1. Introduction
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interaction, challenges in communication, social isolation “loneliness” and repetitive behaviors “sameness” [
1]. Diagnosis is often made as early as 18 months of age, but most patients are not formally diagnosed until they are 5 years old [
2]. Children with ASD may also have other disorders including, intellectual disability [
3], epilepsy [
4], Tourette’s syndrome, difficulty sleeping [
5] and many suffer gastrointestinal dysfunctions [
6].
Because of the lack of biochemical diagnostic tests, it is unclear whether the symptoms of ASD are originated from diverse etiologies with different manifestations of the same genetic or environmental components. Strong genetic association with ASD is supported by data from high monozygotic twin concordance and large-scale genetic screens have revealed numerous autism risk factors [
7], each with relatively low penetrance. This supports the hypothesis that the behavioral manifestations of ASD may be a consequence of genetic or environmental factors. A recent report showed a higher concordance among dizygotic twins that generates a best-fit model that attributes a 55% contribution of environmental factors and a 37% contribution of genetic factors as high risk for autism [
8].
Environmental insults leading to ASD occur early in gestation during first or second trimester [
9,
10]. Prenatally exposed children to the anticonvulsant agent valproic acid early in the first trimester of gestation, significantly increased the incidence of autism in children [
11]. Approximately 5% of individuals exposed
in-utero to thalidomide between day 24 and 36 of gestation [
12], as well as misoprostol [
8], developed ASD. Several studies have suggested that maternal infections associated with fever [
13,
14], immune activation [
15,
16], significant bleeding during the second trimester [
17] or occurrences of cytomegalovirus infection during the third trimesters [
18] are risk factors leading to ASD. Other factors that have been linked with increased risk of ASD in offspring include older paternal age, prenatal stress, maternal diabetes and obesity [
19]. These reports are consistent with the reports that autism is associated with impaired brain development [
20] that affects the amygdala, cerebellum and many other regions of the brain [
21]. The brains of children with autism tend to grow faster than usual just after birth, followed by normal or relatively slower growth in childhood and ASD involves specific regions of the brain [
22].
Although extensive studies have been carried out on autism, there are still controversies regarding its underlying mechanisms. Evidence of neurodegeneration has been seen in some cases of children with ASD who experienced progressive loss of neurological function, in the form of activated microglia and astrocytes, elevated 8-oxoguanine levels, evidence of oxidative stress, the presence of pro-inflammatory cytokines and neuronal cell loss [
23]. The World Health Organization considers ASD to be a developmental disorder exclusively affected by environmental factors and genetics, rather than mere neurodegeneration [
23].
In addition, the human immune cellular and genetic components rely on environmental exposures to induce the expression of genetically encoded signaling influencing effector functions. The maternal immune system control and regulate the health of both mother and fetus. Thus, the components of the maternal immune system that cross the placenta may be considered to be fetal environmental exposures. However, proper fetal neurodevelopment relies on the precise timing, functional levels and anatomic localization of many signaling molecules that may be altered by exogenous factors to which the embryo is exposed [
24].
We, therefore, carried out a study to detect the presence of IgG class circulating autoantibodies against neuronal and glial proteins in the sera of 40 subjects (10 children with ASD and their mothers and 10 control children and their mothers as controls). We determined autoantibodies against neural proteins that are associated with neurogenesis (NFP, tubulin, tau, MAP-2 and alpha-syncline), myelino genesis (MBP and MAG) and astrogliogenesis (GFAP and S100B, both of which are secreted by astrocytes).
7. Discussion
Despite the obvious small sample number of subjects in this preliminary study, the robust similarity between ASD children and their mothers and control children and their mothers further affirm the significance and contribution of maternal autoantibodies being one of the major factors in developing ASD. The higher levels of MAP-2, NFP, MBP, MAG, α-synuclein S100B and GFAP (3.2 to 4.8-fold) autoantibodies reported in ASD children warrant further research for developing autoantibodies screening as a biomarker for detection of early autism. Autoantibodies have been used as biomarkers for brain injury owing to chemical exposures and have been validated in several studies from our laboratory. Serum autoantibodies of IgG class from the mothers can cross the placenta, which may alter brain development [
28]. Although the exact role of these autoantibodies in the development of ASD is not known, it is thought that they may affect key neurons in the cerebellum by binding to and altering their physiological role [
29].
