This study had two primary goals: 1) To further characterize the occurrence of autoantibodies to cerebellum in children with autism spectrum disorders with respect to behavioral outcome, and (2) To ascertain if an association exists between the presence of brain-directed autoantibodies in children and the presence of brain-directed antibodies in their respective mothers. Autoantibody profiles differed between children with autism (AU), the broader phenotype of autism spectrum disorder (ASD), and typically developing (TD) controls. Moreover, we demonstrated for the first time that children harboring these antibodies had more impaired behavioral scores as well as lower cognitive and adaptive function compared to children without the antibodies. In addition, as previously reported, mothers of children with AU and ASD show a unique pattern of antibody reactivity to fetal brain proteins compared to mothers of TD children (Braunschweig et al., 2008
; Braunschweig et al., 2010
; Croen et al., 2008
; Zimmerman et al., 2007
). Familial analysis showed a very limited relationship between anti-brain antibodies in plasma from mothers and their children, though this relationship did not extend to the definitive patterns of maternal autoantibodies associated with an AU or ASD diagnosis. This suggests that while there may be some familial propensity for autoantibody production, autism spectrum disorder-associated autoantibodies observed in mothers and children largely occur in different families.
Independent studies have described the presence of autoantibodies directed against various brain proteins in individuals with an autism spectrum disorder (Enstrom et al., 2009b
). We previously characterized autoantibodies towards cerebellum proteins in a smaller group of AU subjects (Wills et al., 2009
). The results of the present study differ to some extent from the Wills study. First, Wills et al.
originally showed that plasma IgG directed towards a 52 kDa cerebellum protein (rather than 45 kDa protein) correlated with an autism diagnosis. This has now been explained by differences in gel systems as noted in the results section. Second, we observed a lower incidence of IgG reactivity to the cerebellum in children with autism in the present study (10% versus 21%). This difference may be attributable to several factors including 1) an increased sample size, which may have revealed a more accurate estimation of the occurrence of brain-directed antibodies among autism subjects, and/or 2) the use of younger study subjects (mean age of 3.5 years compared to 6 years in Wills et al.
) A longitudinal analysis of the same children over time would help to clarify this issue.
This is the first study to examine specific behavioral phenotypes associated with the presence of brain-targeted antibodies in autism spectrum disorders. We demonstrate that children diagnosed with either AU or ASD tend to have divergent IgG targets in the cerebellum; AU children showed significant IgG reactivity towards a 45kDa protein, while ASD children showed reactivity towards a 62kDa protein. One explanation for this difference may be that these proteins are involved in a pathway that impacts behavioral traits, and that interfering the 62kDa protein may lead to a less severe phenotype than interfering with the 45kDa protein.
In addition to the differences observed between AU and ASD groups, we found that children with these autoantibodies had significantly more impaired behavioral, cognitive and adaptive traits than children without the autoantibodies. In many cases, this difference was observed regardless of diagnosis. This suggests that rather than representing a specific marker of autism, these antibodies might somehow be linked to specific behavioral outcomes associated with, but not specific to, autism spectrum disorders. Future studies should examine the occurrence of these antibodies in neurodevelopmental diseases other than autism disorder. A small number of typically developing children also had the antibodies, and it is possible that additional environmental exposures and/or genetic factors are necessary for the development of full AU or ASD. Previous studies have similarly shown that differences in other immune factors such as cytokines levels and total IgG correlate with behavioral severity among children with an autism spectrum disorder (Ashwood et al., 2008a
; Ashwood et al., 2008b
; Grigorenko et al., 2008
; Heuer, 2008). It is unclear whether these differences in immune measures are responsible for variations in behavioral characteristics, or if they are a secondary manifestation of other factors involved in the disease. Collectively, these studies illustrate that biological factors can be linked to specific behavioral characteristics; an observation that can perhaps help differentiate ontogenic mechanisms specific to the varying phenotypes within the autism spectrum.
The clinical and mechanistic significance of these brain directed antibodies is not known, and further research is necessary to explore this issue. First, it should be made clear that it is likely that the target antigens for these autoantibodies are not restricted to the cerebellum (as noted in the study by Cabanlit, et al(Cabanlit et al., 2007
)), and ongoing studies to further examine this possibility are in progress. In addition, it is not entirely clear how a cerebellum-directed immune responses might relate to behavioral abnormalities common in autism spectrum disorders. It is possible that the antibodies observed herein interfere with normal neuronal processes, or are indicative of abnormal cerebellar function. The study by Wills et al
(Wills et al., 2009
) described a very particular staining pattern for antibodies reactive to the 52kDa antigen also reacted against the Golgi interneurons in the Purkinje layer of the cerebellum. These cells act as down-regulators of the excitatory synapses in the granule cell layer of the cerebellum, which impacts the activity of Purkinje cells, and interfering with this pathway could lead to various motor and behavioral abnormalities (Hirano et al., 2002
). Other studies have described cerebellar abnormalities in individuals with an autism spectrum disorder, including reduced numbers of Purkinje cells in post-mortem brains (Bailey et al., 1998
; Kemper and Bauman, 2002
). Further, injury to the cerebellum and alterations in cerebellar development are associated with reduced cognitive function, impaired language, and increased stereotypic behaviors (Gillig and Sanders, 2010
; Martin et al., 2010
; Steinlin, 2008
). For example, mice lacking Purkinje cells demonstrate increased repetitive behaviors (Martin et al., 2010
). Stereotypic behavior, cognition, and language were all found to be more severely affected in children harboring the cerebellum-directed antibodies.
