Expression and oxidative modifications of plasma proteins in autism spectrum disorders: Interplay between inflammatory response and lipid peroxidation.

PURPOSE: A role for inflammation and oxidative stress is reported in autism spectrum disorders (ASDs). Here, we tested possible changes in expression and/or oxidative status for plasma proteins in subjects with ASDs. EXPERIMENTAL DESIGN: To evaluate protein expression and protein adducts of lipid peroxidation-derived aldehyde, analysis of plasma proteins was performed in 30 subjects with ASDs and compared with 30 healthy controls with typical development, using a proteomic approach. RESULTS: Significant changes were evidenced for a total of 12 proteins. Of these, ten were identified as proteins involved in the acute inflammatory response including alpha-2-macroglobulin, alpha-1-antitrypsin, haptoglobin, fibrinogen, serum transferrin, prealbumin, apolipoprotein A-I apolipoprotein A-IV, apolipoprotein J, and serum albumin. In addition, significant changes occurred for two immunoglobulins alpha and gamma chains. CONCLUSIONS AND CLINICAL RELEVANCE: Our present data indicate that an inflammatory response, coupled with increased lipid peroxidation, is present in subjects with ASDs. This information can provide new insight into the identification of potential plasma protein biomarkers in autism.

MECP2 Duplication Syndrome: Evidence of Enhanced Oxidative Stress. A Comparison with Rett Syndrome.

Rett syndrome (RTT) and MECP2 duplication syndrome (MDS) are neurodevelopmental disorders caused by alterations in the methyl-CpG binding protein 2 (MECP2) gene expression. A relationship between MECP2 loss-of-function mutations and oxidative stress has been previously documented in RTT patients and murine models. To date, no data on oxidative stress have been reported for the MECP2 gain-of-function mutations in patients with MDS. In the present work, the pro-oxidant status and oxidative fatty acid damage in MDS was investigated (subjects n = 6) and compared to RTT (subjects n = 24) and healthy condition (subjects n = 12). Patients with MECP2 gain-of-function mutations showed increased oxidative stress marker levels (plasma non-protein bound iron, intraerythrocyte non-protein bound iron, F2-isoprostanes, and F4-neuroprostanes), as compared to healthy controls (P ≤ 0.05). Such increases were similar to those observed in RTT patients except for higher plasma F2-isoprostanes levels (P < 0.0196). Moreover, plasma levels of F2-isoprostanes were significantly correlated (P = 0.0098) with the size of the amplified region. The present work shows unique data in patients affected by MDS. For the first time MECP2 gain-of-function mutations are indicated to be linked to an oxidative damage and related clinical symptoms overlapping with those of MECP2 loss-of-function mutations. A finely tuned balance of MECP2 expression appears to be critical to oxidative stress homeostasis, thus shedding light on the relevance of the redox balance in the central nervous system integrity.

Cytokine Dysregulation in MECP2- and CDKL5-Related Rett Syndrome: Relationships with Aberrant Redox Homeostasis, Inflammation, and ω-3 PUFAs.

An involvement of the immune system has been suggested in Rett syndrome (RTT), a devastating neurodevelopmental disorder related to oxidative stress, and caused by a mutation in the methyl-CpG binding protein 2 gene (MECP2) or, more rarely, cyclin-dependent kinase-like 5 (CDKL5). To date, it is unclear whether both mutations may have an impact on the circulating cytokine patterns. In the present study, cytokines involved in the Th1-, Th2-, and T regulatory (T-reg) response, as well as chemokines, were investigated in MECP2- (MECP2-RTT) (n = 16) and CDKL5-Rett syndrome (CDKL5-RTT) (n = 8), before and after ω-3 polyunsaturated fatty acids (PUFAs) supplementation. A major cytokine dysregulation was evidenced in untreated RTT patients. In MECP2-RTT, a Th2-shifted balance was evidenced, whereas in CDKL5-RTT both Th1- and Th2-related cytokines (except for IL-4) were upregulated. In MECP2-RTT, decreased levels of IL-22 were observed, whereas increased IL-22 and T-reg cytokine levels were evidenced in CDKL5-RTT. Chemokines were unchanged. The cytokine dysregulation was proportional to clinical severity, inflammatory status, and redox imbalance. Omega-3 PUFAs partially counterbalanced cytokine changes, as well as aberrant redox homeostasis and the inflammatory status. RTT is associated with a subclinical immune dysregulation as the likely consequence of a defective inflammation regulatory signaling system.

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.

Altered erythrocyte membrane fatty acid profile in typical Rett syndrome: effects of omega-3 polyunsaturated fatty acid supplementation.

This study mainly aims at examining the erythrocyte membrane fatty acid (FAs) profile in Rett syndrome (RTT), a genetically determined neurodevelopmental disease. Early reports suggest a beneficial effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on disease severity in RTT. A total of 24 RTT patients were assigned to ω-3 PUFAs-containing fish oil for 12 months in a randomized controlled study (average DHA and EPA doses of 72.9, and 117.1mg/kgb.w./day, respectively). A distinctly altered FAs profile was detectable in RTT, with deficient ω-6 PUFAs, increased saturated FAs and reduced trans 20:4 FAs. FAs changes were found to be related to redox imbalance, subclinical inflammation, and decreased bone density. Supplementation with ω-3 PUFAs led to improved ω-6/ω-3 ratio and serum plasma lipid profile, decreased PUFAs peroxidation end-products, normalization of biochemical markers of inflammation, and reduction of bone hypodensity as compared to the untreated RTT group. Our data indicate that a significant FAs abnormality is detectable in the RTT erythrocyte membranes and is partially rescued by ω-3 PUFAs.

Redox imbalance and morphological changes in skin fibroblasts in typical Rett syndrome.

Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (-43.6%) and GSH/GSSG ratio (-3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.

Beta-actin deficiency with oxidative posttranslational modifications in Rett syndrome erythrocytes: insights into an altered cytoskeletal organization.

Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT) and methyl CpG binding protein 2 (MECP2) gene mutations. RTT, affecting almost exclusively females with an average frequency of 1∶10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs), drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: -1.82±0.15, -2.15±0.06, and -2.59±0.48, respectively) in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, beta-actin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs) as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE). Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization.

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.

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.

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.