The Gut Brain Axis and the Apparent Links to Autism Spectrum Disorders

PUBLISHED IN ISSUE 1 | FALL 2021

The Gut Brain Axis and the Apparent Links to Autism Spectrum Disorders

MacGregor Thomas

Oral Roberts University

MacGregor Thomas is studying pre-med at Oral Roberts University.

ABSTRACT

In recent years, researchers have pushed the boundaries of understanding of the human
microbiome. Studies have pointed towards the involvement of gut bacteria in human health for
decades, but now scientists have begun to acquire a more comprehensive understanding of the
symbiotic relationship that bacteria have with their hosts. Research shows that gut bacteria can
influence an organism's neural and immune system development, behavior, mood, and even
neurotransmitter concentrations. Like other systems in the human body, a delicate balance of
homeostasis, acting through bidirectional communication between the central nervous system
and the gut, keeps this system in check. Studies show that gut bacterial dysbiosis is directly

correlated to changes in an organism's overall health, with deficits in or overrepresentation ofcertain flora even implicated in the development of neurological disorders, specifically AutismSpectrum Disorders (ASD). Interestingly, research points towards the metabolite byproducts ofthese microorganisms influencing the severity of ASD, as well as gastrointestinal inflammationand alterations in the serotonergic system. While the cause of autism is multifaceted and is stillan active area of study, the evidence that this paper examines puts forth a review on the gut-brain axis and how deficiencies in this crucial system influence the course of autism.

INTRODUCTION

The adult human harbors over 100 trillion bacteria, with approximately 80% residing in
the gastrointestinal tract, commonly referred to as the gut. Bacteria outnumber our own cells by roughly ten times and collectively encode 150 times more genes than our own genome (Wang et al. 2011). With such a presence in our own body, the human microbiota has become an increasing field of research over the past two decades. Mounting evidence suggests that these
microbes share a commensal relationship with our bodies, impacting and modulating cognitive,
behavioral, and even the brain's physiological functions, resulting in potential consequences in
human health and disease states (Wang et al. 2011). Many studies demonstrate microbes' alleged role in many mental health disorders, specifically Autism Spectrum Disorder (ASD). This paper will argue the importance of a healthy gut microbiome and highlight the role of the gut-brain axis in modulating neurocognitive disorders.

As part of an ongoing effort to determine the microbiota's exact microbial composition,
the National Institutes of Health Human Microbiome Project provides the most comprehensive
and thorough descriptions to date. While various bacterial species were isolated from individuals in the past, such a comprehensive study showing the natural microbiome population was lacking. Including the latest extended dataset from the project, researchers secured samples from 2,355 healthy participants, sampling a diverse range of 18 different body sites (Lloyd-Price et al. 2017). Results showing varying diversities of taxa and their contributions to various metabolic pathways from the project are shown (Figures 1-3).

Figure 1: The diversity and spatiotemporal distribution of strains. a. phylogenic diversions c. Niche association from participants. Higher values signify greater phylogenic separation at body sites. f. reference genome set (Lloyd-Price et al. 2017)

Figure 2: Distinguishing functions of the microbiome at various body sites (abundance is logscored) a. Total of 28 metabolic pathways shown in all 6 major body sites. Shown is a broad “supercore” pathway character of many bacteria b. 17 additional pathways specific to the human microbiota c. 21 pathways more pronounced at one body site that at others (Lloyd-Price et al. 2017)

Figure 3: Bacterial contigs’ assembly size, maximum length, mean length, and gene count, relative to samples (Lloyd-Price et al. 2017)

PHENOTYPICAL ATTRIBUTES INFLUENCED BY MICROBIOTA

Several pathways connect the gut to the brain; however, the interactions between a host and its microbiome are exceptionally intricate and complex. Bacteria can produce neurotransmitters, hormones, metabolites, and even influence immune signaling, often using the circulatory system to reach the central nervous system. Immune stimulation and modulation, as shown

previously, can be shaped by microbes as well. Lastly, the enteric nervous system or Vagus nerve directly connects the brain and the gut and is stimulated by microbes. These three pathways (Figure 4) provide ways for bacteria to shape physiological and behavioral processes in an organism (Sampson and Mazmanian 2015).

Figure 4: The three pathways that microbes can interact bidirectionally with the central nervous system (Sampson and Mazmanian 2015)

Mood and Behavior

Sudo and colleagues conducted one of the more noteworthy studies that initially demonstrated that the microbiota is an influential regulator in mood and behavior. In the study, researchers compared specific pathogen-free (SPF) mice, germ-free (GF), and a gnotobiotic mice group in which the microbiome contents were known. In response to restraint stress, the GF mice displayed significantly reduced brain-derived neurotrophic factor (BDNF) in the hippocampus and cortex, higher plasma ACTH levels, and increased corticosterone concentrations, when compared to the SPF mice (Figure 5) (Sudo et al. 2004). This is significant because ACTH and corticosterone elevations are direct indicators of a stress response, and because such levels decreased in SPF and gnotobiotic mice, suggesting that gut bacteria play a role in alleviating a

host's stress response. The fact that a reduction in BDNF accompanied these results proves that this is not just an endocrine phenomenon. BDNF is an essential modulator in neuron proliferation, modulation, and survival. Such data adds a new layer of complexity to the hypothalamic-pituitary-adrenal (HPA) axis. Interestingly, Sudo and colleagues reversed elevated stress responses in the germ-free mice by introducing Bifidobacterium infantis, a bacterium commonly found in the gut. However, when the GF mice encountered Escherichia coli instead, the stress response increased (Figure 6). In such cases, fecal transplants from SPF mice early in life partly corrected the elevated response (Sudo et al. 2004). Such results suggest the microbiota participates in a delicate balance for the developing HPA stress response.

