Gut microbiome in neuropsychiatric disorders

ABSTRACT Background: Neuropsychiatric disorders are a significant cause of death and disability worldwide. The mechanisms underlying these disorders include a constellation of structural, infectious, immunological, metabolic, and genetic etiologies. Advances in next-generation sequencing techniques have demonstrated that the composition of the enteric microbiome is dynamic and plays a pivotal role in host homeostasis and several diseases. The enteric microbiome acts as a key mediator in neuronal signaling via metabolic, neuroimmune, and neuroendocrine pathways. Objective: In this review, we aim to present and discuss the most current knowledge regarding the putative influence of the gut microbiome in neuropsychiatric disorders. Methods: We examined some of the preclinical and clinical evidence and therapeutic strategies associated with the manipulation of the gut microbiome. Results: targeted taxa were described and grouped from major studies to each disease. Conclusions: Understanding the complexity of these ecological interactions and their association with susceptibility and progression of acute and chronic disorders could lead to novel diagnostic biomarkers based on molecular targets. Moreover, research on the microbiome can also improve some emerging treatment choices, such as fecal transplantation, personalized probiotics, and dietary interventions, which could be used to reduce the impact of specific neuropsychiatric disorders. We expect that this knowledge will help physicians caring for patients with neuropsychiatric disorders.


INTRODUCTION
Over the past decade, microbiomics have emerged as a new field led by advances in culture-independent methods and next-generation sequencing technologies. These methods have provided a broader understanding of how the interaction between microbes and humans can profoundly influence host homeostasis and different disease states 1 . The literature has reflected the growing number of studies on the putative influence of the microbiome on human health and disease (Figure 1). In humans, bacteria are the most prevalent domain, and it has been estimated that the ratio of microbes to human cells in an adult is nearly 1:1 2 . The human microbiome is defined as the compendium of microbial communities (including bacteria, archaea, viruses, protozoa, and fungi) living in a given body niche 3,4 .
It has been demonstrated that the gut microbiome can be shaped by prenatal conditions, lifestyle, aging, host genetics, antibiotic use, and even geography. Moreover, it has been shown that microbial aggregates on the large intestine can modulate a wide range of host physiological processes related to immune system maturation, food metabolism, energy production, and brain development 5,6 .
The crosstalk between the gut microbiome and the brain is known as the gut microbiota-brain axis (MBA), which involves immunological, neuroendocrine, metabolic, and stress-response pathways 7 . This axis appears to be a cardinal mediator in a repertory of complex traits that range from metabolic to neuropsychiatric conditions 8 . Despite the vast contribution of animal models to elucidate biological mechanisms underlying host-microbiota interactions, there are difficulties in translating the findings in animal experiments to human research. Also, differences in methodologic standards, metadata curation, and reference databases management (https:// portal.hmpdacc.org/; https://www.sanger.ac.uk/resources/ downloads/bacteria/metahit/) can be potential pitfalls for study comparison, interpretation, and reproducibility 9 .
Since cohorts in microbiome studies tend to be small and heterogeneous, it is difficult to know which observations are generalizable to larger patient populations. Metabolomic, proteomic, and big data analyses of microbiome function will be critical to determine if the conclusions of these studies can apply to the clinical setting. Further experimental studies using in vitro or in vivo models are needed to understand the direct impact and causal relationships between host and microbes in order to control both known and potential hidden confounders. In this way, it still not currently possible to determine whether changes in the microbiota could be merely an epiphenomenon rather than the primary cause of the problem. Thus, few clinicians currently recognize that the gut microbiome is relevant to human neurophysiology because the nature of the relationship between microbiota and disease is still unclear.
In this context, we aim to present and discuss the current literature on the putative biological significance of the gut microbiome in neuropsychiatric disorders. We hope to show why this field is emerging as a possible source of therapeutic targets in these disorders and how it can be incorporated into personalized medicine strategies 4 . We will describe only the major studies related to each disease; however, additional references are presented in Tables 1-5.  Antibodies to mimicry peptides from Acinetobacter, P. aeruginosa, myelin basic protein, and myelin oligodendrocyte glycoprotein were significantly elevated in patients compared to controls.
