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.


Introduction
Actins are highly conserved proteins ubiquitously expressed in eukaryotic cells [1]. In vertebrates, three main groups of actin isoforms have been identified, i.e., alpha, beta and gamma. While the alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus, beta and gamma actins coexist in most cell types either as components of the cytoskeleton or mediators of internal cell motility [2]. In particular, beta-actin is a critical player in several cellular functions, ranging from cell motility and the maintenance of cell shape and polarity to transcription regulation. Interestingly, several syndromes associated with mental retardation are characterized by cortical dendritic abnormalities [3], and the neuronal cytoskeleton is essential for dendritic developmental processes. It includes microfilaments, neurofilaments and microtubules, each one being formed by a major protein, i.e., actin, neurofilament and tubulin, respectively [4,5].
Rett syndrome (RTT), the second commonest cause of severe mental retardation in the female gender, is represented by a devastating neurodevelopmental disorder with a wide phenotypical heterogeneity which is caused in the overwhelming majority of cases by loss-of-function de novo sporadic mutations in the X-linked gene encoding the methyl-CpG binding protein 2 (MeCP2) [6]. In its classical phenotype, RTT is characterized by an apparently normal development in the first few months followed by loss of neurodevelopmental milestones in a 4-stage progression [7,8]. Increasing evidence supports the concept that RTT is associated with impairment of dendritic arborization [9,10]. Several studies have described neuronal abnormalities in RTT human brains, and in male Mecp2-mutant mice [9,10,11,12,13]. Overall, this body of research suggests that MECP2 deficiency mutations are involved in cellular mechanisms regulating short term dynamics of dendritic spines early in development within the frame of maximum neural plasticity. However, the underlying cause for the dendritic arborization impairment in RTT is still largely unknown.
While prior studies on Mecp2-deficient brains show striking changes in neuronal maturation [14], recent evidence indicates that MeCP2 deficiency affects microtubule dynamics in RTT astrocytes and impairs microtubule stability in RTT primary fibroblast cultures [15,16]. These data suggest that MeCP2 has a stabilizing role on microtubule dynamics and that its deficiency could lead to impaired microtubule stability which may at least partly underlie the dendritic abnormalities detected in RTT brains. We have previously demonstrated the existence of an abnormal erythrocyte shape in typical RTT patients showing a striking prevalence of circulating leptocytes with enhanced membrane oxidative stress (OS). Therefore, in the present study, we tested the hypothesis that a beta-actin deficiency with an increased oxidative posttranslational modification (PTMs) could underlie the red blood cells (RBCs) shape abnormalities in patients affected by the typical form of the disease [17].

Study Population and Ethical Statement
The study included 20 female patients with clinical diagnosis of typical RTT (median age: 5.0 years inter-quartile range 3-6, values range 3-10 years) with demonstrated MECP2 gene mutations. RTT diagnosis and inclusion/exclusion criteria were based on RTT nomenclature consensus [18]. All the patients were admitted to the Siena Rett Syndrome National Reference Centre. Gender-matched healthy control subjects of comparable age (N = 20, median age: 5.0 years inter-quartile range 3-5.5, values range 3-10 years) with a typical development were selected as a control population. Blood samplings in the patients' group was performed during the routine follow-up study at hospital admission, while the samples from the control group were carried out during routine health checks, sports, or blood donations, obtained during the periodic clinical checks. The subjects examined in this study were on a typical Mediterranean diet.
The study was conducted with the approval by the competent Ethics Committee of the Azienda Ospedaliera Universitaria Senese, Siena. All the informed consents were obtained from either the parents or the legal tutors of the enrolled patients. Parents or legal tutors provided written informed consent to participate to the study, as approved by the Institutional Ethics Committee.