Figure 5: Top: BDNF levels in GF mice (black), compared to SPF mice (white). Bottom: Response to restraint stress (Sudo et al. 2004)

Figure 6: Stress response after colonization with E.coli (EPEC), Bifidobacterium, or E. coli mutant strain (D Tir) (Sudo et al. 2004)

The administration of specific Lactobacillus strains alleviated anxiety-like behavior and
depressive behavior in mice (Bravo et al. 2011). Gamma-Aminobutyric Acid, or GABA, is the
chief inhibitory neurotransmitter in the central nervous system, and alterations in GABA
concentrations are known to influence many psycho-physiological conditions. Specifically,
anxiety and depression's pathogenesis is affected by alterations in the central GABA receptor.
Bravo and colleagues used Lactobacillus rhamnosus to test if bacteria could have a direct impact
on such receptors. They found that in the cingulate and prelimbic cortical regions, GABAB1b
mRNA concentrations increased, while expression levels decreased in the amygdala, hippocampus, and the locus coeruleus, which is responsible for the synthesis of norepinephrine
in the brain. Additionally, GABAAa2 mRNA expression levels in the amygdala and prefrontal
cortex diminished, while expression escalated in the hippocampus. The researchers also found
that the administration of L. rhamnosus reduced stress-induced levels of corticosterone. Lastly,
they demonstrated that the vagus nerve was a key communication pathway between the gut and
brain by treating vagotomized mice with the same bacteria, which had no effect. Behavioral and neurochemical results were not replicated in this group (Bravo et al. 2011). Additionally,

Messaoudi and colleagues showed that anxiety-like behavior significantly decreases in mice during an electric shock test when administered a probiotic blend of B. longum and L. helveticus for 30 days (Messaoudi et al. 2011). These studies from Bravo and Messaoudi add to the evidence that gut bacteria and the brain can communicate bidirectionally and suggest the
possibility of developing treatments for such GABA-dependent disorders using specific probiotic strains.

While many animal studies explore the gut-brain axis, human microbial research is still in
its infancy. One such study, conducted by Tillisch and associates, addressed whether human
consumption of fermented probiotic milk products could affect the neural response to emotional stimuli. Using fMRI analysis of the brain, they found that probiotics could alter the processing of emotion after four weeks. The somatosensory and interoceptive regions of the brain showed less activation in response to emotional stimuli than control groups (Figure 7) (Tillisch et al. 2013).

Figure 7: Brain activity in response to emotional faces attention task. From left to right on scale;no intervention, control, and Fermented Milk Products Probiotic (Tillisch et al. 2013)

Similarly, another study administered L. helveticus and B. longum to rats and humans to determine the impact of probiotics on mood and psychological distress. In rodents, anxiety-like behavior significantly decreased. Human participants were placed in placebo-controlled, doubleblind, and randomized group studies and received probiotics for 30 days. Subjects were assessed using the Hospital Anxiety and Depression Scale, Perceived Stress Scale, Coping Checklist, Hopkins Symptom Checklist, and a urinary free cortisol test. Participants in the probiotic treatment group showed a decrease in self-reported anxiety and reported less psychological stress. Additionally, urinary cortisol levels diminished in this group, confirming the participants' self-reported feelings (Messaoudi et al. 2011).

Once humans reach adult age, gut microbiota composition is usually established. This raises the question of how probiotic supplementation affects the gut to bring about such changes in mood and behavior. McNulty and colleagues showed that in administering fermented milk strains to monozygotic twins and mice does not necessarily change the gut microbiome's composition, but rather the metabolic activity and transcriptional state of the commensal bacteria (McNulty et al. 2011). In addition to previous findings, this study suggests that behavior and mood

improvements may not be a direct consequence of ingesting specific strains of probiotics, but rather a synergistic consequence of interactions among the symbiotic microbiota of the gut.

Bacteria of the gut are also heavily involved in generating and providing essential vitamins and cofactors for the host and react to specific macronutrients that the host consumes and digests (Sampson and Mazmanian 2015). A well-documented phenomenon occurs when the host ingests fermentable complex carbohydrates. Upon digestion, the gut bacteria will produce short-chain fatty acids (SCFAs) from metabolized fiber. Such SCFAs are capable of crossing the blood-brain barrier after transport into the serum from the GI tract. In mice, Frost and colleagues demonstrated that the short-chain fatty acid acetate helps promote satiety by inducing physiological changes in the hypothalamus. Moreover, acetate produced by bacteria

can alter levels of glutamate, GABA, and glutamine in the brain, as well as increase levels of anorectic neuropeptide. The cumulative effects of SCFAs also presented as a decrease in appetite in the study (Frost et al. 2014). Frost’s results suggest that gut microbiota indirectly influences hunger and satiety. It can be hypothesized that these bacteria adapted this mechanism to signal its host to feed, which would pose a strategic survival mechanism; however, further research must validate or test this claim.

Neurophysiology

Remarkably, the microbiota is involved in modulating the host's serotonergic system (Clarke et al. 2013). By investigating germ-free mice, Clarke and colleagues found that the serotonin metabolite concentrations, 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid increased in the hippocampus, compared to control animals. Such results were also sexspecific, as male GF rodents showed increased plasma tryptophan levels, a precursor to serotonin, when compared to females. Male GF mice also showed decreased levels of BDNF in the hippocampus, confirming prior research. The concentrations of such molecules were not rescued when adult mice underwent re-colonization with bacteria (Figure 8) (Clarke et al. 2013). Clarke’s results add to the evidence that certain phenotypical traits are firmly established and modulated by bacterial influence during fetal development. Yano and associates built on this data and showed that gut microbes actively regulate the levels of 5-HT in the blood