Hughes et al. 23 Gut bacteria from multiple sclerosis patients modulate human T cells and exacerbate symptoms in mouse models n=142 (stool samples from 71 patients with MS and 71 controls) Increased Akkermansia muciniphila and Acinetobacter calcoaceticus in patients, which induced pro-inflammatory responses. Reduced Parabacteroides distasonis in patients with stimulated antiinflammatory IL-10-expressing human CD4+CD25+ T cells and IL-10+FoxP3+ Tregs in mice. Microbiota transplants from patients into germ-free mice induced more severe experimental autoimmune encephalomyelitis compared with controls. Cekanaviciute

GUT MICROBIOME STRUCTURE AND FUNCTION: INTESTINAL AND SYSTEMIC IMPACT
The gastrointestinal (GI) tract is considered the organ that harbors the highest amount of commensal microorganisms, reaching 10 13 bacteria/mL 2 . Bacteroidetes, Firmicutes, Actinobacteria, Verrumicrobia, Proteobacteria, and Fusobacteria have been identified as the core enterotypes at the phylum level, which consist of more than 1000 species 3 .
These consortia tend to remain steady throughout adult life, but preclinical evidence in both animals and humans have revealed that pre-and post-natal colonization of the gut has specific signatures and interindividual variations (~20%), depending on critical events such as maternal conditions, perinatal infections, mode of delivery, breastfeeding, diet, antibiotic exposure, and host genetics 5,6 .
Different approaches comparing germ-free mice and conventionally colonized controls demonstrated that the      abnormal composition of the gut microbiota led to functional and structural changes in the gastrointestinal tract.
A variety of morphological defects on mucosa-associated lymphoid tissue and intestinal microvasculature, including reducing the number of Paneth cells, Peyer's patches, and villi size, were already described in mice models 10 . Similarly, the loss of microbial diversity can negatively affect the expression of genes related to nutrient absorption, mucosal cellularity, and barrier fortification 11 . In vitro and in vivo studies have examined the importance of Toll-like receptors (TLRs) present on the surface of intestinal epithelial cells and immune T cells 12 . The signaling transmission mediated by TLRs can also modulate mechanisms encompassing the host's tolerance to commensal bacteria and the inhibition of pro-inflammatory cytokines such as the tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-1B (IL-1B) 13 . These findings have been classically associated with several GI disorders, including inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC), as well as irritable bowel syndrome (IBS), functional dyspepsia (FD), and colorectal cancer (CRC) 14,15 . Besides, observational studies have indicated that the loss of beneficial microorganisms affects ecological interactions among local populations and drive systemic diseases. Some of the metabolic processes that are impaired in patients with obesity, diabetes mellitus, and non-alcoholic fatty liver disease are associated with the synthesis of vitamins, xenobiotic transformation, and bacteria-derived molecules production, including secondary bile acids and short-chain fatty acids (SCFA) production 16,17 .

INTESTINAL MICROBIOTA AFFECTS BRAIN FUNCTION AND HAS IMMUNOMODULATORY PROPERTIES
The human gut contains its own neural system, consisting of more than one hundred million neuron cells (10 8 ) 18 . Crosstalk between the central nervous system (CNS) and the gut microbiome is known as the gut microbiota-brain axis, and it is orchestrated at different anatomical levels. It occurs through an intricate network of afferent and efferent circuits alongside the vagus nerve, enteric nervous system, and hypothalamic-pituitary-adrenal axis. Thus, the gut connectome integrates neuroendocrine, enteroendocrine, neuroimmune, and metabolic signaling pathways responsible for regulating functions associated with digestion, tract motility, and brain development. Furthermore, gut microbes can control host responses to vascular injury and autoimmunity by modifications in both the blood-brain barrier (BBB) and brain lymphatic system 7,15 .
Additionally, evidence from intervention studies in germfree mice, where a diverse microbiota is absent, has also shown the great potential that microorganisms have in regulating microglia differentiation and maturation. Other features commonly displayed in gnotobiotic models are the abnormal expression of proteins related to synaptic plasticity, such as the brain-derived neurotrophic factor and the impairment in global cognition responses. For instance, in a study carried out by Möhle   has been reported that microbial metabolites such as SCFAs, secondary bile acids, precursors of the lipid biosynthesis (propionate), and specific amino acids (tryptophan) are critical in modulating the release of host cytokines and hormones such as peptide YY, vasoactive intestinal peptide, glucagon-like peptide-1 (GLP-1), and melatonin, as well as neurotransmitters such as serotonin (5-HT), catecholamines, and gamma-aminobutyric acid (GABA). All of these affect brain functions related to feeding, stress response, emotional behaviors, aging, and cognition 17,21 . Furthermore, intestinal dysbiosis appears to be linked to the development of brain autoimmunity driven by specific T cell subtypes, pro-inflammatory cytokines, endothelial barrier impairment, and neurodegeneration 7,22 . The next section will explore some of the neuropsychiatric disorders thought to be influenced by microbiome disturbances.

