We have carried out a detailed examination for the presence antibodies that are directed against cerebellar antigen(s) in subjects with autism spectrum disorders (ASD), by western blot and immunhistochemistry. A substantial number (21%) of individuals with ASD were found to have specific plasma antibodies that were directed against proteins of approximately 52kDa molecular weight from human cerebellum. In contrast, such reactivity was absent in the plasma of all but two control subjects: one typically developing control and one with developmental delay without ASD. To determine which neural or glial component of the cerebelleum these antibodies identified, we carried out immunohistochemical analysis of sections from the cerebellum of Macaca fascicularis
monkey brain. We demonstrated a consistent, cell-specific staining in 21% of subjects with ASD, compared to 0% of either control group. Of particular note was the association found between plasma reactivity to a distinct band at approximately 52 kDa as determined by western blot of both human and monkey cerebellar proteins and the presence of reactivity to Golgi cells. This finding raises the possibility that the autoantibodies specific for the Golgi cells in the cerebellum may be recognizing a protein that is approximately 52 kDa. However, it is possible that the approximately 52 kDa protein is not isomorphic with the Golgi cell antigen(s) that are recognized by plasma from children with ASD. The identity of the 52 kDa band is currently unknown. Previous studies have cited autoantibodies to neural proteins in the blood of some children with ASD, including antibodies to glial fibrillary acid protein (GFAP) that has an apparent molecular weight of approximately 50 kDa (Singh, et al., 1997b
). Through absorption studies and western blot analysis, we have determined that the 52 kDa band does not appear to be GFAP (data not shown), Therefore, due to the apparent specificity of autoantibody reactivity to an apparently 52 kDa band in the subjects with ASD, further identification of this protein is currently in progress.
Previous studies published by our group have described the presence of autoreactivity to bands, predominantly in the hypothalamus and thalamus, which appear to be specific for a subpopulation of children with ASD (Cabanlit, et al., 2007
). While such reactivity appears to a lesser extent in the cerebellum and are certainly of interest to us, only the 52 kDa band correlated with the presence of Golgi cell staining.
Our finding of selective Golgi cell immunoreactivity adds to a growing literature on the presence of autoantibodies to neuronal tissue in subjects with ASD. In a recent study by Singer et al, a variety of brain-specific autoantibodies were identified in the serum of subjects with autism spectrum disorders by both ELISA and western blot analysis (Singer et al., 2006
). Interestingly, the authors found that subjects with autism disorder as well as their non-autistic siblings had denser bands of antibody reactivity at 73 kD in the cerebellum and cingulate gyrus when compared with controls (Singer et al., 2006
). More recently, using fetal rat brain, Zimmerman and colleagues reported reactivity by immunoblotting in subjects with autism (Zimmerman et al., 2007
). Siblings of ASD-affected individuals could be differentiated based on their antibody pattern of reactivity. However, the pattern of antibody reactivity observed in subjects with ASD when compared to subjects with other neurodevelopmental disorders, such as developmental delay without autism, did not distinguish between groups.
The potential role of autoantibodies in nervous system disorders has been widely investigated. Elevated levels of circulating autoantibodies to nervous system components have been reported in a number of psychiatric disorders including schizophrenia, obsessive-compulsive disorder, neuropsychiatric symptoms associated with systemic lupus erythematosus (SLE), pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS), and Gilles de la Tourette’s Syndrome (TS)(Huerta et al., 2006
; Jones et al., 2005
; Kiessling et al., 1994
; Pandey et al., 1981
; Rothermundt et al., 2001
; Snider and Swedo, 2004
; Yeh et al., 2005
). While the findings of some of these studies remain controversial, they all point to the potential for autoantibodies to play a role in neural dysfunction.
Autoantibodies, such as those described herein, have the potential to exert their effects through several different mechanisms. For example, some autoantibodies act as ligands and are able to bind to a receptor and induce hyperactivity, or even excitotoxic death through excessive signaling. An example of this mechanism was demonstrated in a recent study of patients with cognitive and neuropsychiatric symptoms associated with systemic lupus erythematosus (SLE)(Kowal et al., 2004
). The authors found that antibodies to dsDNA from the serum of patients with SLE cross-react with the NR2a polypeptide of the NMDA receptor (NMDAR). In a subsequent study by this group, following lipopolysaccharide (LPS) exposure to compromise blood brain barrier integrity, passive intravenous transfer of serum with reactivity to DNA and NMDAR extracted from lupus patients elicited cognitive impairments in the recipient mice. Brain histopathology revealed that the passively transferred human antibodies were capable of causing apoptotic neuronal death in the hippocampus (Huerta et al., 2006
). Alternatively, autoantibodies to neural antigens can act as an antagonist by blocking an essential pathway that may lead to abnormal nervous system development and/or function. Finally, autoantibodies may also mediate tissue destruction through either complement-mediated or cell-mediated cytotoxicity. Any of these scenarios could potentially result in alterations in neuronal function, receptor density and/or distribution, or an increase or reduction in the release of neurotransmitters and/or cytokines. It is currently premature to speculate whether the Golgi cell-reactive autoantibodies are of pathogenic significance or merely an epiphenomenon. Thus, we are pursuing additional studies to determine whether the antibodies that we have identified here may have deleterious effects on brain structure and function in animal models.
The autoantibodies described herein demonstrate intense, defined reactivity for the Golgi cell, which is a large interneuron that participates in the glomeruli of the granule cell layer and acts as a down-regulatory neuron. As discussed above, such antibodies could have an impact, through various potential mechanisms, on down-stream function. For example, Golgi cells modulate the activity of the mossy fiber to granule cell excitatory synapses, thus influencing the input that leaves the cerebellum via the Purkinje cells (De Schutter et al., 2000
; Hirano et al., 2002
). Interference in this neural pathway could have profound effects on cerebellar and ultimately cerebral function.
In order for the Golgi-cell specific autoantibodies to be of pathological significance, they must be able to access their target antigens in the cerebellum. While the brain has historically been described as an immune-privileged site, a more accurate depiction is an immunologically customized site (Cohen and Schwartz, 1999
; Schwartz and Cohen, 2000
). Immune responses are tightly regulated, and as in other parts of the body, immune surveillance also occurs routinely in the central nervous system, even in the presence of an intact blood-brain barrier (Hickey, 2001
). Antibodies in the circulation are capable of reaching the brain when there is a disturbance in the blood brain barrier, such as was noted above in the Kowal LPS model (Kowal et al., 2004
). Further, autoantibodies have been shown to reach intracellular targets, even those in the nucleus, in living cells (Alarcon-Segovia et al., 1978
; Yanase et al., 1997
). This penetration has been shown to result in cell cycle changes, alterations in gene expression, as well as apoptotic cell death.
At present, the pathological significance of elevated levels of autoantibodies to cerebellar protein(s) in ASD is unclear. These autoantibodies may be pathologically relevant, or merely an epiphenomenon of abnormal central nervous system development or brain injury in children with ASD. We are currently investigating the distribution of the target antigen in other regions of the brain. Moreover, further work is under way to both replicate these findings in a larger study cohort, and to determine the protein targets of these antibodies, the identification of which is key to deciphering their potential role in ASD. Once these issues are addressed, we will better understand the pathological significance, for at least some forms of ASD, and of the cerebellum-specific autoantibodies described herein.