and the colon (Yano et al. 2015). Specifically, researchers demonstrated that spore-forming bacteria from both humans and mice stimulated enterochromaffin cells of the colon to produce 5-HT by inducing expression of tryptophan hydroxylase (Tph). The Tph enzyme, produced by EC cells, is responsible for 5-HT biosynthesis. Once induced to produce 5-HT, EC cells provide the surrounding intestinal lumen, mucosa, and circulating platelets with 5-HT to be transported in circulation. The increase in 5-HT induced stimulation of myenteric neurons as well as gut motility. However, when such mechanisms were tested in GF mice compared to SPF mice, the GF mice showed lower plasma 5-HT. When treated with a combination of spore-forming bacteria, GF mouse 5-HT biosynthesis deficits reverted to normal levels (Figure 8) (Yano et al. 2015). Yano’s data regarding 5-HT modulation is consistent with prior findings that suggest that 5-HT, or serotonin, levels are adjusted by the host's microbiota.

Figure 8: Colonic levels of 5-HT for different mice (a), serum levels of 5-HT (b), quantification of 5-HT+ producing cells and EC cells (marked by CgA+) (c), and immunofluorescent staining of the colon for various mice and their various cell types of interest (Yano et al. 2015)

In 1986, Minuk and associates discovered that common bacteria such as H. influenza, E. coli, B. fragilis, P. mirabilis, Enterococcus, S. aureus, P. aeruginosa, and K. pneumoniae produced GABA directly. These bacteria species are present in abundance in intestinal infections (Minuk 1986), and since GABA modulates intestinal motility in the intestines, the stress response in the periphery, and thermoregulation, it suggests that such bacteria could exacerbate such infections (Sampson and Mazmanian 2015). Additionally, Lucas and colleagues discovered that Enterococcus species and Lactobacillus brevis genomes coded for tyrosine decarboxylase, an enzyme that converts tyrosine to tyramine, an amino acid involved in regulating blood pressure

(Lucas et al. 2003). Regarding other neurotransmitters, GF mice exhibit increased turnover levels of dopamine, 5-HT, and noradrenaline in the striatum by Heijtz and colleagues. High turnover rates of these neurotransmitters could influence the rodents to have high motor activity, which is typical of GF mice since all of these molecules are involved in central motor control and modulating blood flow to various muscles (Heijtz et al. 2011). This growing collection of evidence shows the impact that bacterial species have on their host. It is interesting to consider the delicate symbiotic balance that the human microbiota holds.

Microbiota and Immune Modulation

The gut bacteria's influence over the central nervous system may be indirect and more directly modulated by the immune system's interaction with the microbiota. However, Deshmukh and colleagues devised a model demonstrating how gut microbiota may more directly influence neutrophil homeostasis. Prior research showed that neonates' antibiotic exposure is associated with the development of neutrophil-mediated late-onset sepsis (LOS). Knowing this, researchers administered antibiotics to pregnant mice, which showed a decreased number of microbes in the neonatal intestines. The decreased level and diversity of microbes reflected a decrease in granulocyte progenitor cells, macrophage progenitors, and neutrophils in the bone marrow and circulation. The group also found that mice receiving antibiotics exhibited a decreased level of granulocyte colony-stimulating factor (G-CSF) and decreased Interleukin 17 (IL-17) producing cells. Such antibiotic-treated neonatal mice displaying these phenotypes

showed an increased susceptibility to Klebsiella pneumoniae and Escherichia coli sepsis. Interestingly, when researchers transferred typical microbiota species into the neonates following the antibiotic treatment, innate lymphoid cells (ILCs) began to synthesize IL-17 in the intestine. This, in turn, increased plasma G-CSF concentrations and circulating neutrophil populations, thereby restoring IL-17 dependent resistance to bacterial sepsis (Deshmukh et al. 2014). Therefore, a healthy microbiota is involved in regulating neutrophil homeostasis, particularly by influencing G-CSF's gene expression. Granulocyte colony-stimulating factor also plays a role in neurogenesis, helps repair the brain after ischemic injuries, and acts as a protector against Alzheimer's and Parkinson's diseases (Sampson and Mazmanian 2015). Therefore, it is possible that the microbiota influence and protect against such diseases through this model by controlling G-CSF expression.

Epithelial Control in the Intestines

In the immune system of a host, the microbiota has a homeostatic relationship with the host immune system. The gastrointestinal tract is home to the largest concentration of commensal bacteria in the body, which is kept from harming the rest of the body through the cooperative function of immune cells, epithelial cells, and intestinal mucus. These combined mechanisms prevent the translocation of the bacteria to other areas of the body. However, pathogenic microbes can overcome these barriers (McGuckin et al. 2011). In addition, epithelial cells actively secrete antimicrobial peptides that attack and degrade bacterial cell walls. These

epithelial cells employ pattern-recognition receptors to trigger the release of peptides against specific pathogens and actively secrete a-defensins (Hooper et al. 2012). Cash and colleagues demonstrated that by colonizing germ-free mice with microbes, epithelial cells secrete the C-type antimicrobial lectin RegIIIg (Cash et al. 2006). Moreover, intestinal epithelial cells also respond to signals from short-chain fatty acids, Toll-like receptors, and Nod-like receptors to ensure that the epithelial barrier's integrity is maintained and that the gut bacteria remain in homeostasis with the host (Belkaid and Hand 2014).

Immune Development and Function

The presence of the gut microbiota is required for the immune system to fully and properly develop. Specifically, Toll-Like receptors (TLRs) on lymphoid and epithelial cells in the GI tract allow the immune system to distinguish between beneficial commensal bacteria and pathogenic species. TLRs actively suppress inflammatory responses towards gut bacteria by recognizing microbe-associated molecular patterns (MAMPs) and trigger innate intestinal immune responses towards pathogens. During the first few weeks of development, TLR stimulation causes NF-kB release, which activates B cells to proliferate and create immune memory. During the first weeks of life, this TLR activation subsides, following the development of a stable microbiota. Additionally, NOD-like receptors, which trigger inflammasomes that recognize damage association patterns, activate at this time (Lazar et al. 2018).