MULTIPLE SCLEROSIS
Previous studies have linked T helper 17 cells (Th17) to MS pathogenesis through its effect in exacerbating experimental autoimmune encephalomyelitis (EAE). Regulatory T cells (Treg) have an essential role in suppressing inflammation in the CNS in EAE models 22,23 . Cekanaviciute et al. have identified increased EAE disease scores and deficient IL-10+ Treg induction in mice colonized with microbiota from patients with MS 24 . Furthermore, some butyrate-producing bacteria, mainly belonging to the Firmicutes phylum, have also been implicated in the pathogenesis of MS. Butyrate is known to inhibit pro-inflammatory pathways and prevent systemic exposure to intestinal antigens 25 . Bacteria, such as Faecalibacterium from the Firmicutes phylum, were found to be reduced in MS patients 26 .
In a study of adult patients, Jangi et al. found a higher abundance of Methanobrevibacter and Akkermansia with a lower prevalence of Prevotella, Butyricimonas, Colinsella, and Slackia in patients with MS compared to healthy controls. Combining microbiome results with the immunogenetics characteristics of patients with MS, they found a positive correlation between Methanobrevibacter and Akkermansia and a negative correlation of Butyricimonas with MAPK14, MAPK1, LTBR, STAT5B, CASP1, and HLA-DRB1 -genes associated with potentiation of the immune response in MS 27 .
Tremlett et al. studied the microbiome in pediatric MS. A phylum-level analysis found a negative association between Bacteroidetes and CD4+ T cells and Tregs and a positive association between Actinobacteria and CD4+ T cells and Tr1 (IL-10), which represent some of the most common bacterial phyla of the human microbiota. The evenness of the gut microbiome also had a strong and negative association with Th17 and T helper 2 (Th2) response in the control group 28 .
More recently, the interaction between microbiome and diet in MS has attracted attention. A study found an association between a lower number of relapses and a lower disability status scale after one year of a high vegetable/low protein diet (HV/LP diet) when compared with a classical Western diet characterized by regular consumption of red meat, processed meat, refined grains, sweetened food, salt, and saturated and omega-6 fatty acids. The HV/LP group had higher levels of bacteria from the Lachnospiraceae genus, and they were positively related to Treg cells 29 . Table 1 summarizes the most relevant microbiome findings in patients with MS.

AUTOIMMUNE ENCEPHALITIS
Immuno-mediated encephalitis is an emergent group of syndromes characterized by the development of acute or subacute progressive encephalopathy (less than three months onset) that occurs due to an abnormal antibody response against cell-surface, intracellular synaptic, or intraneuronal (nuclear or cytoplasmic) antigens 30, 31 .
Most of what is known about the impact of intestinal dysbiosis in patients with autoimmune encephalitis (AE) comes from studies conducted in rodents. It has been suggested that changes in the intestinal microbiota could increase the susceptibility to AE through different mechanisms. Thus, increases in abundance and richness of specific pathobionts can provoke pro-inflammatory T cell crossreactivity due to molecular similarities with neural proteins 22 . Microbiome products of diet fermentation such as the SCFAs have a major impact on gene expression of transcription factors via epigenetic mechanisms. Indeed, in a case-control study conducted by Gong et al., fecal samples were examined in 30 patients at different phases of anti-N-methyl-Daspartate receptor (NMDAR) encephalitis. Patients in the acute phase had low Firmicutes to Bacteroidetes (F/B) ratios than the control group 32 . These results support the theory that an imbalance in commensal microbes negatively impacts the production of SCFAs. Also, main SCFAs such as acetate (C2), propionate (C3), and butyrate (C4) can alter a variety of cellular mechanisms involving the activity of G-protein coupled receptors (GPRs), the inhibition of histones deacetylases (HDACs), and nuclear factor-jB (NF-jB), and the biosynthesis of retinoic acid, which are all essential in maintaining Treg differentiation and hence reducing neuroinflammation 33,34 .
