According to the genotype determinations, three groups of mice were selected: wild-type control mice (n = 13), heterozygotes mice (carriers of GALC mutations, n = 14) and homozygous twitcher mice (n = 13). Measurement of F2-dihomo-IsoP and F4-NeuroP levels were performed on whole brain tissue obtained at day 15 and day 35 of the life cycle. Brain isoprostanoid levels were significantly higher in the twitcher mice compared to the heterozygous and wild-type control mice. However, F2-dihomo-IsoP and F4-NeuroP levels did not differ in brain of day 15 compared to day 35 of the heterozygote mice. Interestingly, isoprostanoid levels were proportionally enhanced with disease severity (F2-dihomo-IsoPs, rho = 0.54; F4-NeuroPs, rho = 0.581; P values ≤ 0.05; n = 13). Our findings are the first to show the key role of polyunsaturated fatty acid oxidative damage to brain grey and white matter in the pathogenesis and progression of KD. This shed new insights on the biochemical indexes of KD progression, and potentially provide information for novel therapeutic targets.

Isoprostanoids in Clinical and Experimental Neurological Disease Models.

Isoprostanoids are a large family of compounds derived from non-enzymatic oxidation of polyunsaturated fatty acids (PUFAs). Unlike other oxidative stress biomarkers, they provide unique information on the precursor of the targeted PUFA. Although they were discovered about a quarter of century ago, the knowledge on the role of key isoprostanoids in the pathogenesis of experimental and human disease models remains limited. This is mainly due to the limited availability of highly purified molecules to be used as a reference standard in the identification of biological samples. The accurate knowledge on their biological relevance is the critical step that could be translated from some mere technical/industrial advances into a reliable biological disease marker which is helpful in deciphering the oxidative stress puzzle related to neurological disorders. Recent research indicates the value of isoprostanoids in predicting the clinical presentation and evolution of the neurological diseases. This review focuses on the relevance of isoprostanoids as mediators and potential biomarkers in neurological diseases, a heterogeneous family ranging from rare brain diseases to major health conditions that could have worldwide socioeconomic impact in the health sector. The current challenge is to identify the preferential biochemical pathways that actually follow the oxidative reactions in the neurological diseases and the consequence of the specific isoprostanes in the underlying pathogenic mechanisms.

Relevance of 4-F4t-neuroprostane and 10-F4t-neuroprostane to neurological diseases.

F4-neuroprostanes (F4-NeuroPs) are non-enzymatic oxidized products derived from docosahexaenoic acid (DHA) and are suggested to be oxidative damage biomarkers of neurological diseases. However, 128 isomers can be formed from DHA oxidation and among them, 4(RS)-4-F4t-NeuroP (4-F4t-NeuroP) and 10(RS)-10-F4t-NeuroP (10-F4t-NeuroP) are the most studied. Here, we report the identification and the clinical relevance of 4-F4t-NeuroP and 10-F4t-NeuroP in plasma of four different neurological diseases, including multiple sclerosis (MS), autism spectrum disorders (ASD), Rett syndrome (RTT), and Down syndrome (DS). The identification and the optimization of the method were carried out by gas chromatography/negative-ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) using chemically synthesized 4-F4t-NeuroP and 10-F4t-NeuroP standards and in oxidized DHA liposome. Both 4-F4t-NeuroP and 10-F4t-NeuroP were detectable in all plasma samples from MS (n = 16), DS (n = 16), ASD (n = 9) and RTT (n = 20) patients. While plasma 10-F4t-NeuroP content was significantly higher in patients of all diseases as compared to age and gender matched healthy control subjects (n = 61), 4-F4t-NeuroP levels were significantly higher in MS and RTT as compared to healthy controls. Significant positive relationships were observed between relative disease severity and 4-F4t-NeuroP levels (r = 0.469, P <0.0001), and 10-F4t-NeuroP levels (r = 0.757, P < 0.0001). The study showed that the plasma amount ratio of 10-F4t-NeuroP to 4-F4t-NeuroP and the plasma amount as individual isomer can be used to discriminate between different brain diseases. Overall, by comparing the different types of disease, our plasma data indicates that 4-F4t-NeuroP and 10-F4t -NeuroP: i) are biologically synthesized in vivo and circulated, ii) are related to clinical severity of neurological diseases, iii) are useful to identify shared pathogenetic pathways in distinct brain diseases, and iv) appears to be distinctive for different neurological conditions, thus representing potentially new biological disease markers. Our data strongly suggest that in vivo DHA oxidation follows preferential chemical rearrangements according to different brain diseases.

