CHAPTER V
SUMMARY
Clinical Issues
Autism Spectrum Disorders (ASDs) are relatively common neurological disorders; the most common ASDs are Autistic Disorder ("classical" autism), Asperger Disorder, and PDD-NOS (Pervasive Developmental Disorder-Not Otherwise Specified). Current estimates from the Centers for Disease Control and Prevention indicate an overall ASD prevalence of 1 per 110 children. The three essential criteria for an ASD diagnosis are: (a) impaired verbal and nonverbal communication; (b) impaired social interaction; and (c) restricted, repetitive and stereotyped patterns of behavior, interests and activities. Family studies (including studies of twins) have shown a strong hereditary basis for ASDs, apparently involving a large number of genes, but it is also clear that environmental factors play a significant role in the etiology and severity of these disorders.
An international effort to identify "autism-related" genes has demonstrated an association with autism (in some families) of mutations in genes encoding synaptic proteins. It is now generally accepted that mutations affecting structural components of synapses provide a significant risk factor for the development of ASDs. Thus far, the best-studied of these autism-associated proteins are the neuroligins: post-synaptic adhesion/signaling proteins that bind specifically to a set of presynaptic membrane proteins called neurexins. There are 4 neuroligin encoding (NLGN) genes in humans, and mutations disrupting NLGN3 and NLGN4 are associated with autism. However, it remains unclear how disruptions of widely expressed synaptic proteins can contribute to the relatively specific behavioral deficits associated with ASDs.
Results
We investigated the effects of neuroligin disrupting mutations in the nematode Caenorhabditis elegans. Using worms to study the effects of autism-related mutations is not as far-fetched as it might seem. Numerous studies have shown that C. elegans neuronal proteins are structural and functional homologs of the corresponding mammalian proteins, and it is now well established that C. elegans provides a powerful model for analyzing synapse structure, function and development. We characterized C. elegans neuroligin, and showed that the protein is quite similar in structure to its mammalian homologs. Worms have a single neuroligin gene (nlg-1), and mutants completely lacking the neuroligin protein have superficially normal growth and behavior, and apparently normal nervous systems. However, nlg-1 mutants have several sensory deficits; for example, they lack the normal (wild-type) response to specific chemicals and they are insensitive to changes in temperature. The mutants also have deficits in the processing of conflicting sensory inputs.
The other important results follow from the observation that nlg-1 mutants have increased levels of oxidative stress. Oxidative stress is caused by an excess of free radicals and reactive oxygen species (ROS) that are toxic and can cause damage to cellular components (e.g., proteins, lipids, and DNA). The elevated oxidative stress was demonstrated in two ways. First, nlg-1 mutants are hypersensitive to the toxic effects of paraquat (an herbicide that produces excess free radicals and ROS), implying that the mutants have elevated levels of endogenous free radicals. In addition, the extent of oxidative damage to proteins in nlg-1 mutants was much greater than that found in wild-type animals. Another consequence of the oxidative stress caused by loss of neuroligin in nematodes is that nlg-1 mutants are hypersensitive to the toxic effects of copper and mercury-containing compounds.
Implications and future directions
Although sensory issues are not part of the official diagnostic criteria for ASDs, problems of sensory hypersensitivity, insensitivity or habituation are commonly reported, as well as difficulties with the processing and/or integration of simultaneous sensory inputs. It is therefore particularly intriguing that nlg-1 mutants have specific sensory deficits, as well as deficits in the processing of conflicting sensory inputs.
Another possible similarity between nlg-1 mutants and individuals with ASDs involves oxidative stress. Oxidative stress is caused by an excess of free radicals and ROS, and there are many examples of mutations in detoxifying enzymes or mitochondrial proteins that result in oxidative stress. However, the elevated oxidative stress present in nlg-1 mutants is a completely unexpected phenotype for a synaptic protein mutant.
A number of studies have been published demonstrating elevated markers of oxidative stress in individuals with ASDs, although the methods of patient selection, the sample sizes and the designs of these studies may have some limitations. More importantly, however, the nature of the possible relationship between autism and oxidative stress has never been clear. At best, these studies provide a correlation between oxidative stress and ASDs, yet a model often proposed is that oxidative stress (resulting from environmental toxins) somehow causes or contributes to the development of ASDs. Some of these studies and putative models have migrated from the scientific literature to the popular press, and unfortunately, the limitations and caveats associated with these investigations have not always migrated with the models themselves.
An important result is that loss of the synaptic protein neuroligin is not merely correlated with oxidative stress, but it actually causes the oxidative stress. If oxidative stress is a consequence of aberrant synapticstructure and/or function in nematode neurons, then it seems plausible to expect that similarly aberrant synapses between human neurons would have similar consequences. This raises the possibility that specific types of neuronal disruption might be the cause, rather than the result, of oxidative stress. In addition, these data demonstrate a clear connection between an autism-associated synaptic mutation in C. elegans, and hypersensitivity to environmental toxins (e.g., paraquat, mercury compounds, etc.), and this provides an important example of how both genetic and environmental contributions to a neurological disorder can have a single underlying basis.
Hopefully, future experiments will elucidate the precise mechanism by which genetic disruptions of synapse proteins can trigger oxidative stress. Such mechanisms would provide a molecular framework for future biochemical and metabolic studies, initially using mouse models of autism and then individuals with ASDs. It is likely that some of the symptoms associated with ASDs are consequences of increased oxidative stress; therefore, progress in the cell biology of oxidative stress and the biochemistry of antioxidants may make it possible to design rational antioxidant strategies for the treatment of such symptoms.