Enrichment of the genus Fusobacterium was also reported in the anti-NMDAR AE. Fusobacteria species comprise gram-negative anaerobic bacilli, which are considered normal microbiota in oral cavity, GI, and urogenital tract. However, recent studies report that F. nucleatum can present pathogenic properties that have been implicated in oral and extraoral diseases, including neurological disorders 32 . It is believed that adhesion and invasion via hematogenous translocation are the primary strategies used by F. nucleatum to activate inflammatory and oncogenic genes, thus contributing to disease development and progression 35 .

MICROBIOME AND STROKE
Studies have shown that up to 50% of patients with stroke suffer from GI complications, which has a strong association with patient recovery, deterioration of neurological functions, and mortality 36 . These features regarding stroke make it an interesting condition to look for associations between brain and gut microbiota. Indeed, several studies have shown a link between stroke outcomes and microbiota regulation of the immune system and metabolism [37][38][39] (Table 2).
The bidirectional communication between the brain and gut after stroke involves the vagus nerve, release of damageassociated molecular patterns (DAMPs), cytokines from the injury site of the brain and gut, and migration of inflammatory or immune cells from the gut to the injury site 40 . This communication occurs by complex signaling pathways from the vagus nerve to the enteric nervous system, the neuronal-glialendothelial interactions, and DAMPs and cytokines-induced activation of gut inflammatory and immune cells 40 .
Several studies show significant changes in the microbial diversity in fecal samples of patients after an ischemic stroke, leading to gut alterations including dysbiosis, dysmotility, hemorrhage, and sepsis 40,41 . Furthermore, Singh et al. showed a reduction in microbiota diversity after stroke events that might be associated with stress response, impaired motility, and tissue necrosis 42 . These changes may cause gut permeability modifications and the increase of circulating lipopolysaccharides (LPS) molecules, which may influence systemic inflammation and immune response after stroke.
Circulating IL-17 released by γδ T cells and IL-10 released by regulatory T cells (Tregs) were associated with increased ischemic brain injury and neuroprotective properties, respectively, after ischemic brain injury 43 . There is some evidence that these inflammatory molecules are regulated by gut microbiota, promoting a strong interrelation between brain and gut and influencing several neurological diseases 40,43 . After a stroke, dysbiosis leads to an imbalance of T-cell subpopulations (Th1, Th2, Th9, Th17, Treg, and follicular T helper cells) that trigger several types of autoimmune and inflammatory disease 40 . For example, Th1 (production of IL-2 and interferon-gamma) and Th2 (IL-4, IL-5, and IL-13) induce inflammation; Th9 (IL-9 and TGF-β) and Treg (IL-10 and IL-35) have a neuroprotective function; Th17 activates matrix metalloproteinases and causes blood-brain barrier breakdown by secreting IL-17A, IL17-F, and IL-22 40 . Benakis et al. demonstrated that antibiotic-treatment-induced dysbiosis could influence stroke outcome in vivo models by regulating T cells in the small intestine 43 . Some studies also observed improved stroke outcomes by fecal microbiota transplantation to control post-stroke dysbiosis 43 . Most interestingly, treatments involving reduction of Clostridiaceae and S24-7 spp. showed to be relevant to neuroprotection after stroke in mice 43 .
Zeng et al. recently raised the possibility that the microbiome might be a novel risk factor for stroke 44 . With a risk stratification approach and comparing higher-versus lower-risk patients, they found an increased risk of stroke associated with enrichment of opportunistic pathogens (e.g., Proteobacteria, Bacilli, Enterobacteriaceae), low abundance of butyrate-producing bacteria (e.g., Lachnospiraceae, Ruminococcaceae), and reduced concentrations of fecal butyrate 44,45 .
Xia et al. developed a stroke dysbiosis index (SDI) based on the patient' s gut taxonomic differences compared to healthy individuals 46 . They observed that high SDI values predicted severe brain injury in patients with stroke. Furthermore, to investigate a putative causal effect of intestinal dysbiosis, the authors performed experiments using a middle cerebral artery occlusion model in animals colonized with microbiota from affected individuals. They observed that mice transplanted with intestinal microbiota from high-SDI patients also developed an exacerbated inflammatory response, hence, worsening the acute brain injury associated with stroke 46 .
Although the results are encouraging, as described above, additional studies with larger samples and different ethnic backgrounds are needed to validate these findings. However, if further confirmed, a careful approach, including microbiome screening as a possible preventive target for stroke management is needed.

