Regulation of common neurological disorders by gut microbial metabolites

The gut is connected to the CNS by immunological mediators, lymphocytes, neurotransmitters, microbes and microbial metabolites. A mounting body of evidence indicates that the microbiome exerts significant effects on immune cells and CNS cells. These effects frequently result in the suppression or exacerbation of inflammatory responses, the latter of which can lead to severe tissue damage, altered synapse formation and disrupted maintenance of the CNS. Herein, we review recent progress in research on the microbial regulation of CNS diseases with a focus on major gut microbial metabolites, such as short-chain fatty acids, tryptophan metabolites, and secondary bile acids. Pathological changes in the CNS are associated with dysbiosis and altered levels of microbial metabolites, which can further exacerbate various neurological disorders. The cellular and molecular mechanisms by which these gut microbial metabolites regulate inflammatory diseases in the CNS are discussed. We highlight the similarities and differences in the impact on four major CNS diseases, i.e., multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, and autism spectrum disorder, to identify common cellular and molecular networks governing the regulation of cellular constituents and pathogenesis in the CNS by microbial metabolites.


INTRODUCTION
The central nervous system (CNS) is known as an immuneprivileged tissue system but remains susceptible to inflammatory responses. While well protected by the blood-brain barrier (BBB), nevertheless the CNS hosts many types of inflammatory responses. A mounting body of evidence indicates the presence of close interactions between the neural system and gut microbes. These interactions involve endocrine, neurological, metabolic and immunological communication. The CNS and enteric nervous system (ENS) communicate via neurotransmitters, hormones, and metabolites 1,2 . Gut microbial metabolites are transported to the CNS, wherein they regulate CNS cells and immune cells. This review will examine our current understanding of how gut microbial metabolites impact four major CNS diseases and highlight the common disease-modifying functions of major microbial metabolites.
Multiple sclerosis (MS) is the most common form of inflammatory disease in the CNS and is associated with autoimmunemediated demyelination 3,4 . Other less frequent inflammatory diseases include Rasmussen's encephalitis 5 and neuromyelitis optica (NMO) 6 . These diseases involve immune cell infiltrates in the brain and spinal cord, resulting in neuritis and myelitis 7,8 . Antigen-specific T and B lymphocytes are frequent immune cell infiltrates in CNS lesions. Inflammatory responses in the CNS are induced or suppressed by different types of T lymphocytes, such as Th1 and Th17 cells and Tregs 9,10 . Antibodies and B cells can augment or alter the pathogenesis of CNS inflammation 11 . Other cell types, such as microglia, macrophages and astrocytes, actively participate in immune cell regulation and neuroinflammation 12 . An increasing body of evidence indicates that other neurological diseases, such as Parkinson's disease (PD), Alzheimer's disease, and autism spectrum disorder (ASD), also involve significant inflammatory responses [13][14][15] . Therefore, we focus this review on these four CNS diseases, which have distinct tissue involvement, inflammatory responses and pathogenesis.
In recent years, we have witnessed a plethora of research on the functions of gut microbiota and their metabolites in regulating inflammatory responses in the CNS [16][17][18][19] . The intestine is rich in dietary materials and host secretions such as bile acids and mucins, which are subsequently metabolized by the cooperative activities of host enzymes and then by the gut microbiome. Various dietary fibers and complex carbohydrates can reach the terminal ileum and colon, which host the majority of the gut microbiome in the body. Microbial fermentation of sugars derived from carbohydrates in the colon produces short-chain fatty acids (SCFAs) such as acetate (C2), propionate (C3) and butyrate (C4) 20 . Another major group of metabolites includes tryptophan (Trp) metabolites. Dietary tryptophan is converted into indole derivatives, including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-aldehyde (IAld), indole-3-acetaldehyde (IAAld), and indoleacrylic acid. Serotonin (5-HT), a neurotransmitter, is also produced in the gut lumen from Trp [21][22][23][24] . Primary bile acids are produced from cholesterol in the liver and are dehydroxylated by gut microbes to generate secondary bile acids 25 . These gut microbial metabolites function as agonists for various host receptors and intracellular molecules, such as G-protein-coupled receptors (GPCRs) and transcription factors, including GPR43, GPR41, TGR5, aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), farnesoid X receptor (FXR), vitamin D receptor (VDR), and others 26 . Moreover, many of these metabolites, particularly SCFAs, are metabolized in host cells to generate energy. Microbial metabolites regulate numerous host enzymes, such as histone deacetylases (HDACs). In general, microbial metabolites regulate host gene expression and metabolic activity in both immune cells and tissue cells. Importantly, these metabolites directly and indirectly affect the CNS to exert their regulatory functions 19,27,28 .