Another critical issue is whether these antibodies are pathogenic on their own or if they are secondary to pathology. In order to be pathogenic, the antibodies must gain access to the central nervous system (CNS). Under normal circumstances, large molecules such as IgG and other immune components are largely excluded from the CNS by the blood-brain-barrier (BBB). However, infectious and environmental factors can increase permeability of the BBB allowing immune components to enter the CNS. Examples of exposures that compromise the integrity of the BBB include pertussis toxin, extreme stress, sub-clinical infection, and exposure to nicotine or epinephrine (Hawkins et al., 2004
; Kuang et al., 2004
; Kugler et al., 2007
; Theoharides and Konstantinidou, 2007
). It is possible that TD children with the autoantibodies may not have had the required insult that would allow passage of the autoantibodies to the neuronal targets.
Once in the brain, autoantibodies can act through various pathogenic mechanisms. First, they can mimic receptor ligands and induce excitotoxic death through excessive signaling. This has been demonstrated in individuals with systemic lupus erythematosus (SLE) accompanied by cognitive and neuropsychiatric symptoms (Kowal et al., 2004
). In these subjects, DNA-specific antibodies are able to cross-react with the NMDA receptor for glutamate, and administration of these antibodies to mice with a compromised blood brain barrier (BBB) leads to cognitive impairments and apoptotic neuronal death (Huerta et al., 2006
). Autoantibodies to neural antigens might also block vital pathways and compromise the development and function of the nervous system. Alternatively, autoantibodies can cause tissue destruction by fixing complement or inducing cell-mediated death. Studies involving passive transfer of these antibodies in animal models will be essential to explore their pathogenic significance.
Alternatively, it is entirely possible that these antibodies were produced as a result of previous neuronal injury, and may not have pathogenic significance on their own. An event in the CNS (perhaps an infection or injury) could have altered the course of neurodevelopment leading to an autism disorder; and simultaneously spurred an immune reaction that caused a break in immune tolerance towards CNS antigens. The antibodies produced in the course of such an event may or may not have pathogenic properties on their own. If these antibodies are not pathogenic, they still represent a potentially valuable biological marker for a subset of autism spectrum disorders and/or autism-associated behaviors. Since the underlying biology of behavioral disorders like autism remains poorly understood, the discovery of any biological connection could be of interest; even one that is secondary to pathology/etiology. Further, if autism behaviors are the result of neuronal injury, and antibodies are produced as a secondary result of this neuronal injury, the antibodies may serve to point researchers towards specific neuronal components/processes that may be involved in the neuropathology. Identification of the 45kDa and 62kDa antigens will be an important step towards understanding their role in autism spectrum disorders.
Independent studies have shown that a subset of mothers of children with autism harbor circulating IgG directed towards fetal brain proteins (Braunschweig et al., 2008
; Croen et al., 2008
; Singer et al., 2008
; Zimmerman et al., 2007
). This is in contrast to children with AU or ASD, who demonstrate IgG reactivity to the mature brain rather than the fetal brain (Cabanlit et al., 2007
; Morris et al., 2009
; Singer et al., 2006
; Wills et al., 2009
). The mothers included in this study demonstrated IgG reactivity to fetal brain proteins as previously described (Braunschweig et al., 2008
; Braunschweig et al., 2010
; Croen et al., 2008
). Previous primate and murine studies have demonstrated the potential pathogenic significance of maternal anti-fetal brain antibodies, where prenatal exposure to purified IgG from mothers of children with autism resulted in behavioral alterations that were not observed in controls (Martin et al., 2008
; Singer et al., 2009
). Our final analysis in the current study was designed to determine if there was a familial (maternal/child) relationship for brain-directed antibodies. Our results show very limited relationships between the anti-brain autoantibodies in AU, ASD, and TD mother-child pairs. Interestingly, we found that the overall presence of brain-directed antibodies in maternal plasma has a higher degree of association with an AU or ASD diagnosis than brain directed antibodies in children. It may be that exposure to anti-brain antibodies during gestation is more detrimental to neurodevelopment than exposure to anti-brain antibodies in early childhood. Additionally, the presence of the autoantibodies found in the children may not be sufficient on their own to cause a pathologic insult, or may simply be the result of previous damage through other mechanisms.
In summary, we describe the presence of autoantibodies to proteins in a large well-characterized population of children and their mothers. We further demonstrated that reactivity to specific proteins within the cerebellum was associated with a diagnosis of AU or ASD. Additionally, the presence of these antibodies was linked with more aberrant behaviors and lower cognitive and adaptive function regardless of diagnosis, suggesting a potential association with some features of autism rather than an autism spectrum disorder specifically. Finally, we found limited familial associations for specific patterns of anti-brain antibodies in AU, ASD, and TD mother-child pairs. Future studies will strive to identify the autoantibody targets in the cerebellum and fetal brain, characterize their pathologic significance with respect to autism, and determine whether the development of therapeutic measures would be warranted.
- Plasma autoantibodies for 45kDa cerebellar protein associate with an autism diagnosis.
- Plasma autoantibodies for 62kDa protein associate with broader diagnosis of ASD.
- Children with autoantibodies had worse behavioral scores than children without them.
- Few familial associations found for brain-targeted antibodies in children and mothers.
- Antibodies may be markers for specific behavioral characteristics of autism.