GUT MICROBIOTA: THE RELATIONSHIP WITH DEMENTIA
Studies in germ-free animals exposed to microbial infections, human post-mortem brain samples, and microbiome analysis of living humans have revealed that disorder of the gut microbiota may underlie the development or exacerbation of Alzheimer's disease (AD) pathology 47 . Also, available data suggest that the gut microbiota in AD is characterized by a substantial reduction in beneficial microbial diversity and presence of pathogenic species such as Proteobacteria phylum, especially the Enterobacteriaceae family 48 . These shifts in microbial diversity may activate immune cells and stimulate overproduction of toxic metabolites or pro-inflammatory cytokines, which contribute to the destruction of the GI mucosa. It is well known that chronic inflammation and immune dysregulation precede cognitive decline by years 49 . Increased inflammation makes gut microorganisms move from the GI tract through cells overlying the Peyer's patches into blood and other tissues (a process known as atopobiosis) 47 . Likewise, systemic inflammation can increase BBB permeability, exposing the brain to cytokines that can lead to neuroinflammation and neuronal cell death, promoting neurodegenerative diseases 48,50 . Initially, the brain can resist, but the regenerative capacity, together with the microglia's ability to clear toxic metabolites, decreases with time 49 .
The outer membrane component of Gram-negative bacteria is LPS, capable of triggering systemic inflammation by increasing pro-inflammatory cytokines 48,51 . Lipopolysaccharides may also modify gut homeostasis and promote gut inflammation and permeability. The abundance of Gram-negative intestinal bacteria, such as the Enterobacteriaceae family in individuals with AD, results in increased translocation of LPS from the gut into the circulation, which in turn may contribute to AD pathology 52 . Indeed, a study involving post-mortem brain tissue from patients with AD showed that LPS fragments co-localized with amyloid plaques in the hippocampus and neocortex 53 .
Surprisingly, bacteria can produce their own amyloid, which maintains cellular junctions, promotes the formation of biofilms, and confers resistance against physical or immune destruction 47 . Microbial and cerebral amyloids are structurally similar and can be recognized by the same TLR2/TLR1 receptor system 50 . They might activate signaling pathways known to play a role in neurodegeneration and AD pathogenesis 50,51 . The hypothesized mechanism is that bacteria-derived amyloids leak from the GI tract and accumulate in the brain, resulting in an increase of reactive oxygen species and activation of nuclear factor-κB, which upregulates the pro-inflammatory microRNA-34a 51 . Subsequently, microRNA-34a downregulates the expression of TREM2 (triggering receptor expressed on myeloid cells 2), leading to impairment of phagocytosis and contributing to the peptide accumulation β-amyloid1-42 51 . Both amyloids and LPS are potent activators of the receptor for advanced glycation end-products (RAGE) and Tolllike receptors (TLR), and their co-activation amplifies pro-inflammatory signaling leading to sustained chronic inflammation in AD 50,51 .
Commensal microbiota produces an assortment of neuroactive molecules, such as serotonin, kynurenine, GABA, catecholamines, histamine, and acetylcholine, among others 50 . The consequence of a dysbiotic bowel in the metabolism of tryptophan and kynurenic pathways is documented in AD 50 . Gut microbes may regulate the serotonergic system directly by producing serotonin or degrading the serotonin precursor, tryptophan 49 . Escherichia coli, an Enterobacteriaceae member, plays an essential role in regulating production and availability of serotonin, acting as a transmitter both in the CNS and in the enteric nervous system 51 . Nevertheless, gut-derived serotonin only exerts indirect effects on brain functions. Despite that, the gut is the only source of tryptophan, derived either from the diet or microbial production. Tryptophan crosses the BBB to become available for serotonin synthesis in the brain 49 . Gulaj et al. found reduced plasma concentration of tryptophan and kynurenic acid in 34 patients with AD, suggesting that dysregulation of the kynurenine route is present in AD 54 .
Lactobacillus and Bifidobacterial genera can metabolize glutamate to produce GABA. Changes in gut microbiota might compromise the endogenous production of this inhibitory neurotransmitter 51 . Alterations in GABA signaling are linked to cognitive impairment and AD neuropathy 49 . Likewise, Lactobacillus, Lactococcus, Streptococcus, and Enterococcus may produce histamine, which acts as a neurotransmitter essential for modulating neuroinflammation through TNF-α expression in the brain 55 . Furthermore, an N-methyl-D-aspartate (NMDA)targeting neurotoxin that was observed to be raised in AD brains may be produced by gut cyanobacteria 49 .