Proteomic analysis of the Rett syndrome experimental model mecp2Q63X mutant zebrafish.

Rett syndrome (RTT) is a severe genetic disorder resulting from mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Recently, a zebrafish carrying a mecp2-null mutation has been developed with the resulting phenotypes exhibiting defective sensory and thigmotactic responses, and abnormal motor behavior reminiscent of the human disease. Here, we performed a proteomic analysis to examine protein expression changes in mecp2-null vs. wild-type larvae and adult zebrafish. We found a total of 20 proteins differentially expressed between wild-type and mutant zebrafish, suggesting skeletal and cardiac muscle functional defects, a stunted glycolysis and depleted energy availability. This molecular evidence is directly linked to the mecp2-null zebrafish observed phenotype. In addition, we identified changes in expression of proteins critical for a proper redox balance, suggesting an enhanced oxidative stress, a phenomenon also documented in human patients and RTT murine models. The molecular alterations observed in the mecp2-null zebrafish expand our knowledge on the molecular cascade of events that lead to the RTT phenotype. BIOLOGICAL SIGNIFICANCE: We performed a proteomic study of a non-mammalian vertebrate model (zebrafish, Danio rerio) for Rett syndrome (RTT) at larval and adult stages of development. Our results reveal major protein expression changes pointing out to defects in energy metabolism, redox status imbalance, and muscle function, both skeletal and cardiac. Our molecular analysis grants the mecp2-null zebrafish as a valuable RTT model, triggering new research approaches for a better understanding of the RTT pathogenesis and phenotype expression. This non-mammalian vertebrate model of RTT strongly suggests a broad impact of Mecp2 dysfunction.

Erectile dysfunction and diabetes: Association with the impairment of lipid metabolism and oxidative stress.

OBJECTIVES: To test the hypothesis that exists an association of non-diabetic and diabetic patients suffering from erectile dysfunction (ED) with lipid metabolism and oxidative stress. DESIGN AND METHODS: Clinical and laboratory characteristics in non-diabetic (n = 30, middle age range: 41­55.5 years; n = 25, old age range: 55.5­73), diabetic ED patients (n = 30, age range: 55.5­75 years) and diabetic patients (n = 25, age range: 56­73.25), were investigated. Proteomic analysis was performed to identify differentially expressed plasma proteins and to evaluate their oxidative posttranslational modifications. RESULTS: A decreased level of high-density lipoproteins in all ED patients (P < 0.001, C.I. 0.046­0.10), was detected by routine laboratory tests. Proteomic analysis showed a significant decreased expression (P < 0.05) of 5 apolipoproteins (i.e. apolipoprotein H, apolipoprotein A4, apolipoprotein J, apolipoprotein E and apolipoprotein A1) and zinc-alpha-2-glycoprotein, 50% of which are more oxidized proteins. Exclusively for diabetic ED patients, oxidative posttranslational modifications for prealbumin, serum albumin, serum transferrin and haptoglobin markedly increased. CONCLUSIONS: Showing evidence for decreased expression of apolipoproteins in ED and the remarkable enhancement of oxidative posttranslational modifications in diabetes-associated ED, considering type 2 diabetes mellitus and age as independent risk factors involved in the ED pathogenesis, lipid metabolism and oxidative stress appear to exert a complex interplay in the disease.