Dietary tryptophan is metabolized through three main pathways: the serotonin, kynurenine (KYN), and indole metabolic pathways. Over 95% of tryptophan is oxidized and degraded to yield metabolites along the KYN pathway
[69]. Importantly, tryptophan is also the sole precursor to the neurotransmitter serotonin (5-HT) in the brain and gut (synthesized by action of enzyme tryptophan hydroxylase [TPH])
[69]. Whereas gut microbiota play a modulatory role in the balance between serotonin and KYN production, the biosynthesis of indoles and indole derivatives (e.g., indole-3-aldehyde, indole-3-acetic acid, indole-3-propionic acid) from tryptophan is fully dependent on the enzyme tryptophanase, only found in select microbes
[70]. How changes in the relative abundance of certain gut microbiota contribute to modifications of these pathways, central tryptophan metabolism, and ultimately brain function and behavior, is a crucial and ongoing area of research
[71]. Many of these findings also require analysis of how this relationship modulates clinical symptoms characteristic of neurodevelopmental disorders, such as ASD.
4.1.1. Indole Pathway and ASD
Indole synthesis is driven by certain bacterial taxa that convert undigested tryptophan from the gut lumen into indole and indole derivatives, constituting an exclusively microbe-dependent pathway
[70]. Many of the phyla, genera, and species associated with the production of indoles and altered indole products involved in tryptophan metabolism have been linked to the development of ASD and related neuropsychiatric disorders
[33][55][72][73][74][75]. Mice from the MIA model have shown abnormally high levels of key serum metabolites produced by gut microbes, including 4-ethylphenylsulfate, serum indolepyruvate, and indole-3-acryloylglycine, all of which were readjusted by treatment with
B. fragilis [49]. In comparison, a human study also found that urinary metabolites of ASD and TD children significantly differed along the tryptophan and purine metabolic pathways, suggesting that the gut microbiome contributes to abnormal tryptophan metabolism in ASD
[73]. Specifically, gut bacteria-derived metabolites indolyl-3-acetic acid and indolyl-lactate were more numerous in the ASD group compared to controls
[73], consistent with the findings from Xiao et al.
[55] in the cecal matter of mice that had received FMT from ASD donors. These altered pathways overlapped with those of rodent models which displayed ASD-like behaviors, demonstrating a potential pathophysiological explanation for many behavioral symptoms of ASD
[73]. Similarly, De Angelis et al.
[72] found increased indole and 3-methylindole in the fecal samples of ASD children. After indole is absorbed in the gut, it is oxidized and sulfated by liver enzymes into indoxyl and indoxyl sulfate metabolites, respectively. Interestingly, these indole metabolites have been identified as potential metabolic markers for ASD, as well
[76][77].
4.1.2. Kynurenine Pathway and ASD
The KYN pathway, also derived from tryptophan and modulated by gut microbes, largely depends on indoleamine-2,3-dioxygenase (IDO) and, to a lesser degree, tryptophan-2, 3-dioxygenase (TDO) for metabolization
[78]. IDO, expressed in all body tissues, is typically activated in the presence of pro-inflammatory cytokines, whereas TDO, expressed primarily in liver tissues, is activated by glucocorticoids
[78][79]. Once transformed from tryptophan, KYN metabolizes into two downstream metabolites, neuroprotective kynurenic acid (KA) and neurotoxic quinolinic acid (QA)
[78]. Recent evidence suggests that altered KYN metabolism is indicative of greater tryptophan depletion and an impaired serotonergic pathway in ASD
[80]. In a study investigating the role of the KYN pathway in ASD, Bryn et al.
[81] showed that children with ASD had significantly lower KA serum levels, higher KYN/KA ratios, and higher QA serum concentrations than TD children. These findings are consistent with those of Gevi et al.
[73], who found that tryptophan was disproportionately metabolized into QA, with significantly decreased levels of KA, in children with ASD. Both studies demonstrate an increased potential for neurotoxicity in children with ASD, which is thought to be involved in the pathophysiology of the disorder
[73][81]. Interestingly, Xiao et al.
[55] found increased KA in mice following FMT from children with ASD. These levels correlated with specific bacteria (e.g., genera in the orders
Clostridiales and
Bacteroidetes), supporting their modulatory role in tryptophan metabolism, but demonstrating a need for further research on how microbiota alter KYN-pathway products
[55].
Although there is minimal literature associating gut microbiota with the KYN pathway in human ASD populations, studies in other clinical and normative populations have provided evidence to support this relationship
[78]. Interestingly, Luna et al.
[82] found in their study of ASD microbiome-neuroimmune signatures that along with tryptophan and serotonin levels, inflammatory cytokine levels correlated with certain bacterial species in children with ASD and functional GI disorder comorbidities. No direct link was made to the KYN pathway; however, because IDO is typically activated in response to cytokines
[78][79], there is reason to investigate whether the abnormal microbial profile of individuals with ASD may be implicated in the dysregulation of the KYN pathway.