The role of diet in these mechanisms is still poorly understood but probably extremely important 47 . Dietary patterns similar to the Mediterranean diet and Dietary Approaches to Stop Hypertension have been associated with a reduced risk of AD 56 . In contrast, a Western-style diet represents a risk factor. The variety and composition of a diet and long-term dietary habits may influence the gut microbiota composition and shape the microbial community 51 . A newly proposed insight is that the transplantation of fecal microflora from healthy people to patients with AD can help restore the intestinal microbiota and reduce the negative impact of the dysbiotic microbiome on the gut and brain functions 47 . The influence of gut microbiota on brain function is being investigated continuously. Table 3 presents additional studies on the role of gut microbiota and dementia.

EPILEPSY, KETOGENIC DIET, AND THE INTESTINAL MICROBIOME
About one-third of patients with epilepsy have seizures refractory to anti-seizure drugs (ASD) 57 . Non-pharmacological approaches, especially the ketogenic diet (KD), are alternatives in cases of pharmacoresistant epilepsy. The KD has been used for about one hundred years, demonstrating efficacy in reducing seizure frequency, mostly in children with difficultto-control epilepsy 58 .
The KD appears to be a powerful contributor in modulating downstream effects on an individual' s gut metagenomic composition and metabolomic profile 59 . In a dietary intervention study conducted by Ang et al., two cohorts composed of obese patients and mice were followed up during short-and long-term periods. In humans, a baseline diet and a KD with only 5% of carbohydrates content were used. Mice were submitted to three different diet types: low-fat diet (LFD), highfat diet (HFD), and KD. Results from a 16S amplicon-based metagenomic approach of stool samples and metabolomic profile in humans demonstrated a significant reduction in the relative abundance of Actinobacteria phylum and a marked decrease in different bifidobacterial species, suggesting that the KD is sufficient to shift the gut microbiota composition. Similar findings were seen in a mice model, where ketone bodies like βHB had a direct effect in suppressing microbial proliferation of bifidobacteria. Moreover, fecal microbial transplantation from patients into germ-free mice demonstrated that the colonization of KD-associated microorganisms drove the reduction of selected members of Bifidobacterium adolescentis, therefore modulating the induction of Th17 cells.
These findings reveal a separate pathway whereby carbohydrate restriction, rather than high-fat intake, is the main contributor to gut microbiome structure and immune response 60 .
Olson et al. demonstrated that the taxonomic composition of the gut microbiome in mice is altered after treatment with KD. They also raised the possibility that some KD-associated species such as Akkermansia muciniphila and Parabacteroides merdae and particular molecules predicted seizure protection with high accuracy and were necessary to reduce brain electrical activity 61 . Taken together, these findings are likely to be correlated with a decrease in the metabolism of ketogenic gamma-glutamylated (GG) amino acids and, therefore, to low concentrations of these amino acids in the colon, blood, and CNS. Likewise, this phenomenon can boost GABA bioavailability on the hippocampus, increasing seizure threshold in mice and contributing to the anti-seizure effect. Finally, based on these bacterial species and molecules, researchers could identify microbiome-based treatments such as microbiome transplant, live biotherapeutic products, and targeted pharmacological approaches that protect against seizures in mice 61 .
To further examine the potential of KD in shaping the intestinal microbiome in patients with epilepsy, Lindefeldt et al. analyzed the taxonomic and functional profile in children with difficult-to-control epilepsy using whole metagenomic sequencing 62 . The study consistently showed that i) there were differences in the composition of the patient' s gut regarding healthy controls before starting the intervention; ii) those differences were reflected in the reduction of relative abundances of butyrate-producing organisms such as Eubacterium rectale and Dialister during and after treatment with KD where variations were more noticeable; iii) KD decreased Bifidobacterium and Dialister had an impact not only on the production of nondigestible carbohydrates (e.g., lactate) but also on the final conversion to SCFAs (mainly acetate), which are crucial in brain physiology; finally, iv) shifts on gut microbiome associated with KD promoted the growth of Escherichia coli, which could trigger gut inflammation in epileptic patients 62 . These results are in line with a study carried out by Zhang et al., who explored the potential of microbial biomarkers in patients with refractory epilepsy who followed a KD for six months 63 . Overall, they observed low diversity and richness ratios in individuals undergoing a KD as well as an increase in the relative abundance of Bacteroidetes and reduction of Firmicutes and Actinobacteria phyla. They also reported a high abundance of Clostridia class organisms in non-responders. This class has been associated with tryptophan catabolites, which in turn are responsible for hormone secretion, neurotransmission, gut motility and permeability, and anti-oxidative effects 63 . Another work involving epilepsy and KD had a similar outcome and reported significant gut dysbiosis in the refractory group. Nonetheless, the enterotype Bacteroidetes was accumulated in both the healthy and epileptic groups after at least one week of high-fat diet therapy. This phylum has been related to seizure modulation by secretion of inflammatory cytokines, including IL6 and IL17.
