Detection of autoantibodies to neural cells of the cerebellum in the plasma of subjects with autism spectrum disorders

Abstract

Autism spectrum disorders (ASD) are a group of heterogeneous, behaviorally defined disorders characterized by disturbances in social interaction and communication, often with repetitive and stereotyped behavior. Previous studies have described the presence of antibodies to various neural proteins in autistic individuals as well as post-mortem evidence of neuropathology in the cerebellum. We examined plasma from children with ASD, as well as age-matched typically developing controls, for antibodies directed against human cerebellar protein extracts using Western blot analysis. In addition, the presence of cerebellar specific antibodies was assessed by immunohistochemical staining of sections from Macaca fascicularis monkey cerebellum. Western blot analysis revealed that 13/63 (21%) of subjects with ASD possessed antibodies that demonstrated specific reactivity to a cerebellar protein with an apparent molecular weight of approximately 52 kDa compared with only 1/63 (2%) of the typically developing controls (p = 0.0010). Intense immunoreactivity, to what was determined morphologically to be the Golgi cell of the cerebellum, was noted for 7/34 (21%) of subjects with ASD, compared with 0/23 of the typically developing controls. Furthermore, there was a strong association between the presence of antibodies reactive to the 52 kDa protein by Western blot with positive immunohistochemical staining of cerebellar Golgi cells in the ASD group (r = 0.76; p = 0.001) but not controls. These studies suggest that when compared with age-matched typically developing controls, children with ASD exhibit a differential antibody response to specific cells located in the cerebellum and this response may be associated with a protein of approximately 52 kDa.

Introduction

Autism spectrum disorders (ASD) are a group of heterogenous, behaviorally defined disorders characterized by disturbances in social interaction and communication (verbal and non-verbal) and often with repetitive and stereotyped behavior which is typically apparent between 2 and 3 years-of-age. The prevalence of ASD has been estimated at 1 in 150 in the total population and affects approximately four times as many males as females (MMWR, 2007). The potential exists for numerous etiologies, including those of a genetic and/or environmental nature, which could contribute to the development of ASD.

Immune system-related irregularities associated with ASD involve the innate as well as the adaptive arms of the immune system. Immune findings in ASD are varied and include increased numbers of circulating monocytes, decreased natural killer cell lytic activity, abnormal cytokine, and immunoglobulin levels and decreased peripheral lymphocyte numbers, and responsiveness (Ashwood et al., 2006, Croonenberghs et al., 2002, Molloy et al., 2006, Stubbs and Crawford, 1977, Sweeten et al., 2003, Trajkovski et al., 2004, Warren et al., 1987, Warren et al., 1990, Yonk et al., 1990). The presence of several putative autoantibodies to various elements of the nervous system has also been reported in ASD, including antibodies directed against myelin basic protein (MBP), brain serotonin receptor, neurofilament proteins, brain endothelial cell proteins, heat shock protein as well as autoantibodies directed against epitopes within the cerebellum (Cabanlit et al., 2007, Connolly et al., 1999, Evers et al., 2002, Plioplys et al., 1994, Silva et al., 2004, Singer et al., 2006, Singh et al., 1998, Singh et al., 1997a, Singh et al., 1997b, Singh et al., 1993, Todd et al., 1988, Vojdani et al., 2002). It is important to note that many of these autoantibodies are not unique to individuals with ASD, nor are they found in all subjects with ASD. MBP self-reactive antibodies, for example, are often identified in individuals with multiple sclerosis (Egg et al., 2001).

The neuropathology of ASD is still in its infancy (Amaral et al., 2008). Yet, cerebellar irregularity is one of the most consistent findings in the brains of subjects with ASD. For example, in a review by Brambilla et al. of the 24 post-mortem cases of autism reported in which the cerebellum was studied, 19 demonstrated a lower number of Purkinje cells (Brambilla et al., 2003). The lack of cells is most commonly observed in the cerebellar hemispheres. Other abnormalities observed in the cerebellum or cerebellum-related brain regions, such as changes overtime in the inferior olive, deep cerebellar nuclei, and increases in white matter have also been described in post-mortem tissue (Bailey et al., 1998, Kemper and Bauman, 1998). Interestingly, very little information is available with respect to the other neuronal cell types found in the cerebellum, although Yip et al., recently described reduced GAD 67 mRNA in the basket and stellate cells of the molecular layer in post-mortem autism tissue (Yip et al., 2007).

Previous studies describing the presence of antibodies to various neural proteins as well as neuropathological indications for a reduced number of Purkinje cells led us to examine plasma from a cohort of extremely well-characterized children with ASD as well as age-matched typically developing and developmentally delayed controls using a two-pronged approach. First, to look for the presence of specific autoantibodies to brain tissue, we examined plasma from children with ASD and controls for reactivity to human brain protein extracts using Western blot analysis. Second, to identify specific autoantibodies that were directed to neural structures using immunohistochemistry, plasma of subjects with ASD were examined for their ability to bind to sections from the Macaca fascicularis monkey cerebellum. These approaches enabled us to determine both the apparent molecular weight and the cellular location of the target molecule(s).