BASIC FUNCTIONS OF GUT MICROBIAL METABOLITES
The most abundant metabolites in the colon are SCFAs, which have a combined luminal concentration of greater than 0.1 M in the human colon 29 . The three major SCFAs, C2, C3 and C4, function through four G-protein-coupled receptors (GPCRs), GPR41, GPR43, GPR109A and Olfr78 [30][31][32] . In addition, SCFAs are natural HDAC inhibitors, and therefore, they can increase the acetylation and phosphorylation of host proteins such as histones, leading to increased gene expression and cell signaling 33,34 . SCFAs directly inhibit Type I and II HDACs and can also indirectly affect certain Type III HDACs, such as Sirtuin 1 35 . Moreover, SCFAs are metabolized in host cells for energy production and thus have significant regulatory effects on host cell metabolism, leading to increased ATP levels, mTOR activity and fatty acid synthesis 36 . SCFAs are known to boost the differentiation of Th1 and Th17 cells and Tregs from naïve CD4 T cells, depending on the immunological milieu 36 . SCFAs increase IL-10 expression by lymphocytes (T and B cells) and macrophages 37,38 in a manner largely mediated by their HDAC inhibitory activity. SCFAs regulate the activity of innate lymphocytes; they increase the number and activity of Group 3 innate lymphoid cells (ILC3s) but suppress Group 2 innate lymphoid cells (ILC2s) through GPCR activation and HDAC inhibition, respectively [39][40][41] . SCFAs also induce tolerogenic macrophages and dendritic cells by both GPCR activation and HDAC inhibition 42 .
Certain amino acid metabolites produced by microbes are also important regulators of host cells. Phenylalanine and tyrosine are precursors of dopamine, a key neurotransmitter, in the host, and some commensal bacteria produce amines related to these amino acids that activate host GPR56 43 . Trp is metabolized by both host cells and certain gut bacteria to serotonin (5hydroxytryptamine), kynurenine (Kyn) and indole derivatives, which function as neurotransmitters and metabolic regulators 24 . Certain indole derivatives, such as IAA, IPA, and IAld, function as ligands for AhR, a member of the basic helix-loop-helix transcription factor family. These metabolites can also regulate host cells through EIF5a, IR76B, and PXR 44 . Certain microbial species, such as Peptostreptococcus russellii 45 and Lactobacillus species 46,47 , can effectively produce indole derivatives [45][46][47] . Dietary indole derivatives, such as indole-3-carbinol (I3C) and diindolylmethane (DIM), can increase the activity of Tregs but suppress the generation of myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells 48 . Polyamines such as putrescine and spermidine are produced by intestinal bacteria and are present at 0.5 to 1 mM in the human colon 49 . Polyamines support the hypusine modification of eukaryotic translation initiation factor 5A (eIF5A) and promote polypeptide translation and cell proliferation 50 . Polyamines also have suppressive effects on Th17 cells 51 . Similarly, phytochemical metabolites, such as ginsenosides, resveratrol, and I3C, activate AhR to exert some of their regulatory functions 52,53 . Ginsenosides suppress Th1 and Th17 cells but increase Treg activity 54 . Resveratrol suppresses Th17 activity 55 .

REGULATION OF EXPERIMENTAL NEUROINFLAMMATION BY MICROBIAL METABOLITES
The regulatory effects of microbiota on experimental neuroinflammation have been reported [71][72][73][74] . Rodent experimental autoimmune encephalitis (EAE) models are most frequently used 75 because they largely mimic human MS. Antigen-induced EAE can be induced by immunization with several neuronal cell antigens, such as MOG, myelin basic protein (MBP), and proteolipid protein (PLP), along with potent adjuvants, such as complete Freund's adjuvant and pertussis toxin 75 . Demyelination similar to that in MS is also induced by chemicals such as cuprizone (a copperchelating agent) and lysolecithin [76][77][78] . Infection with Theiler's murine encephalomyelitis virus induces demyelinating encephalitis 79,80 . Moreover, the engineered expression of T and B cell receptors specific for CNS antigens causes spontaneous EAE responses in mice 81,82 .