Also, Cronobacter was the predominant genus identified only in affected children, which decreased over the treatment period 64 .
It has been observed that patients with epilepsy are presumably prone to gut dysbiosis and, hence, to chronic inflammation of the intestinal epithelium [65][66][67] . In a cohort of 91 individuals, Peng et al. revealed that patients with more than four seizures per year had a predominance of Ruminococcus and rare bacteria genera compared to a drug-sensitive group. These findings lead to various hypotheses: i) the use of several antiepileptic drugs (AED) induces intestinal dysbiosis; ii) the prevalence of rare microorganisms modulate metabolic pathways involving ABC transporters, therefore conferring chemoresistance to the treatment; iii) Bifidobacterial and Lactobacilli genera stimulate the production of GABA and are prevalent in the drug-sensitive group 68 .
So far, all of the studies involving different epilepsy phenotypes either in humans or mice report changes in the intestinal microbiome at baseline or after a KD 69 . Still, there is little overlap across these studies in the exact microbial signatures that have been identified (Table 4). Thus, additional highpowered and well-controlled studies are needed to explore the issue better and propose new treatment options 70,71 .

PARKINSON'S DISEASE AND MICROBIOME
It is well known that patients with Parkinson' s disease (PD) present severe non-motor symptoms at the prodromal phase of the disease, which are determinants of the quality of life in these individuals. Sensorial, neuropsychiatric, sleep dysfunction, and GI symptoms (constipation) are the most common phenotypes describe in this category 72 . Several studies using breath testing demonstrated that a considerable proportion of patients with PD have intestinal bacterial overgrowth and absorption issues, leading to intestinal constipation. On the other hand, 16S ribosomal RNA analysis from colonic biopsies and stool samples of adults with PD showed decreased SCFAs-producing bacteria such as Blautia, Coprococcus, and Roseburia 73,74 . Furthermore, depletion or increase in Prevotella and Lactobacillus genera was reported in several case-control studies performed in these patients 75,76 . This class of beneficial microorganisms is involved in BBB integrity, permeability, and neuronal inflammatory signaling 73 . Moreover, the increase in Enterobacteriaceae members was directly proportional to the severity of symptoms like stability, gait, and rigidity 77 . Conversely, in a study conducted in individuals with PD, the oral microbiome was analyzed. Male patients exhibited an increase in the abundance of Prevotella, which is considered an opportunistic pathobiont on the mouth, suggesting a strong role of these genera in periodontal disease 78 . Taken together, these findings support that modifications in bacteria density, taxonomic levels (dysbiosis), and mucin production may, in turn, boost the neuroglia system, triggering damage to the intestinal and brain barriers, leading to alpha-synuclein protein misfolding and finally aggregation. Likewise, chronic systemic exposure to LPS leads to the selective death of dopaminergic neurons in the substantia nigra 73,79,80 .
The vagus nerve (VN) has also been implicated in the pathogenesis of PD. Some researchers suggest that the VN can modulate neuroimmune and inflammatory signals either via top-down or through the microbiota-gut axis 79 . Thus, it has been proposed that the VN could transport alpha-synuclein to the CNS and vice versa. After examining 9,430 vagotomies in Swedish patients, Liu et al. demonstrated that truncal, but not selective vagotomy, had a protective effect against PD development 80 . In contrast, recent studies in mouse models mimicking motor and non-motor symptoms of early and late stages of the disease point out that changes in the immune response to gut bacteria could affect motor symptoms in PD 81 . In an experimental study performed by Sangjune et al., the authors demonstrated that pathologic species of a-synuclein could spread from the gut muscles to the brain through connections of the vagus nerve. These mice also showed neuropsychiatric symptoms, including anxiety, depression, olfactory dysfunction, and spatial learning and memory abnormalities. They also assessed another group of animals that were submitted to an injection of a-synuclein and vagotomy. The authors observed that no transmission of pathologic α-synuclein occurred in these animals, which were also free of the cardinal symptoms of PD 82 .