The intestinal microbiota modulates differentiation and cytokine expression of T cell populations during an individual's lifetime. Mazmanian and colleagues noted that imbalances in gut bacteria are involved in the pathophysiology of GI disorders such as inflammatory bowel

disease. Researchers began by inducing colitis through Helicobacter hepaticus, a pathogenic bacterium which causes GI abnormalities and promotes proinflammatory cytokine profiles when overrepresented. They also demonstrated that the administration of a purified polysaccharide from Bacteroides fragilis suppressed proinflammatory IL-17 production. Specifically, this microbial metabolite from B. fragilis induced CD4+ T cells to secrete IL-10, an antiinflammatory cytokine (Mazmanian et al. 2008). Such regulatory T cells are essential for immune homeostasis; thus, it is interesting to consider how gut bacteria can modulate such cells' expression via their dysbiosis. In a mouse study, Salzman and colleagues demonstrated that Bacteroides thetaiotaomicron actively triggers the production of a-defensins, an antimicrobial peptide, to provide protection from pathogenic species implicated in the etiology of colorectal cancer and inflammatory bowel disease (Salzman et al. 2010). Gut dysbiosis has been suggested to play a role in numerous conditions and diseases, such as diabetes, rheumatoid arthritis, celiac disease, allergies, lupus erythematosus, Alzheimer's disease, various cancers, and of particular interest, Autism Spectrum Disorders (Lazar et al. 2018).

THE GUT-BRAIN LINK TO AUTISM

Autism spectrum disorders (ASD) refer to a group of dysfunctions characterized by repetitive behaviors and communication impairments, which appear early in life due to genetic and environmental factors. Individuals with autism will need lifelong support or care, depending on the varying severity of their disorder. Autism varies significantly from person to person, but several criteria taken from the Diagnostic and Statistical Manuel of Mental Disorders (DSM-5) are often used to diagnose individuals. To be diagnosed, a person must have deficits in the three communication categories of affective reciprocity, the theory of mind, joint attention (Robertson et al. 1999), and show difficulty in at least two of the sensory-motor repetitive behaviors. Additionally, autism can be comorbid with psychiatric conditions and genetic

disorders (Lord et al. 2018). While scientists are unsuccessful in formulating a comprehensive etiology for ASD, research demonstrates risk factors, genetic links, and other linkages. Specifically, the gut microbiota has been hypothesized to modulate the outcome of ASD, and its dysbiosis is implicated in its etiology. Attention was drawn to this possibility since many individuals with autism report experiencing severe gastrointestinal symptoms, the intensity of which strongly correlates with the degree of ASD severity that they are diagnosed with. By examining the microbiota's apparent dysbiosis, gastrointestinal abnormalities, the serotonin pathway, gutmicrobial metabolites pathway, dietary interventions, and potential probiotic therapies, the gutbrain link to autism will be explored.

Dysbiosis of Microflora

Research shows that the mother's own microbiota and overall health play a large role in modulating the offspring (Sharon et al. 2016). Conolly et al. researched a link between maternal obesity, gestational diabetes mellitis (GDM), and autism. Through analyzing hospital records, they found statistically significant evidence that mothers who were previously diagnosed with obesity and GDM had a 1.5-fold increased chance of giving birth to an autistic child (Connolly et al. 2016). Conversely, breastfeeding for six months lowers the risk of ASD (Schultz et al. 2006). Tanoue and associates demonstrated that out of a sample of 145 autistic individuals and 224 normal children, 24.8% of those with ASD, compared to 7.5% of the control population, were weaned after only one week (Tanoue and Oda 1989). Several factors, such as socioeconomics and a lack of maternal education, could play into this, but such data shows that breastfeeding is an essential part of development. Additionally, Azad and colleagues examined the microbiota profiles of healthy infants. They found that children born naturally and breastfed showed sufficient colonization by Actinobacteria and Firmicutes, while infants raised on formula had an increase in species and presented with colonization by Clostridium defficile species. The increase in C. defficile is important because this bacteria species is implicated in inflammatory diseases of the colon, specifically colitis (Azad et al. 2013).

As previously established, antibiotic treatment is known to cause long-lasting alternations even if taken for short times during neurodevelopment. In a separate study, Azad investigated the impact of maternal intrapartum antibiotics, birthing methods, and breastfeeding on an offspring's gut microbiome. Using a prospective pregnancy cohort of Canadian infants born in 2010-2012, researchers examined fecal samples of 198 healthy babies taken at 3 and 12 months. In the study, mothers received intrapartum antibiotic prophylaxis (IAP) for group B Streptococcus infections, pre-labor rupturing, or emergency C-sections. They found that genera Bacteroides and Parabacteroides were underrepresented for those administered IAP, and Enterococcus and Clostridium were overrepresented just after three months. Such prevalence is significant because while Enterococcus and Clostridium are part of the normal microflora, their overrepresentation is associated with gastrointestinal disease states (Table 1) (Azad et al. 2016).