Blood Sample Collection and Preparation
All samplings from RTT patients and healthy controls were carried out around 8 AM after overnight fasting. Blood was collected in heparinized tubes and all manipulations were carried out within 2 h after sample collection. The blood samples were centrifuged at 24006g for 15 min at 4uC; after plasma and buffy coat removal, RBCs were washed twice with physiological solution (150 mM NaCl) and lysed in Dodge buffer containing 5 mM of potassium phosphate buffer, 0.5 mM EDTA, pH 8 and 1 mM of phenylmethanesulfonyl fluoride (Sigma). Erythrocyte membranes were freshly prepared, according to Dodge [19], by repeated washing until the ''ghosts'' were pearly white. Samples were kept frozen at 270uC until used. An aliquot of each blood sample (1 ml) was centrifuged at 8006g for 10 min at 4uC and washed twice with physiological solution for confocal microscopy analysis of erythrocytes.

Immunoprecipitation of Erythrocyte Beta-actin
Ghosts (200 mg of protein determined using the protein assay; BioRad, Hercules, CA) were incubated with 5 mg of rabbit monoclonal anti-beta-actin antibody (cod. 04-1116; Millipore Corporation, Billerica, MA, USA) overnight at 4uC on a rotator. Then, immune complex was incubated with 50 ml of Protein A-Sepharose (Sigma-Aldrich, Milan, Italy) and rotated at 4uC for 2 h. Samples were centrifuged at 10,0006g for 5 min and washed three times with 1 ml ice-cold PBS. The pellet was mixed with 2 6 reducing sample buffer, boiled and loaded on electrophoretic gels for silver staining or western blotting (WB) analysis. Samples processed equally but with normal rabbit IgG (Sigma-Aldrich, Milan, Italy), instead of anti-beta-actin antibody, were used as negative control.

Polyacrylamide Gel Electrophoresis Analysis
Ghosts (20 mg of protein) and immunoprecipitated beta-actin were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%, SDS-PAGE) according to Laemmli [20]. Resulting gels were stained with silver nitrate (Sigma-Aldrich, Milan, Italy). Gel images were acquired using LabScan software (GE Healthcare, version 6.0) by Image Scanner and the bands were automatically analyzed using TotalLab software (nonlinear dynamics, version 1.0). The application of the software allows calibration and normalization of the gel.

Western Blot Analysis
Ghosts (40 mg of protein) and immunoprecipitated beta-actin were resolved on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (GE Healthcare Europe GmbH, Milan, Italy).
Following washed in TBS Tween, membranes were incubated with specific secondary antibodies for 1 h at RT and, finally, with ECL reagent (BioRad, Hercules, CA, USA). Images were digitized (ChemiDoc XRS, BioRad, Hercules, CA). Optical densities were quantified using a computerized imaging system (Quantity One Imaging system).

Confocal Microscopy
Erythrocytes purified as described before were smeared onto slides and after drying were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at RT for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min at 4uC and washed three times in PBS. Cells were blocked with 1% BSA for 30 min at RT and incubated overnight at 4uC with a monoclonal rabbit beta-actin antibody (Millipore Corporation, Billerica, MA, USA) diluted 1:50 in PBS. Following washed in TBS, cells were incubated with specific secondary antibody goat anti-rabbit (cod. A-11008; Alexa Fluor 488,) diluted 1:100 in PBS for 1 h at RT. After three washes in PBS, fluorescent RBCs cells, mounted with a drop antifade, were visualized by confocal microscopy LSM-700 (Zeiss, Jena, Germany). Cell signal semiquantitative analysis and cells 3D visualization were carried out with ImageJ an open source program (rsb.info.nih.gov/ij).

Two-Dimensional Gel Electrophoresis Analysis
Two-Dimensional gel electrophoresis (2-DE) was used to separate ghost proteins as previous described [21]. Samples (60 mg of protein determined using the Bradford method) [22], were denatured a solution containing 10% of SDS, 2.3% of dithiothreitol (DTT) heated to 95uC for 5 min. The samples were then combined with solubilizing buffer containing 8 M urea, 2% of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), 0.3% DTT, 2% immobilized pH gradient (IPG) buffer, and a trace of bromophenol blue and loaded into 18 cm IPG strips 3-10 non linear on an Ettan IPGphor (GE Healthcare) apparatus system and rehydrated for 7 h. Isoelectric focusing (IEF) was carried out for a total of 32 kV h. The strips were first equilibrated with a buffer containing 50 mM Tris-HCl, pH 8.8, 6 M urea, 2% w/v SDS, 30% v/v glycerol, and 1% w/v DTT for 15 min; then they were equilibrated again with the same buffer described above, except it contained 4% w/v iodoacetamide instead of DTT. The second dimension was performed on an EttanDalt Six Electrophoresis system (GE Healthcare). IPG strips were embedded at the top of a 1.5 mm thick vertical polyacrylamide gradient gel (8-16%T)

