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

Abstract

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 β-actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and β-actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.

Figure 1

Abnormal erythrocyte shapes in classical autism at the scanning electron microscopy (SEM). (a): normal discocyte shape; (b) to (g): main shape-altered RBCs observed in autistic patients; (h): healthy controls; (i): a typical morphological pattern in nonautistic neurodevelopmental disorders (NA-NDDs); (j): typical picture in an autistic patient with predominant elliptocytosis. Symbols indicate intermediate-shaped RBCs: the arrow indicates a disco-echinocyte shape, while the arrowhead indicates the presence of a knizo-echinocyte shape in autistic patients, bars correspond to 2 μm in (a) to (g) upper panels and to 10 μm in the (h), (i), and (j) lower panels.

Figure 2

RBC morphology distribution in autistic patients, subjects with nonautistic neurodevelopmental disorders (NA-NDDs) and healthy controls. Statistically significant differences are denoted by single asterisk, P > 0.05. P values above each erythrocyte shape classification refer to one-way ANOVA.

Figure 3

Intraerythrocyte NPBI, along with erythrocyte membrane esterified F₂-IsoPs and 4-HNE protein adducts in autistic patients, subjects with nonautistic neurodevelopmental disorders (NA-NDDs), and healthy controls. NPBI was reported as nmol/mL erythrocyte suspension, esterified F₂-IsoPs as pg/mg of erythrocyte membrane proteins, and 4-HNE protein adducts as arbitrary units. The data are expressed as means ± SD. Statistically significant differences are denoted by asterisks, P < 0.05. NPBI: non protein-bound-iron; F₂-IsoPs: F₂-isoprostanes; 4-HNE PAs: 4-HNE protein adducts.

Figure 4

(a) Representative SDS-PAGE analyses of RBC ghosts (silver staining) in a healthy control subject (lane C⁻), a positive control subject (see text for definition; lane C⁺), and two autistic patients (lanes A₁ and A₂): visible reduction of intensity for β-actin in autistic patients (lanes A₁ and A₂) is evidenced; the electrophoretic position of bands 4.1 and 4.2 is indicated by arrowheads; (b) SDS-PAGE of immunoprecipitated β-actin (silver staining) from RBC ghosts of a healthy control subject (lane C⁻), a positive control individual (lane C⁺), and an autistic patient (lane A): visible reduction of intensity for β-actin in autistic patients (lane A); (c) immunochemical detection in the 4-HNE protein adducts in RBC ghosts: representative western blot from a healthy control subject (lane C⁻), a positive control individual (lane C⁺) and from an autistic patients (lanes A₁ and A₂). An increase in the 4-HNE PAs signal is evident in RBC ghosts from autistic patients (lanes A₁ and A₂), in particular as it concerns the β-actin band; the 4-HNE binding to bands 4.1 and 4.2 is indicated by arrowheads; and (d) immunochemical detection in the 4-HNE protein adducts in immunoprecipitated β-actin from RBC ghosts: representative western blot from a healthy control subject (lane C⁻), positive control individual (lane C⁺), and from an autistic patient (lane A): a visible increase in the 4-HNE PAs signal in β-actin from autistic patient (lane A) is evident. Molecular weight marks are indicated on the right side.