Table 1: Abundance of various taxa in fecal microbiota at 3 and 12 months, in regards to various conditions (Azad et al. 2016)

Cox and colleagues' further research showed that administering low-dose penicillin (LDP) to young mice during critical developmental windows resulted in lasting metabolic consequences. Researchers demonstrated a microbe-induced obesity (MIO) model in such mice, which showed that an increase in adipose tissue resulted from primary dysbiosis of gut bacteria. Even with exposure of LDP limited to infancy, MIO still developed. Additionally, researchers transferred the microbiota of such mice treated with LDP into germ-free organisms, displaying the same MIO phenotype, proving that dysbiosis was causal. Interestingly, Cox and associates also showed that LDP lowered intestinal integrity and suppressed antibacterial responses. Specifically, TH17 cell differentiation, bacterial killing, and antigen presentation processes diminished, building on prior evidence that linked intestinal luma integrity and immune function as essential modulators in the etiology of various metabolic syndromes. Perhaps most interesting, after LDP treatment, gut microflora returned to normal; however, the MIO phenotype still presented and developed (Cox et al. 2014). Such data adds to the evidence that there is a critical period when gut bacteria are involved in modulating developmental outcomes for an organism.

Besides studying antibiotic medication, research conducted on valproic acid (VPA) exposure, reveals much considering the gut microflora's role in autism. While prior research shows that exposure to VPA is strongly associated with ASD symptoms (de Theije et al. 2014), the exact mechanism is largely unknown. Theije et al. elucidated how microflora are affected by exposure to VPA, which showed the implications for ASD. Offspring of mice treated with VPA exhibited higher concentrations of the short-chain fatty acid (SCFA) butyrate, a consequence more pronounced in males. Increased butyrate levels were associated with the genera Rikenella, Bacteroidales, Mucispirillum (Deferribacterales), and Erysipelotrichales. In VPA treated male offspring, an increased number of Erysipelotrichales and a decreased abundance of Bacteriodales presented. Theije and colleagues also demonstrated that VPA-treated male offspring increases in Alistipes, and Erysopelitrichales was significantly correlated with a decrease in intestinal levels of serotonin. The decrease in serotonin was accompanied by fewer enterochromaffin cells, which produce roughly 90% of the body's serotonin in the intestinal epithelial layer (de Theije et al. 2014). Such results agree with data that De Angelis and associates collected. The genus Alistipes showed overrepresentation in children with autism, accompanied by increased levels of indole in the intestines, which is a metabolite of the amino acid tryptophan (de Angelis et al. 2015). The increased indole concentration is worth mentioning because tryptophan is also a precursor of serotonin, and a male-specific overrepresentation of Alistipes species may contribute to male-specific disturbances in the serotonergic system. Microbial metabolites such as SCFA's can cross the blood-brain barrier

and induce changes to the developing brain (Braniste et al. 2014), and Theije et al. corroborated this.

Additionally, Finegold and colleagues pyrosequenced the fecal microflora of 33 individuals, containing autistic and control children. The data collected indicated for those diagnosed as severely autistic, populations of Bacteroidetes increased while Firmicutes diminished. Also, within the phyla, Actinobacterium and Proteobacterium, Bacteroides vulgatus, and Desulfovibrio species were found to be overrepresented in the severely autistic compared to the control children (Finegold et al. 2010). This is interesting because Bacteroidetes are SCFA-producing bacteria, whose metabolites can modulate the central nervous system during neurodevelopment (Fattorusso et al. 2019). Additionally, Desulfovibrio and Bacteroides vulgatus are both gram-negative bacteria species. Such bacteria release lipopolysaccharide (LPS) toxins from their cell walls. Zhu and colleagues demonstrated that mice exposed to LPS prenatally have much lower glutathione concentrations, an essential antioxidant in the brain (Zhu et al. 2007). Such deficits in glutathione, caused by increased dysbiosis in gram-negative species, could be part of the etiology of ASD. Further sequencing by Kang and associates demonstrated that less diverse gut microbiomes were significantly associated with autistic symptoms in children. By investigating fecal samples from 20 neurotypical children and 20 autistic children, they found that autistic individuals presented with significantly lower abundances of Prevotella, Coprococcus, and Veillonellaceae species. Such bacteria are heavily involved in carbohydrate metabolism and fermentation in the gastrointestinal tract. Lower representation of these bacteria could influence the characteristic, unusual diet patterns of autistic children, though only further research can confirm this hypothesis (Kang et al. 2013). Additionally, various studies demonstrate a lower abundance of Clostridium, Lactobacillus, Akkermansia, Desulfovibrio, Sutterellaceae, Alistipes, and Sarcina, Caloramator, and Enterobacteriaceae in autistic children as well; however, other studies demonstrate contradictory data (Fattorusso et al. 2019). Lastly, sequencing conducted by Iovene and colleagues demonstrated that yeasts reside in the gastrointestinal tract of autistic toddlers. Specifically, Candida albicans was twice as abundance when compared to control children. This increase in C. albicans was accompanied by a decreased quantity of Lactobacillus and Clostridium species (Iovene et al. 2017). Such data, taken as a whole, demonstrates the incredibly complex microscopic community that resides in the gut. Research concerning the microbiota and its multifaceted role as a modulator on human health is still in its infancy, and more definitive studies are needed to show causality or define dysbiosis' role in ASD better. Studies with homogenous people groups are necessary for accomplishing this.

The Serotonin Pathway

Elevated plasma serotonin levels in ASD was first discovered by Hanley and associates in 1977, who showed that children with autism showed statistically significant blood elevations (Hanley et al. 1977). Since then, efforts have been made to attempt to elucidate how the microbiota influences the serotonergic system. As previously discussed, Yano and associates conducted a significant study that investigated how spore-forming bacteria upregulate concentrations of 5-hydroxytryptamine (5-HT) in colonic enterochromaffin cells (ECs). In one part of the study, they used knockout mice lacking the TPH1 enzyme, which is responsible for catalyzing the first step of serotonin biosynthesis. Such mice, which were colonized with SP bacteria, had a 90% decrease in serotonin levels. This decrease is interesting because it shows that in the absence of the TPH1 enzyme, 10% of the control levels of serotonin were still present, meaning that SP bacteria in the intestines account for nearly 10% of serotonin production directly and can influence EC cells to produce the rest under normal conditions via metabolites. Metabolomic