Tryptic Digestion and Proteins Identification by Mass Spectrometry
A spot-picking list was generated and exported to Ettan Spot Picker (GE Healthcare). The spots were excised and delivered into 96-well microplates where they were destained and dehydrated with acetonitrile (ACN) for subsequent rehydration with trypsin solution. Tryptic digestion was carried out overnight at 37uC. Each protein spot digest (0.75 ml) was spotted into the MALDI instrument target and allowed to dry. Then 0.75 ml of the instrument matrix solution (saturated solution of a-cyano-4hydroxycinnamic acid in 50% ACN and 0.5% v/v trifluoroacetic acid) was applied to dried samples and dried again. Mass spectra were obtained, as described [23], using an ultrafleXtreme MALDI-ToF/ToF (Bruker Corporation, Billerica, MA, United States). After tryptic peptide mass acquisition, mass fingerprint searching was carried out in Swiss-Prot/TREMBL and NCBInr databases using MASCOT (Matrix Science, London, UK, www. matrixscience.com).

Image and Statistical Data Analysis
2-DE gel images were analyzed using ImageMaster 2D Platinum v7.0 software (GE Healthcare). Spot intensity was expressed as protein percentage volume (%V). Comparative analysis between healthy controls and RTT patients of differently expressed proteins were evaluated using either Mann-Whitney rank sum test or Kruskal-Wallis test. Statistical significance was indicated by a two-tailed P-value ,0.05. Data were expressed as mean 6 standard deviations (mean 6 SD) from triplicate determinations obtained in five separate experiments. The MedCalc version 12.1.4 statistical software package (MedCalc Software, Mariakerke, Belgium) was used.

Erythrocytes from RTT Patients Show a Decreased Betaactin Band Intensity
In the SDS-PAGE analysis (silver staining), a visible significant reduction (P,0.01) of the beta-actin (band 6) intensity was detected in the erythrocyte ghosts from RTT patients with an average decrease of 21.9360.09 fold as mean 6 SD (range: 2 2.09 to 21.78 fold), as compared to control values ( Figure 1 and Table 1).

Beta-actin Decrease in RTT Erythrocytes is Independent from the Monoclonal Antibodies
Western blot analyses confirmed that the detected changes were specifically attributable to beta-actin, with a signal intensity decrease in the patients as compared to controls (Figure 2). Observed reduction in beta-actin band intensity is independent of the employed monoclonal antibodies (Figure 2A-D). These findings are compatible with a true decrease in beta-actin expression in the RTT patients erythrocytes membrane.

RTT Erythrocytes Show Underexpressed Beta-actin Isoforms
The 2-DE/MALDI-TOF analysis of RBC ghosts evidenced three differentially expressed beta-actin peptide spots both in RTT patients ( Figure 3A, right panel) and in healthy controls ( Figure 3A, left panel). A decrease in protein expression was detectable in each of the beta-actin RTT RBCs spots, as identified on the basis of their MW and pI values. Beta-actin spots identified by mass spectrometry, as well as peptide matches, sequence coverage, and the probabilistic score obtained using the MASCOT software are reported in Table 2. Quantitative analysis of the identified betaactin spots changes (spot 1: 21.8260.15 fold; spot 2: 22.1560.06 fold; spot 3: 22.5960.48 fold) are reported ( Figure 3B).

Differential Expression and Distribution of Beta-actin in RTT Erythrocytes
In the confocal microscopy analysis, beta-actin appears to be decreased and differently distributed in the RTT erythrocyte membranes ( Figure 4A, right panel) as compared to healthy control RBCs ( Figure 4A, left panel). As shown in Figure 4B, quantitative analysis of fluorescence signal intensity confirmed the decrement of beta-actin in RTT erythrocytes. These results indicate the existence of an altered cytoskeletal organization.

