De novo Blood Biomarkers in Autism: Autoantibodies against Neuronal and Glial Proteins

Abstract

Autism spectrum disorders (ASDs) are the most common neurodevelopmental disorders with unidentified etiology. The behavioral manifestations of ASD may be a consequence of genetic and/or environmental pathology in neurodevelopmental processes. In this limited study, we assayed autoantibodies to a panel of vital neuronal and glial proteins in the sera of 40 subjects (10 children with ASD and their mothers along with 10 healthy controls, age-matched children and their mothers). Serum samples were screened using Western Blot analysis to measure immunoglobulin (IgG) reactivity against a panel of 9 neuronal proteins commonly associated with neuronal degeneration: neurofilament triplet proteins (NFP), tubulin, microtubule-associated proteins (tau), microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), α-synuclein (SNCA) and astrocytes proteins such as glial fibrillary acidic protein (GFAP) and S100B protein. Our data show that the levels of circulating IgG class autoantibodies against the nine proteins were significantly elevated in ASD children. Mothers of ASD children exhibited increased levels of autoantibodies against all panel of tested proteins except for S100B and tubulin compared to age-matched healthy control children and their mothers. Control children and their mothers showed low and insignificant levels of autoantibodies to neuronal and glial proteins. These results strongly support the importance of anti-neuronal and glial protein autoantibodies biomarker in screening for ASD children and further confirm the importance of the involvement of the maternal immune system as an index that should be considered in fetal in utero environmental exposures. More studies are needed using larger cohort to verify these results and understand the importance of the presence of such autoantibodies in children with autism and their mothers, both as biomarkers and their role in the mechanism of action of autism and perhaps in its treatment.

Keywords: Autism Spectrum Disorder; Control children; autoimmune disorder; maternal autoantibodies; neuronal and astroglial biomarkers; neuronal autoantibodies.

Figure 1

Specificity of the serum autoantibody. The specificity of the serum autoantibody against all tested neural proteins was carried out by protein/peptide competitive or absorption assay. Upper panel) Western blot from ASD-C following the absorption of the sera with S100B, GFAP, Tau, MAP2, NFP, Tubulin, MBP, SNCA and MAG proteins. The levels of autoantibodies against neuronal and glial proteins are negligible supporting the specificity of the assay. Lower panel) Commassie stain showing the proteins signals on the blot indicating that the sera were depleted from the autoantibodies by the adsorption reaction.

Figure 2

Representative panel of Western blotting from three cases of the (ASD-C)—ASD children (A), (Cont-C)—control children (B), (ASD-M)—mothers of ASD children (C) and (Cont-M) the mothers of control children (D) are shown with the molecular weight marker. Total IgG was used as a reference protein. Note that the IgG fractionates as 150 kDa on a native gel consisting of an intact IgG molecule, however, when fractionated on a denaturing gel, it resolves as two heavy (H) chains and two light (L) chains showing thick bands at 50 kDa and light bands at 25 kDa. Bands in each lane were quantified on digitized images in the mid-dynamic range and normalized to the value of IgGband density.

Figure 3

Histogram representing the mean levels of autoantibodies against neural proteins relative to total IgG with the bar indicating standard error. Comparison between (A) control children (Cont-C) and ASD children (ASD-C); (B) ASD Children (ASD-C) and ASD children’s mothers (ASD-M); (C) ASD children’s mother (ASD-M); (D) control children (Cont-C) and control children’s mothers (Cont-M). Significant differences with P < 0.05 are indicated in asterisks and the actual p-values are presented in Table 3.

Figure 4

Histogram representing the profiles of individual levels of serum autoantibodies in ASD children (ASD-C) represented in red color bars; ASD children’s mothers (ASD-M) represented in light rose color bars; Control children (Cont-C) represented in royal blue color bars and Control children’s mothers (Cont-M) represented in light blue color bars. (A) S100B; (B) GFAP; (C) Tau; (D) MAP2; (E) NFP; (F) Tubulin; (G) MBP; (H) SNCA and (I) MAG.

Figure 5

Scatter Plot showing the distribution of neural autoantibodies in Control Children’s mothers (Cont-M), Control children (Cont-C), ASD children’s mothers (ASD-M), ASD children (ASD-C) relative to the total IgG levels. Autoantibodies against (A) GFAP. (B) MAP2, (C) MBP, (D) NFP, (E) Tau, (F) SNCA, (G) MAG, (H) Tubulinand, (I) S100B. The cross line on the dot plot represents the mean values for each protein. The vertical line represents SEM. ^† P > 0.05 or * P < 0.01 is considered significant.