profiling was also utilized to determine which specific metabolites spore-forming bacteria (SP) released that increased 5-HT levels. Such bacteria also produce the SCFAs propionate, butyrate, and acetate, among 16 other tested metabolites. Yano et al. demonstrated that butyrate, cholate, propionate, tyramine, p-aminobenzoate, a-tocopherol, and deoxycholate all elevate 5-HT levels in chromaffin cell cultures (Figure 9). Specifically, researchers showed that such metabolites induce EC cells to upregulate TPH1, which then is directly responsible for increasing the production of 5-HT (Yano et al. 2015). Autistic individuals show high concentrations of SCFAs, which is associated with dysbiosis in certain SP bacteria (MacFabe 2012). Therefore, evidence suggests that overrepresentation of such bacteria leads to an increase in SFCAs, which, in addition to their other potentially harmful effects, increases 5-HT levels in the intestines, which could be implicated in the development and progression of ASD.

Figure 9: Various metabolites affecting 5-HT release (left), and Tph1 expression measured by mRNA transcripts (right) (Yano et al. 2015)

Previous studies showed the importance of serotonin on neurodevelopment. For example, Cote et al. used knockout mice to show that mothers who were homozygous for lacking the TPH1 gene gave birth to heterozygous offspring with severe brain structure abnormalities. This indicated that serotonin is critical in directing CNS development, as observations suggested that the lack of serotonin influenced morphogenesis even before serotonergic neurons appeared in offspring (Cote et al. 2007). Additionally, Bonnin and colleagues showed that serotonin from the placenta directed thalamocortical axon guidance (Bonnin et al. 2011). Research concerning individuals with ASD has shown that they are characterized by brain abnormalities and dysfunction. Specifically, Courchesne et al. demonstrated that MRI scans taken from 60 autistic boys, compared to 52 scans from healthy boys, showed unusual brain growth patterns. Analyzing scans taken from the ages 2 through 16 years, they found that autism was characterized by periods of increased growth early in life, followed by abnormally slowed growth. The intensified growth was evident from apparent hyperplasia in cerebral grey matter and white matter early in life (Courchesne et al. 2001). Brain volume augmentations are worth mentioning because Wassink and associates analyzed a possible link to increased brain volume and the serotonergic system. When analyzing the SERT gene (SLC6A4), which codes for the SERT serotonin integral membrane transporter protein, they found a polymorphism (5-HTTLPR) in the promotor, which was significantly associated with ASD. Individuals with this polymorphism showed low expression of SERT and an increased cerebral cortex grey matter volume (Wassink et al. 2007). Examining the same 5-HTTLPR genotypes, Wiggins and colleagues reported that children and adolescents with ASD showed stronger connectivity of the default neural network, which is most active during periods of wakeful rest; the increased activity found here in autistic children was a trend opposite to control children (Wiggins et al. 2012). Later in a separate study, Wiggins et al. examined several SERT genotypes and demonstrated that autistic children presenting with low-expressing SERT genotypes exhibited decreased amygdala habituation regarding facial stimuli (Wiggins et al. 2013). In other words, the fewer SERT membrane transporters, reflected by low expression of SERT genotypes, were associated with ASD, meaning that these individuals would have less bioavailable serotonin to be used in various pathways.

Additional studies show that lower levels of tryptophan and tryptophan depletion cause worsening autistic symptoms. Specifically, McDougle et al. demonstrated in multiple studies that decreasing tryptophan, which is a precursor in 5-HT synthesis, leads to reduced levels of 5- HT. This then leads to exacerbated autistic symptoms, such as irritability and repetitive behaviors (McDougle et al. 1993; McDougle et al. 1996). Perhaps most intriguing are the results that Veenstra-VanderWeele and colleagues collected when using a genetic knock-in mouse model for the most common SERT amino acid variant found in autistic individuals, the Ala56 allele. Mice, which expressed the Ala56 variant, showed increased plasma levels of 5-HT but increased 5-HT clearance in the brain and basal p38-MAPK-dependant SERT phosphorylation. When 5-HT receptors in the CNS were analyzed, they showed hypersensitivity to different serotonin agonists. Researchers also showed that compared to wild-type controls, such mice presented with greater inhibition of neuronal firing (Figure 10), which was shown to correlate with the increased 5-HT receptor sensitivity. Such heightened sensitivity could be an adaptive response to compensate for decreased levels of synaptic 5-HT, resulting from decreased SERT expression (Veenstra-VanderWeele et al. 2012). Intriguingly, SERT Ala56 mice presented with hyperserotonemia and displayed significant changes in repetitive and social behaviors, reflecting ASD symptoms. Many knockout studies also add to the growing body of research indicating the importance of 5-HT homeostasis. In particular, mice lacking tryptophan hydroxylase 2, as demonstrated by Kane et al., Del'Guidice et al., and Mosienko et al., display cognitive inflexibility, deficits in social function and memory, decreased ultrasound vocalizations and decreased social odor sniffing (Kane et al. 2012; Del’Guidice et al. 2014; Mosienko et al. 2015). Such results mimic autistic-like behavior. Additionally, knocking out the monoamine oxidase-A (MAOA) enzyme, which is responsible for the breakdown of 5-HT, induced decreased social behavior, impaired reversal learning, and other autistic-like features (Bortolato et al. 2013). Such studies point towards the vital role the serotonin pathway plays in ASD.