4.1.3. Serotonin Pathway and ASD
Serotonin (also referred to as 5-HT) is important for mood regulation, higher order cognition, and neurodevelopment of both the CNS and ENS
[83][84]. Although the majority (>90%) of serotonin comes from enterochromaffin cells in the GI tract, serotonin is also synthesized in the neurons of the ENS and CNS, particularly the raphe nuclei in the brainstem
[85]. Gut microbiota and their metabolites can influence central and peripheral serotonin production and metabolism through a variety of mechanisms
[71][86]. Because only a small percentage of tryptophan is converted into serotonin, any alterations to its metabolism and availability can pose a significant risk to one’s health
[87].
Approximately 30% of ASD patients have hyperserotonemia, or elevated whole-blood serotonin levels
[88], which is believed to be due in part to increased serotonin production in enterochromaffin cells in the gut
[89]. Based on similar and replicated findings, it has been postulated that hyperserotonemia may represent a highly heritable biomarker of ASD and that the serotonin pathway as a whole may be dysfunctional in at least a subgroup of ASD individuals
[89][90]. In preclinical models, hyperserotonemia has been linked to social-behavioral deficits characteristic of ASD
[49][91][92]. Tanaka et al.
[92], for example, found that a tryptophan-depleted diet, which decreases brain serotonin levels and regulates gene expression inside the serotonin system, improved social impairments of genetically modified ASD mouse models. Lim et al.’s
[91] report of elevated serum serotonin levels in environmental risk factor mouse models of ASD that were associated with changes in bacteria known to stimulate serotonin production suggests that alterations in serotonin and hyperserotonemia itself may have a microbial origin. The connection between serotonin and the microbiome has been made in humans as well, as demonstrated by a link between increased GI symptom severity and hyperserotonemia in ASD youth
[93]. Other studies investigating serotonin-related dysfunction in children with ASD and co-occurring GI symptoms have implicated fecal metabolites in the metabolic network of various neurotransmitters, including serotonin
[33], and have found increased levels of serotonergic metabolites, including 5-HIAA, the main metabolite of serotonin, in the rectal tissue of ASD youth with co-occurring functional GI disorders
[82]. These metabolite levels correlated with the dysbiosis of several bacterial species, demonstrating a potential microbiome profile for ASD
[82].
SERT Ala56, the most common variant of the serotonin-selective transporter responsible for serotonin reuptake in both the brain and intestines, has been found to be overexpressed in ASD patients and linked to neurobiological and GI symptoms in a genetically modified murine ASD model
[94]. SERT Ala56 mice are also known to exhibit serotonin-related dysfunction, including excess clearance of central serotonin, augmented serotonin receptor sensitivity, and hyperserotonemia
[95]. Research supporting connections between an altered serotonin system and ASD pathophysiology has demonstrated a positive relationship of serotonin and SERT levels with autism symptom severity in humans
[96]. Furthermore, numerous animal studies have implicated gene polymorphisms of SERT, as well as genetic and surface transporter expression and function, in the underlying repetitive behaviors and social behavior deficits of ASD, for a review, see
[97].
Contributing to the link between serotonin, gut microbiota, and ASD, the BTBR inbred strain has been shown to display (1) reduced SERT density and binding throughout the brain and increased serotonin activity in the hippocampus (a brain region involved in learning, social, and emotional processing, and found to be abnormal in ASD)
[98][99][100]; (2) changes in intestinal microbiota associated with slowed GI motility and impaired intestinal serotonin production
[48]; and (3) increased sociability following brief exposure to serotonin reuptake inhibitors
[98][101] and tryptophan supplementation
[102]. Taken together, these studies support the hypothesis that altered gut microbiota are involved in the tryptophan-serotonin metabolic pathway in ASD and provide a framework for future studies aiming to alleviate the GI and, consequently, behavioral symptoms of ASD patients.
A recent study by Fung et al.
[103] showed that the gut bacterium
Turicibacter sanguinis expresses a neurotransmitter sodium symporter-related protein with sequence and structural homology to mammalian SERT. This microbe imports serotonin through a mechanism that, like its host homologue, is inhibited by the selective serotonin reuptake inhibitor, fluoxetine. Serotonin reduces expression of sporulation factors and membrane transporters in
T. sanguinis, which is reversed by fluoxetine exposure. Treating
T. sanguinis with serotonin or fluoxetine modulates its competitive colonization in the GI tract of antibiotic-treated mice. In addition, fluoxetine reduces membership of
T. sanguinis in the gut microbiota of conventionally colonized mice. One may speculate that genetic variants exist for the microbial SERT-like mechanism and that alterations in the bidirectional host-microbe interactions in tryptophan metabolites play a role in ASD pathophysiology, including gut symptoms.