Increased 4-HNE-beta-actin Adducts in RTT RBC Membranes
SDS-PAGE (silver stain) of immunoprecipitated beta-actin from RBC ghosts showed a visible reduction of intensity for the protein band in RTT patients (21.8460.05 fold; range: 21.9 to 21.7 fold) as compared to controls ( Figure 5A). The absence of betaactin in negative control IgG immunoprecipitates demonstrated that beta-actin was specifically precipitated from RBC ghosts. Immunochemical detection for 4-HNE adducts of immunoprecipitated beta-actin from RBC ghosts showed a significant increase in the signal intensity for aldehyde adducts in RTT patients (2.0660.04 fold; range: 1.98 to 2.11 fold) as compared to controls ( Figure 5B). 2-DE/Western blot for 4-HNE on RBC ghosts evidenced a significant increase in the signal intensity for aldehyde adducts in the three differentially expressed beta-actin peptide spots ( Figure 5C).

Discussion
The data of the present study demonstrate, for the first time, the occurrence of a beta-actin deficiency and its oxidative PTMs in the inner interface of the erythrocytes membrane from patients affected by RTT. The combination of these adverse factors can lead to an altered erythrocyte cytoskeletal network organization. Therefore, it is likely that a cytoskeletal disorganization is ubiquitously present in the cells of RTT patients. It is well known that beta-actin is an essential protein in the RBCs cytoskeleton which represents a major structural component for the horizontal tensile forces in the inner membrane [24]. The resulting imbalance between horizontal and vertical tensile forces could lead to the abnormal RBCs shape detectable in RTT.
On the other hand, a systemic OS is well established in RTT [17,25,26,27,28,29,30]. 4-HNE, formed by arachidonic acid or other unsaturated fatty acids following free radicals attack, can bind to proteins by Michael addition, with preferential amino acidic binding sites to cysteine (C), hystidine, (H), or lysine (K) residues [31]. Therefore, 4-HNE protein adducts could be considered a reliable OS markers and have a biological impact on protein function.
In this regard, the amino acid sequence of the human beta-actin shows several potential 4-HNE binding sites on all the major conformational subdomains of the protein (Figure 6). A total of 6 C, 8 H and 19 K residues are present in the sequence, and potential 4-HNE binding amino acid residues are present in all the relevant conformational subdomains of the protein, for a total of 2 C, 4 H and 7 K in the subdomain 1, a total of 1 H and 3 K in the subdomain 2, a total of 2 C, 2 H and 5 K in the subdomain 3, and a total of 3 C and 4 K in the subdomain 4 [32]. In particular, the subdomain 2 of the molecule is of critical importance for DNAase 1 binding and protein polymerization, while a very crucial C 374 residue and potential binding site for 4-HNE is present in the conformationally critical hydrophobic pocket of the molecule [32].
Our findings indicate that the observed reduction in beta-actin band intensity is independent of the employed monoclonal antibodies and are compatible with a real decrease in the betaactin expression in the RTT patients erythrocytes membrane. Moreover, the reported evidence exhaustively addresses the Table 2. question of whether the observed decrease in beta-actin protein expression might be related to oxidative PTMs potentially preventing antibody recognition. Taken together, our data indicate that major alterations exist in the beta-actin of RTT erythrocytes which results both from changes in expression and as a consequence of oxidative damage. To this regard, beta-actin is a well-known major target for OS processes [33], while erythrocytes represent ''cellular detectors'' revealing the co-existence of major cytoskeletal changes in this key genetic model for neurodevelopmental disorders. In particular, this work suggests a novel role for MeCP2 as a stabilizing protein in microtubule dynamics.
To date, no definitive cure for RTT exists, although several approaches to a potential therapy have been either attempted or hypothesized, including activation of the silenced Mecp2 gene [34,35], gene therapy [36], modulation of some of the downstream effects from MeCP2-deficiency [37]. Our study indicates that a proteomic approach in RTT is able to reveal additional downstream effects of the MECP2-deficiency, and therefore could identify potentially novel therapeutical targets for the disease.