The study reports a significant elevation of the autoantibodies against neuronal and glial proteins in the serum of ASD children and their mothers, compared to age-matched control children and their mothers. The results of this study suggest that the profile of the peripheral autoantibodies against the neural proteins is unique to ASD children as compared to the profiles found in other brain disorders [
30]. Our previous studies documented the presence of IgG autoantibodies against neuronal and glial proteins in sera of aircrews and farm workers who were exposed to organophosphates (OP) and developed OP-induced delayed neurotoxicity(OPIDN) as well as the following exposure to molds [
26,
31,
32]. A recent study from another laboratory-confirmed our finding in another cohort of aircrews using Magnetic Resonance Imaging (MRI) examination [
33]. The MRI results showed deficits in white matter brain microstructures and cerebral perfusion that are potentially causative to cognitive impairments and mood deficits reported by aircrew [
33]. The extent of cognitive impairment was strongly associated with white matter integrity. These results are consistent with the present results where there are increased autoantibody levels against MBP, MAG and NFP reflecting, myelin degeneration. ASD is characterized by increased brain volume and in some cases, there is an altered ratio of gray/white matter. The affected areas of the brain include the cerebellum, cortex, nuclei of the amygdala, the fusiform face area and parts of the limbic system [
34]. Brain abnormalities in ASD include excess white matter neurons and decreased numbers of cerebellar Purkinje cells [
35,
36]. Axonal transport is an essential process for the continuous delivery of materials from the cell body to the nerve terminal. Protein components of the axonal transport include neurofilament triplet proteins (NFP), which is also associated with maturating brain, tubulin and microtubule-associated proteins, MAP-2 and tau [
37]. Neuronal tau proteins are more abundant in white matter than gray matter and are elevated in cerebrospinal fluid (CSF) and serum after traumatic brain injury (TBI) [
38]. Aggregation of Tau has been reported and used as a diagnostic marker for Alzheimer’s disease [
39]. Myelinated axons contain myelin basic protein (MBP) and myelin-associated glycoprotein (MAG). MBP may aid in the clinical evaluation of multiple sclerosis and stroke [
40]. The most abundant microtubule-associated protein in the mammalian brain is MAP-2, found in the dendrite-rich Purkinje cell. α-synuclein functions as a neuroprotective protein against oxidative stress [
41]. The GFAP astrocytic protein contributes to white matter architecture, myelination and integrity of the blood-brain barrier [
42]. The S100B, another astrocytic protein interacts with and stabilizes proteins associated with microtubules, such as tau and MAP-2 and exerts deleterious effects on neutrophils, depending on its concentration in the brain tissues [
43].
The results show that MAP-2 autoantibody levels were the highest that is consistent with reports showing the lower density of Purkinje cells coupled with decreased dendritic MAP-2 neurons in ASD children [
44,
45]. Loss of MAP-2 is a reliable indication of irreversible neuropathological alterations, such as brain damage [
46]. The increase of autoantibodies against neurofilaments (NFP) in ASD subjects is consistent with the destruction of neurofilaments in neurodegeneration [
47]. Also, the presence of NFP in cerebrospinal fluid (CSF) and serum has been suggested to reflect the destruction of axons and rupture of the BBB further causing axonal injury [
48]. Autoantibodies to MBP showed the next highest levels in ASD subjects compared to control children. These results are also in agreement with an increase of autoantibodies against the myelin-associated glycoprotein protein MAG and the involvement of white matter in autism [
40].
Although autoantibodies to astrocyte proteins increased significantly in ASD children compared to controls, they were lower than those in neuronal proteins. GFAP and S100B may reflect specific CNS changes and axonal damage, thus serving as biomarkers for ASD and that serum concentrations of autoantibodies against specific brain proteins obtained from children with ASD are related to the severity contributing to the deficit associated with this disorder. The action of S100B is related to its serum concentration, it is at a nanomolar level at the onset of the injury and the concentration is at a micromolar level during apoptotic phase [
43]. Acute traumatic brain injury resulting in large destruction of astrocytes leads to a massive (50- to 100-fold) release of S100B in serum, whereas levels of S100B in psychiatric disorders were only three times higher in patients compared to the controls [
49], correlating well with their neuroprotective ability in the present study. These findings have been documented in cases of dementia, particularly Alzheimer’s disease, schizophrenia and major depression [
50] and mania [
51]. It has been hypothesized that the serum concentration of S100B may also be a predictive value of the autistic brain. However, because of its short half-life in serum, approximately 2 hours [
43,
52], its assessment is limited for short-term acute brain injury cases.
The role of the immune system in the development of ASD has been explained by dysregulation of immune function [
53], neuroinflammation [
54] and maternal autoantibodies [
55]. The present results agree with previous studies that detected serum autoantibodies against some human unidentified brain proteins using ELISA and Western immunoblot in children with ASD and their non-autistic siblings [
14,
24,
55,
56,
57,
58,
59,
60,
61,
62,
63,
64,
65,
66,
67,
68,
69] and [
24,
56,
57,
64,
65,
69]. Although these studies did not specifically identify these proteins, we can extrapolate based on their reported masses, that some were probably neuronal and glial proteins. We have listed in
Table 5, how the masses of these unidentified proteins, can likely correspond to masses of known neuronal and glial proteins that we have used in our study, with the most notable identical matches being MBP, tau, tubulin, NFL and MAG.
Our results are consistent with the opinion that maternal neuronal cell death could contribute to the formation of autoantibodies against neuronal and glial proteins. Further, modern synthetic pesticides are usually neurotoxicants that are designed to target the nervous system [
58], and recent studies, confirmed that the pesticide exposure can cause neuronal cell death [
30,
31,
32]. Also, studies reported the development of autoantibodies against neuronal and glial proteins in patients exposed to pesticides who developed neurological symptoms characteristic of those caused by the insecticides [
31]. Epidemiological evidence showed that mothers exposed to pesticides near conception increased their likelihood of having children with ASD [
59]. Living near agricultural fields, ASD has been linked to the following pesticides: organophosphorus (OP) insecticides such as diazinon and chlorpyrifos [
60,
61], the OP herbicide glyphosate [
62], pyrethroid insecticides [
19] and the organochlorine pesticides dicofol and endosulfan [
63].Our present study supports the assumption that ASD may be an autoimmune disease that involves maternal perturbations in B-cell development and subsequent formation of IgG autoantibodies against neuronal and glia-specific proteins.