Figure 10: Neuronal firing (left), and % inhibition of firing in relation to 5-HT concentration (right) (Veenstra-VanderWeele et al. 2012)

Even though roughly 50 years of research plays into understanding how varying serotonin levels associate with the development and outcomes of ASD in children, little is still known regarding the specific mechanism of modulation. Gastrointestinal inflammation, which will be discussed shortly, is comorbid with autism in 40% of patients (Wang et al. 2011). Zhu and colleagues showed that sickness behavior and depressive disorders could be attributed to proinflammatory cytokine signaling. Specifically, increases in Interleukin-1b and TNFa treatment stimulate 5-HT uptake through upregulating SERT through p-38 MAPK-linked pathways. This, in-turn, decreases synaptic 5-HT concentrations (Zhu et al. 2006). Additionally, IFN-a also decreases neural 5-HT receptors, which further contributes to the loss of serotonergic signaling (Cai et al. 2005). Since this data contributes to depressive disorders' etiology, varying synaptic serotonin concentrations likely play a role in behavioral deficits in ASD. Increased levels

of plasma serotonin and decreased availability of synaptic 5-HT are associated with a greater risk for ASD (Muller et al. 2016). Additionally, hyperserotonemia is linked to exacerbation of ASD symptoms, and the intestinal microbiota plays a significant role in regulating the serotonergic system (O’Mahony et al. 2015). Therefore, it can be hypothesized that gut dysbiosis can lead to the upregulation of genes involved in the 5-HT synthesis. While the exact mechanism of serotonin regulating ASD has yet to be elucidated, it is evident that the overrepresentation of specific spore-forming bacteria populations produces metabolites that cause increases in serotonin levels. The dysbiosis of bacteria in the gastrointestinal tract is a known indicator of autism and likely explains the GI symptoms that those with ASD complain of. Therefore, it is interesting to consider the role of inflammation and other abnormalities in Autism Spectrum Disorder.

GI Inflammation

According to multiple studies and meta-analyses conducted with autistic children, roughly 40% of those with ASD suffer from GI abnormalities such as constipation, vomiting, diarrhea, reflux, and abdominal pain (Wang et al. 2011; Mazurek et al. 2013). Compared to normal, healthy children, those with ASD experience significantly more gastrointestinal symptoms and abnormalities, with higher rates of such symptoms (McElhanon et al. 2014). Interestingly, Rose

and associates demonstrated that children with ASD that also suffer from gastrointestinal symptoms present with an imbalanced immune response. Upon Toll-like receptor (TLR)-4 stimulation in ASD populations with and without GI symptoms, those with GI symptoms displayed increased levels of cytokines IL-5, IL-15, and IL-17 (Figure 11).

Figure 11: Comparing the cytokine profiles of children with ASD and GI symptoms (ASDGI), ASD without GI symptoms (ASDNoGI), and normal controls (TDNoGI) (D.R. Rose et al. 2018)

Additionally, those with GI symptoms showed greater GI epithelial permeability, as evident from the upregulation of the gene encoding zonulin, which regulates gut permeability (D.R. Rose et al. 2018). To determine the genetic markers for autistic GI symptoms, Walker et al. sequenced the gene expression profile for those suffering from this group and compared them with those without autism, but that suffered from, Chrohn's disease and ulcerative colitis. While ASD patients suffering from GI abnormalities shared genetic markers with known markers for inflammatory bowel disease, autistic patients expressed distinctive traits as well (Walker et al. 2013). The similarity in genetic markers is interesting because this suggests that chronic GI inflammation could be part of the pathophysiology of autism in specific populations.

Adding to the characterization of ASD gastrointestinal inflammation, researchers showed specific inflammation markers in children with autism. Ashwood and colleagues took colonic, ileal, and duodenal biopsies from 52 affected children and compared them with biopsies from unaffected children and those who were histologically inflamed. They found that autistic children with GI inflammation specifically showed increased levels of CD3(+) and CD3(+)CD8(+) IEL as well as CD3(+) LPL markers. This cytokine profile is significant because such features indicate the presence of proinflammatory infiltrate immune cells. Additionally, Ashwood et al. found increased epithelial eosinophils, which suggests that there could be an autoimmune component to ASD-linked GI abnormalities (Ashwood et al. 2003). Another study by Ashwood and associates investigated dysregulated mucosal immunity by examining lymphocyte cytokine profiles in children with autistic GI abnormalities. Increased levels of CD3+ lymphocytes, TNF-alpha, and IFN-gamma were present in inflamed GI states, as well as a decrease of the anti-inflammatory cytokine, IL-10 (Ashwood et al. 2004). IL-10

often works as a regulatory counter-inflammatory signaling molecule in healthy individuals, so such data suggests that increased inflammation and decreased regulatory activity could influence ASD outcomes in children.

While it is evident that those with autism suffering from gastrointestinal symptoms experience significant inflammation, it is hypothesized that such a proinflammatory state could be linked to neuroinflammation in ASD and thus be a part of autism etiology. Specifically, research shows that autistic brains are characterized by excess concentrations of advanced glycation end products (AGEs), which interact with receptors for advanced glycation end products (RAGE) to cause oxidative stress, neuroinflammation, and neuronal degeneration (Fattorusso et al. 2019). Boso and colleagues measured levels of S100A9, RAGE's proinflammatory ligand, as well as levels of plasma endogenous secretory RAGE (esRAGE) in autistic young adults and compared them to healthy individuals. They found that those with ASD demonstrated increased concentrations of S100A9 but a decrease in esRAGE, causing them to hypothesize that variation in RAGE and inflammatory responses could lead to systemic inflammation throughout the body. The increase in inflammation, mediated by RAGE receptors, is associated with the amplification of microglia and astroglia (Boso et al. 2006). Since microglia and astroglia are involved in neurodevelopment, such inflammation could alter CNS development in ASD. Additionally, it is possible that the GI abnormalities, characterized by immune dysregulation, are linked and influenced by the RAGE mediated inflammatory response, and that both are part of ASD's etiology. However, further research is needed to confirm or disprove such a hypothesis.