Several studies have been published exploring the gut microbiome in PD (Table 5). However, the need for welldesigned clinical studies exploring the role of the gut microbiome in PD in the clinical setting is still lacking.

Autism Spectrum Disorder
Clinical observations indicate that patients with Autism Spectrum Disorder (ASD) have GI disturbances that include diarrhea, constipation, and abdominal pain 83 . Likewise, a growing number of studies have shown that patients with ASD have an altered gut microbiota composition compared to neurotypical individuals 84 . Moreover, GI disturbances strongly correlate with the severity of ASD symptoms, and GI disturbances are markedly associated with GI dysbiosis 83,84 . Therefore, it has been suggested that gut microbiota alterations may contribute to the pathogenesis of ASD (69).
Although several studies have shown that adult rodents prenatally exposed to VPA, a model for ASD, exhibit gut dysbiosis 85,86 , there is a lack of evidence of a causal link between abnormal microbiota and ASD-like behaviors.

Schizophrenia
Patients with schizophrenia commonly have GI disturbances, such as constipation and GI hypomotility and inflammation 87,88 . A growing body of evidence indicates that altered gut microbiota may account for the GI disturbances and the severity of symptoms in schizophrenia patients suggesting a key role of the gut microbiota in promoting the pathogenesis of schizophrenia. Interestingly, this suggestion has been recently proved by Zheng and colleagues. In this study, the authors found that the gut microbiota from patients with schizophrenia induced behavioral alterations and modulated brain excitability when transferred to mice 89 . Other studies have also suggested that GI disturbances and gut microbiota alterations in schizophrenia may be related to the use of antipsychotic medication 90 . However, gut microbiota seems to be altered in patients even before medication 91 . Hence, to better understand the gut microbiota alterations in schizophrenia, experimental validation of clinical findings seems necessary. Furthermore, animal models of schizophrenia reinforce the clinical data showing that gut dysbiosis may be implicated in schizophrenia, mainly pointing to alterations in the Firmicutes phylum bacteria.

ANXIETY AND DEPRESSION DISORDERS
Preclinical studies have shown that the intestinal microbiome seems to play a crucial role in the pathophysiology of both neuropsychiatric disorders. Also, fecal microbiota from patients diagnosed with anxiety and depression induce behavioral and physiological features of these disorders when transplanted to microbiota-deficient animals, including anhedonia, anxiety-like behaviors, and altered tryptophan metabolism [92][93][94] .
Park and colleagues showed that anxiety-and depression-like behaviors induced by olfactory bulbectomy (OBx) are related to colonic motility alterations and gut microbiota composition changes. In addition, the expression of hypothalamic corticotropin-hormone (CRH) was elevated in OBx mice, suggesting that GI disturbances and gut dysbiosis may be due to the recruitment of the hypothalamic-pituitaryadrenal axis 95,96 . Murakami et al. analyzed the gut microbiota composition of fecal samples from Wistar rats submitted to maternal separation when neonates. This early-life stressor leads to anxiety-and depression-like behaviors in adulthood. The authors found that maternal separation induced a specific reduction in Bifidobacterium, Bacteroidetes, and Prevotella genera 97 . Li et al. analyzed the gut microbiota of fecal samples from mice submitted to the chronic unpredictable mild stress (CUMS) model. They found that the gut microbiota of CUMS-treated mice exhibits drastic alterations in microbiota composition, including an increased α-diversity and changes in the abundance of specific microbial phyla, such as Verrucomicrobia and Proteobacteria. At the genus level, animals exposed to CUMS exhibit an increased abundance in Helicobacter, Turicibacter, Parasutterella, Alistipes, Odoribacter, and Akkermansia, but a decrease in Barnesiella, Bifidobacterium, Lactobacillus, and Olsenella 98 .

FUTURE DIRECTIONS
Reports using cutting-edge technologies such as metagenomics and metabolomics are changing some of the established paradigms regarding the physiopathological mechanisms behind neuropsychiatric diseases. Using the potential of microbial profiles as biomarkers of neurological and mental health disorders may maximize the efficacy of existing therapies. Current challenges remain in establishing causation rather than association and translating basic science studies into clinical practice with the potential of targeting the microbiome for therapeutic purposes.