Detection of plasma autoantibodies to brain tissue in young children with and without autism spectrum disorders

Abstract

Autism spectrum disorders (ASDs) are characterized by impaired language and social skills, often with restricted interests and stereotyped behaviors. A previous investigation of blood plasma from children with ASDs (mean age = 5½ years) demonstrated that 21% of samples contained autoantibodies that reacted intensely with GABAergic Golgi neurons of the cerebellum while no samples from non-sibling, typically developing children showed similar staining (Wills et al., 2009). In order to characterize the clinical features of children positive for these autoantibodies, we analyzed plasma samples from children enrolled in the Autism Phenome Project, a multidisciplinary project aimed at identifying subtypes of ASD. Plasma from male and female children (mean age = 3.2 years) was analyzed immunohistochemically for the presence of autoantibodies using histological sections of macaque monkey brain. Immunoreactivity to cerebellar Golgi neurons and other presumed interneurons was observed for some samples but there was no difference in the rate of occurrence of these autoantibodies between children with ASD and their typically developing peers. Staining of neurons, punctate profiles in the molecular layer of the dentate gyrus, and neuronal nuclei were also observed. Taken together, 42% of controls and subjects with ASD demonstrated immunoreactivity to some neural element. Interestingly, children whose plasma reacted to brain tissue had scores on the Child Behavior Checklist (CBCL) that indicated increased behavioral and emotional problems. Children whose plasma was immunoreactive with neuronal cell bodies scored higher on multiple CBCL scales. These studies indicate that additional research into the genesis and prevalence of brain-directed autoantibodies is warranted.

Introduction

Autism spectrum disorders (ASDs) are characterized by impaired social functioning and language development combined with a restricted or stereotyped set of behaviors that emerge by three years of age. The most recent estimate of their prevalence indicates that one in 110 children in the United States is diagnosed with an ASD (2009). Males are affected about four times more frequently than females (Fombonne, 2005). Higher concordance rates for these disorders in monozygotic twins compared to dizygotic twins suggest that ASDs are highly heritable (Bailey et al., 1995), and multiple different genetic variations are known to be associated with ASDs (Folstein and Rosen-Sheidley, 2001). Currently, 10–20% of cases are associated with defined mutations, genetic syndromes, or de novo copy number variations, and further cases will likely be attributable to genetic variations upon further study (Abrahams and Geschwind, 2008). However, none of the variations discovered so far consistently results in an individual developing an ASD, suggesting other factors must be involved (Abrahams and Geschwind, 2008).

Numerous studies have identified abnormalities in the immune systems of some individuals with an ASD (Ashwood et al., 2006, Pardo et al., 2005). Postmortem studies point to increased microglial activation in the cerebellum, cortex, and white matter of some individuals with ASD compared to typically developing controls (Morgan et al., 2010, Vargas et al., 2005). Brain tissue and cerebrospinal fluid of children and adults with autism display an increased level of the cytokines macrophage chemoattractant protein-1, tumor growth factor-β1, tumor necrosis factor α, interleukin-6, granulocyte–macrophage colony-stimulating factor, and interferon γ (Li et al., 2009, Vargas et al., 2005). In a study involving 80 subjects with ASD, a screen for anti-nuclear antibodies in serum found that more children with autism possess anti-nuclear antibodies than typically developing children (Mostafa and Kitchener, 2009). Using western blots, several groups have identified increased levels of autoantibodies to proteins in the central nervous system in the plasma of some children with an ASD (Cabanlit et al., 2007, Singer et al., 2006, Singh et al., 1997, Singh and Rivas, 2004, Wills et al., 2009) and in some mothers of children with ASD (Braunschweig et al., 2008, Croen et al., 2008, Singer et al., 2008, Zimmerman et al., 2007). Immunohistochemical studies, in which plasma from affected children is tested for immunoreactivity to sectioned brain tissue, demonstrate that at least some of these autoantibodies can bind to cells in the brain (Connolly et al., 1999, Dalton et al., 2003, Wills et al., 2009, Wills et al., in press).

Our laboratories recently showed that plasma from 21% of a group of 34 children with ASD contained autoantibodies that reacted intensely with GABAergic Golgi neurons in primate cerebellar tissue (Wills et al., 2009). Analysis of the same plasma in different brain regions showed that individuals whose plasma reacted intensely with cerebellar Golgi cells also showed reactivity to interneurons in several other brain regions, including the cerebral cortex and the hippocampus (Wills et al., in press). It is currently unknown whether children with interneuron-reactive antibodies can be distinguished behaviorally from children without these antibodies.

The clinical presentation of behaviors varies considerably across individuals diagnosed with an ASD, leading to the suggestion that there may be many different forms of autism and that these forms may arise from different causes (Geschwind and Levitt, 2007). Studies linking biological findings in subjects with ASD to behavioral measures will be instrumental in identifying these multiple forms of ASD. In the cohort for which intense plasma reactivity to cerebellar Golgi neurons was first observed, there was relatively little clinical information to correlate with the presence of autoantibodies. In order to further investigate the clinical characteristics of individuals whose plasma contains autoantibodies to interneurons, we sought to extend our earlier observations by examining the plasma of individuals for which detailed clinical information is available. Thus, we examined autoantibodies in plasma collected as part of the Autism Phenome Project (APP), a multidisciplinary project at the Medical Investigation of Neurodevelopmental Disorders (M.I.N.D) Institute aimed at identifying subtypes of ASD. Subjects participating in the APP undergo a blood draw as part of a comprehensive multidisciplinary analysis that includes diagnostic, behavioral, and neuropsychological assessments, a medical examination, structural magnetic resonance imaging (MRI), electroencephalogram (EEG) analysis, and characterization of immune function. As participants in the APP are mostly in the 24–42 month age range, the children investigated in the APP represent a younger subject group than those examined in the initial description by Wills et al. (2009).

We tested plasma from 129 subjects with either typical development or ASD for autoantibodies to Golgi cells of the cerebellum and other interneurons throughout the brain. By enrolling this group of age-matched, preschool-aged children for whom we have high quality multidisciplinary data we aimed to investigate associations between autoantibody status, development, behavior, and other characteristics of ASD.