While dysbiosis exacerbates EAE responses 83,84 , studies with germ-free (GF) animals or broad-spectrum antibiotics revealed that the microbiota is required for optimal EAE pathogenesis 85,86 , perhaps because of the general adjuvant effect of the microbiota in inducing immune responses. When specific microbial species or their products are suppressed, EAE activity may either increase or decrease, depending on the function of the affected microbial species 87,88 . For example, segmented filamentous bacteria (SFB), S. aureus and P. heparinolytica promote the generation of Th17 cells and exacerbate EAE responses [89][90][91] . Elimination of these Th17inducing microbial species ameliorated EAE inflammation 85,86 . In contrast, treatment with SCFA-producing bacteria such as Clostridium tyrobutyricum ameliorated EAE immune responses 92 . Thus, the impact of the microbiota on CNS inflammation depends on the function of the altered microbial species in the gut 84 .
It is thought that the microbiota affects neuroinflammation by producing immune regulatory products and metabolites. For example, polysaccharide A (PSA), a capsular polysaccharide produced by Bacteroidetes fragilis, has significant immune regulatory function 93 . Oral administration of PSA suppressed demyelination and delayed the onset of EAE activity 94 . In this previous study, PSA increased the number of CD103 + dendritic cells (DCs) in draining lymph nodes and induced IL-10-producing T cells. PSA promoted Toll-like receptor 2 (TLR2) activation, which induced CD39 + regulatory T cells, in part by modulating DCs. In addition, poly-γ-glutamic acid (γ-PGA) has suppressive effects on EAE activity 95 . This finding is in line with decreased Th17 cell but increased Treg activity in the CNS following the administration of γ-PGA, which activates TLR4-MyD88-dependent and TLR4-MyD88independent pathways to regulate T cell activity 95 . In contrast, some bacterial products can exacerbate CNS inflammation. Lipopolysaccharide (LPS) treatment accelerated inflammationmediated demyelination in rats 96 . It is well established that peptidoglycans, the major cell wall component of gram-positive bacteria, promote DC maturation through TLR2 activation, which increases the secretion of proinflammatory cytokines and promotes effector T cell generation during EAE development 89 . Thus, microbial TLR ligands can have both inflammatory and suppressive roles in neuroinflammation.
BBB disruption is observed in EAE and MS patients, and this can cause harmful exposure of CNS tissues to microbial products and microbial invasion following gut dysbiosis, leading to amplified inflammatory responses 97,98 . Under normal conditions, the microbiota and SCFAs support the integrity of the BBB 99 ; it is important to protect the CNS from the harmful effects of inflammatory responses and toxicants. The BBB integrity is weakened in GF mice, and this defect is normalized by SCFA treatment in drinking water 99 (Fig. 1). SCFAs, such as C2, C3, or C4, administered in drinking water ameliorated EAE activity. Interestingly, SCFAs appear to support CNS development beyond their immunoregulatory roles 100,101 . Microglial cells in GF mice had defective microglia with altered cell proportions and an immature phenotype, which were corrected by SCFA feeding in a GPR43dependent manner 100 . In addition, C4 promoted oligodendrocyte maturation in the brain 101 . Thus, SCFAs support the functional maturation of CNS cells (Fig. 1). Increased levels of secondary bile acids, such as DCA, can weaken BBB function, perhaps because these compounds are amphiphilic steroids that loosen the cell membrane 102,103 . Thus, BBB function and associated CNS pathogenesis can be altered by changes in microbial metabolites.
Multiple groups have reported that SCFAs have EAEsuppressing activities (Table 1). Mice with elevated levels of SCFAs after feeding with SCFA water or dietary fiber had increased numbers of regulatory T cells in the intestine and CNS upon MOG immunization 104,105 . In addition, when mice were fed C4, cuprizone-mediated demyelination was ameliorated. 18,74,101,105 . The protective effect of C4 was further validated in a lysolecithininduced demyelination model 101 . In addition to the major SCFAs (i.e., C2, C3, and C4), valerate (C5) can also regulate EAE activity 27 . C5 increased both regulatory B cells (Bregs) and T cells (Tregs). C5treated B cells showed increased expression of IL-10, and adoptive transfer of these B cells ameliorated EAE activity. The protective effect was accompanied by decreased Th17 cell activity in the small intestine. C5, well known for its potent HDAC inhibitory activity, increased mTOR activation and glycolysis in lymphocytes 27 . Thus, both epigenetic (HDAC inhibition) and metabolic (mTOR) regulation appears to be involved in the protective effects of SCFAs in EAE pathogenesis.
