Kilee Patchell-Evans Autism Research Group (KPEARG) - Using Multidisciplinary Methods to Study the Role of the Gut Microbiome in Autism Spectrum Disorders:
Autism spectrum disorders (ASD) are an increasing problem in Western society. Originally thought to be a relatively rare disorder of approximately one in 10,000, current studies of ASDs are showing ASDs to be as common in one in 68, and may rapidly be on the increase. Although heritable factors may a major role in ASD, recent research is examining ASD as a systemic encephalopathic condition involving immune, digestive and metabolic dysfunction exacerbated by environmental triggers in genetically sensitive subpopulations. Clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in ASD. Furthermore, pre- and post-natal infectious processes, hospitalization, antibiotic exposure, and alteration of bacterial populations of the gut microbiome, have been implicated as possible risk factors for ASD.
Propionic acid (PPA) is a short chain fatty acid and an important intermediate of cellular metabolism. PPA is also a fermentation by-product of a subpopulation of opportunistic enteric bacteria (i.e. Clostridia, Desufovibrio, Propionibacteria, Bacteriodetes, Sutterella ssp.), a putative risk factor for ASD, and is also a common food preservative. PPA and other short chain fatty acids (i.e. butyrate and acetate), affect diverse physiological processes such as cell signaling, neurotransmitter synthesis and release, mitochondrial function, lipid metabolism, immune function, and gene expression. Furthermore, genetic or acquired human subpopulations exist (organic acidurias, biotin/carnitine deficiency, ethanol/valproate exposure, mitochondrial disorder/dysfunction) which have impairments in PPA metabolism and produce neurodevelopmental conditions with ASD features. Thus PPA and related short chain fatty acids are ideal compounds linking the disparate brain, behavioral, dietary, gut, metabolic and immune factors implicated in ASD.
We have found that intracerebroventricular infusions of PPA in adult rats through chronic indwelling brain cannula induces predictable and enduring changes on brain neocortical, hippocampal, and striatal electrical activity. PPA infusion immediately produces reversible repetitive dystonic behaviors, hyperactivity, turning, retropulsion, object fixation, perseveration, caudate spiking, the progressive development of limbic kindled seizures, and the impairment of social behavior, suggesting that this compound has central effects. Examination of brain tissue from PPA treated rats (brain sections, homogenate, ToF-SIMS imaging, gene arrays) reveals an innate neuroinflammatory response (reactive astrogliosis and activated microglia, CREB activation), an increase in oxidative stress markers, reductions in cholesterol, altered phospolipid/acylcarnitine profiles, citric acid and mitochondrial dysfunction, and a reduction of glutathione which is a broad spectrum xenobiotic detoxifier. Current studies in our laboratory are finding similar effects with systemic infusions of PPA at the post-natal and adolescent times and also with infusions of butyric acid, a related gut short chain fatty acid. Emerging studies in the animal model, and human/animal cell lines, are finding short chain fatty acids produce complex alterations in 1) autism associated mitochondrial activity or 2) genes or biochemical pathways. Collectively, these findings are consistent with those found inpersons with ASDs.
Collectively, these central systemic and in vitro effects of PPA and related enteric short chain fatty acid metabolites suggest a possible link between diet, seizure, movement disorder, social impairment, neuroinflammation, increased oxidative stress, mitochondrial dysfunction, altered gene expression, microbiome diversity and environmental sensitivity found in ASD. We propose that some types of ASD may be partial forms of genetically inherited or acquired disorders of altered short chain fatty acid metabolism, resulting in increased exposure to these enteric metabolites at critical times during the life cycle. This study also suggests the novel concept that the gut microbiome may have evolved to utilize production of these compounds to influence behavior in the host to ensure survival. These findings may also lead to the development of novel therapies and biomarkers to screen, predict, reduce, or treat the condition. Collaborations are underway to determine human populations (Canadian Somalis, neonatal populations) at risk for increased exposure to elevated short chain fatty acid exposure at critical neurodevelopmental periods, and specific biochemical mechanisms responsible for these changes.
The Kilee Patchell-Evans Autism Research Group (KPEARG) is examining the neurobiology of gut short chain fatty acids and related compounds in rodents throughout the lifecycle at a multidisciplinary level. Techniques include:
- Automated neurobehavioral assays (locomotor activity, social behavior, anxiety, perseveration, learning and memory)
- Electrophysiology (cortical and subcortical EEG of behaving animals),
- Pathology (neurodevelopment, neuroinflammation, fatty acid transport, blood brain and gut blood barrier integrity, via immunohistochemistry and in situ hybridization),
- Molecular biology (protein, lipid, gene expression, metabolomics, 16S ribosomal RNS) of brain homogenates, tissue culture (PC12., human lymphoblasts), or stool samples
- Time of Flight Secondary Ion Mass Spectroscopy (ToF-SIMS) is a novel imaging technique which we are modifying to allow us to simultaneously examine multiple molecules (i.e. free radical modified biomolecules, lipid profiles, solvent content, neurotransmitter, metals) in brain or gut tissue sections
- Lipid analysis (mass spectroscopy, electrospray, metabolomics, free fatty acid G coupled receptor assays)
We frequently structure our studies to examine multiple behavioral, electrophysiological, in situ/immunohistochemical, and molecular biological parameters in the same animal. Tissue libraries of the above studies are available for future collaborative efforts.