Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes (intracerebroventricular, subcutaneous, intraperitoneal) or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as increased levels of SCFA's can epigenetically modulate cell function further supporting their role as environmental contributors to ASD.

Stress stmulates the sympathoadrenal sys-

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The precise mechanisms by which beneficial responses to acute stress are transformed into long-term pathological effects of chronic stress are largely unknown. Western blot analyses revealed that members of the AP1 transcription factor family are differentially regulated by single and repeated stress in the rat adrenal medulla, suggesting distinct roles in establishing stress-induced patterns of gene expression in this tissue. The induction of c-fos was transient, whereas marked elevation of long-lasting Fos-related antigens, including Fra2, was observed after repeated immobilization. We investigated DNA protein interactions at the AP1-like promoter elements of two stress-responsive genes, tyrosine hydroxylase and dopamine beta-hydroxylase. Increased DNA-binding activity was displayed in adrenomedullary extract from repeatedly stressed rats, which was predominantly composed of c-Jun- and Fra2-containing dimers. The induction of Fra2 and increased AP1-like binding activity was reflected in sustained transcriptional activation of tyrosine hydroxylase and dopamine beta-hydroxylase genes after repeated episodes of stress. The functional link between Fra2 and regulation of tyrosine hydroxylase and dopamine beta-hydroxylase transcription was confirmed in PC12 cells coexpressing this factor and the corresponding promoter-reporter gene constructs. These studies emphasize the potential importance of stress-evoked increases in the expression of the Fra2 gene for in vivo adaptations of the adrenal catecholamine producing system.

Immobilization stress elevates adrenal mRNA for several catecholamine biosynthetic enzymes. Phenylethanolamine N-methyltransferase (PNMT) mRNA is elevated in response to as little as 5 minutes of immobilization, which appears to be a direct consequence of the large rise of glucocorticoids. Tyrosine hydroxylase (TH) mRNA levels are not elevated under these conditions, but are maximally induced by 30 min of a single immobilization. Dopamine beta-hydroxylase (DBH) requires repeated stress for maximal induction. Transcriptional inhibitor actinomycin D prevented the elevation of TH and PNMT mRNA with a single immobilization. Oligonucleotides corresponding to the AP-1 like element in the TH promoter and an important regulatory element in the DBH promoter were used in binding assays with adrenomedullary extracts of rats exposed to stress. The complexes were UV cross-linked and analyzed by gel electrophoresis. The complex bound to the TH AP-1 site contained c-fos and other proteins (including cJun). The complex to the DBH-1 element contained several proteins, probably including fos related proteins but lacked c-fos. Immunoblots found that c-fos is induced in the adrenal medulla near maximally by one hour immobilization and cJun and JunD are present in higher constitutive levels and are only moderately regulated by immobilization stress. The results indicate that different mechanisms of transcriptional activation are used by several members of the catecholamine biosynthetic pathway.

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