Microbial Therapies

Due to the increasing body of research indicating that gut bacteria dysbiosis is involved in the pathophysiology of autism spectrum disorder, multiple treatment methods proposed could directly alter the microbiota. One of the more promising treatments is microbiota transfer therapy (MTT), where a fecal sample from a healthy individual is transplanted into a recipient's gut. Kang et al. conducted a clinical trial that examined the effect of MTT on microbiota composition, GI symptoms, and ASD symptoms in 18 autistic children. The treatment group showed promising results, as participants rated an 80% reduction in GI symptoms at the end of the treatment. Additionally, such improvements and less severe ASD symptoms lasted eight weeks after the treatment ended, as well as an increase in bacterial diversity of gut flora (Kang et al. 2017). A follow-up report conducted by Kang and associates showed that such improvements persisted even after two years (Kang et al. 2019). Additionally, many studies explore the potential that probiotics hold in rescuing ASD deficits hypothesized to be caused by gut bacteria dysbiosis. In once such study, El-Ansary and associates induced autistic-like behavior in hamsters through administration of antibiotics and PPA. The ASD traits exhibited in

the hamsters were ameliorated by probiotic supplementation (El-Ansary et al. 2018). Several other large-scale studies have been conducted regarding probiotic supplementation in children with ASD. A randomized, double-blind, placebo-controlled study conducted by Parracho and associates tracked symptom regression over three weeks, resulting from probiotic administration (Parracho et al. 2010). Also, Partty and colleagues tracked the long-term effects of Lactobacillus rhamnosus supplementation during the first six months of life and observed that children lacking probiotic supplementation had a higher chance for developing Asperger’s or ADHD, which are similar to ASD (Partty et al. 2015). Lastly, Shaaban and associates further demonstrated the beneficial potential of probiotic treatment for ASD and GI symptoms in autistic children, showing an increased bacteria abundance in subject stool samples as well as a significant improvement in ASD symptoms. While these trials show promising results, they are limited in participants and are not comprehensive in nature. To conclusively evaluate the therapeutic nature of probiotic supplementation in ASD, further research is necessary.

CONCLUSION

While significant strides have been taken to characterize the human microbiome, such as the Human Microbiome Project, much remains unknown regarding the mechanisms by which bidirectional communication between the gut and the brain modulates autism spectrum disorders. Over the past few decades, research demonstrated the importance and various roles of the human microbiota regarding development. Gut bacteria are involved with an offspring’s neurodevelopment (Ogbonnaya et al. 2015) and modeling its immune system (Erny et al. 2015), and numerous responses (Basu-Roy et al. 2012). Gut bacteria are also known to modulate host behavior and mood (Sudo et al. 2004) and neurotransmitter concentrations (Bravo et al. 2011), (Yano et al. 2015), making its dysbiosis a prime suspect in the development of several neurological disorders, specifically Autism Spectrum Disorder. Several studies point towards the probability of microbial metabolites influencing ASD severity (MacFabe 2012), while substantial evidence implicates alterations in the serotonergic system and gastrointestinal

inflammation (Yano et al. 2015), (S. Rose et al. 2018). Of particular interest is the potential to alleviate ASD symptoms through microbial transfer therapy (Kang et al. 2019) and probiotic supplementation treatments (Hsiao et al. 2013). The future of treating autism spectrum disorders through targeting the gut microflora is bright. While smaller clinical trials show successful outcomes (Parracho et al. 2010), few large-scale longitudinal studies have been conducted. In order to prove that probiotic treatments or fecal transplant therapies are beneficial for the majority of those with ASD, rather than a sub-population, studies must be completed that sample individuals from different demographics, showing decreases in ASD and GI symptoms. Furthermore, it may one day be possible to use microbiome sequencing as a diagnostic tool for clinical purposes. It is not difficult to imagine a future where the gut microbiome is better defined and understood to a greater degree in regard to autism, but it is essential to remember that increased information requires an added level of responsibility.

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Band-Aids Don't Fix Bullet Holes: A Pentadic Analysis of Emma Gonzalez's 'We Call B.S.' Speech

Joel Reyes

Lone Star College - Tomball

On Wednesday, February 14, 2018, Nikolas Cruz murdered seventeen individuals and injured seventeen others at Marjory Stoneman Douglas High School using an automatic rifle. Traumatized by this event and angered by the notion that this situation could have been avoided, survivor Emma Gonzalez would recite a speech, titled “We Call B.S.,” at a Ft. Lauderdale anti-gun rally, only three days after the incident, to speak on the issue of United States gun violence...

Submit to Sin: Contextualizing the Roman Empire in Augustine's Detailing of Sexuality in Confessions

Joseph Flores

Lone Star College - CyFair

Compiling historical studies of the Roman Empire’s concepts of family, sex, and social status, this work compares the greater society’s ideologies to Augustine’s retrospective narrative. By covering Augustine’s experiences with his abusive father, ascension in social status through Romanianus, conversion to Manichaeism, and strictly sexual relationship with his concubine, this essay engages with the social underpinnings of the Bishop’s articulation of the role of sex and Catholicism...

Hypersexualization at Middle School Dances: An Analysis of Self-Presentation in Pen15 and Big Mouth

PEN15-S01E10-3232d17e817827cac04f9381f90

Alissa Boxleitner

Lone Star College - Montgomery

With an aim to understand the relationship between school dances and the sexualization of children, this research compares the “ideal selves” that present day American middle schoolers attempt to put forward at school dances versus the “actual selves” that they put forth. Erving Goffman’s work on the dramaturgical perspective and E. Tory Higgin’s self-discrepancy theory are utilized as tools to analyze middle schoolers expectations of dances in cringe comedies Pen15 and Big Mouth...