Beta-Actin Deficiency with Oxidative Posttranslational Modifications in Rett Syndrome Erythrocytes: Insights into an Altered Cytoskeletal Organization

Abstract

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.

High predictive values of RBC membrane-based diagnostics by biophotonics in an integrated approach for Autism Spectrum Disorders

“…An aliquot of the same blood sample (250 μL) of healthy and ASD children was processed for fatty acid analysis of membrane phospholipids, in order to evaluate statistically these results for the first time in combination with the HDFM data. The RBC membrane fatty acids of healthy and ASD subjects are reported in Supplementary Table 4 , evidencing a significant DHA decrease in disease ( P value = 0.0424), as already reported for previously studied cohorts 7 , 8 . Using ANOVA test (see Methods and Supplementary Table 5S ) to compare the two groups, the DHA decrease in ASD was found to be significant ( P value = 0.0344).…”

“…Equipment settings and handling protocol for hyperspectral imaging are reported in Methods. Optical images of RBC were acquired for all samples, and for uniformity of the sampling procedure, we choose to have the prevalent RBC round shape in the optical field, excluding echinocyte and stomatocyte forms, which are known to be increased in autism 7 , 8 , but can also depend from pH and other blood parameters, or even be artefacts during blood storage or oxidative transformations. Accurate collection of the spectral reflectance from RBCs gave rise to 8 spectra in the 430–800 nm wavelength region (Supplementary Fig.…”

Glutathione attenuates nitric oxide‐induced retinal lipid and protein changes

“…Moreover, cytoskeletal dynamics can be altered by actin oxidative modification in which the polymerisation ability is reduced . Our findings indicate that the observed reduction in β‐actin band intensity is compatible with a decrease in β‐actin expression (spots 4 and 5) in SNP‐treated retina . As noted, the inhibitory effects of SNP was prevented after GSH treatment.…”

“…Reports have shown that β‐actin is one of the cellular targets in oxidative stress, and it is vulnerable to cellular damage in certain diseases. For example, alteration of cytoskeletal organization has been found in Rett syndrome (RTT) where β‐actin has been found to be down‐regulated and post‐translationally modified in red blood cells . Moreover, cytoskeletal dynamics can be altered by actin oxidative modification in which the polymerisation ability is reduced .…”

Na+, K+‐ATPase activity in children with autism spectrum disorder: Searching for the reason(s) of its decrease in blood cells

“…Beta‐actin is the most widely used loading control for Western Blotting, however, we chose TGX gel fluorescence based on the observation reported by Cortelazzo et al [], who found that erythrocytes from children affected by Rett syndrome (a monogenic disease where subjects display autistic features) had decreased β‐actin content, as compared to healthy children. However, our results showed no difference in β‐actin content between TD and ASD children (Fig.…”