The effects of SCFA-producing fiber-rich diets on EAE are mixed, and this appears to be due to considerable variation in the dietary fiber composition of high-and low-fiber diets used by different groups. Some groups used a diet containing no fiber (soluble or insoluble) as the control diet [106][107][108] . In general, soluble fibers such as pectin and inulin produce high levels of SCFAs, whereas insoluble fibers such as cellulose are not efficient sources of SCFAs. Other groups, including ours, compared the effects of high-and low-fiber diets containing the same amount of cellulose but different levels (0, 5, and 20%) of soluble fiber. When a high-fiber diet was compared with a zero-fiber diet, the protective effect was significant 105 . However, when the animal group fed a diet high in soluble fiber (pectin and inulin) was compared with that fed a diet containing zero soluble dietary fiber but the same level of cellulose, the difference was not significant 18 . This finding suggests the involvement of mechanisms other than SCFA production in the beneficial effects of dietary fiber. Despite its low bioavailability and SCFA-producing ability, cellulose seems to have a suppressive effect on EAE activity 84 . Cellulose decreased the number of neutrophils and Th2 cells in a spontaneous opticospinal encephalomyelitis mouse model expressing MOG-specific T and B cell Fig. 1 Microbial metabolites regulate CNS development, integrity, and inflammation. Microbial metabolites positively and negatively influence CNS development and inflammatory responses. In the best case, beneficial metabolites are produced in symbiosis with a balanced population of diverse microbes in the gut. Together, these microbes produce myriad metabolites that are beneficial for the host. In dysbiosis, the production of harmful metabolites is increased while that of beneficial metabolites is decreased. In general, beneficial metabolites, such as SCFAs and Trp metabolites, reinforce the integrity of the gut barrier and BBB and support the functional maturation of CNS cells such as microglia, oligodendrocytes and astrocytes. Thus, these metabolites support CNS formation, neurological function, and the development of regulatory immune cells for immune tolerance. Moreover, these metabolites suppress harmful immune responses, such as the generation of pathogenic Th17 cells. These functions are mediated in part by various host receptors, such as GPCRs, transcription factors, nuclear ligand receptors (PXR and FXR), and TLRs. Conversely, harmful metabolites weaken the gut barrier and BBB and cause systemic inflammatory responses, neuronal cell death and tissue injury (e.g., demyelination), leading to inflammatory conditions that exacerbate CNS diseases. Not only harmful microbial metabolites but also pathogenic bacterial cells and T cells travel from the gut to the CNS to increase inflammation under pathological conditions. antigen receptors 84 . While cellulose did not significantly change SCFA levels, it changed the microbiome composition in the gut, indicating that the protective effect of certain dietary fibers, such as cellulose, appears to be more dependent on microbial changes than on SCFA production 108 .
T cells, which differentially regulate inflammatory responses. Several cell types can produce IL-10 in response to SCFAs. Major cell types that produce IL-10 are T cells and macrophages, and this production can be further increased by SCFAs 38,109-111 (Fig. 1). In our own study, SCFAs increased IL-10 production by microglial cells, and IL-10 production was required for the protective effect of SCFAs on EAE activity 18 . While SCFAs have suppressive effects on EAE activity, their Gprotein-coupled receptors, such as GPR43, can function unexpectedly to exacerbate EAE activity 18 . The mechanism is not entirely clear at this point, but the data imply that the protective function of SCFAs is perhaps mediated mainly through non-GPCRdependent mechanisms. In this regard, the HDAC inhibitory activity of SCFAs may be important. GPCR activation by SCFAs can increase immune activity, including invigorating epithelial responses and activating inflammasomes in the gut 112,113 . We speculate that GPCR activation by SCFAs in the CNS could promote similar inflammatory responses. In this regard, the function of SCFAs in regulating EAE activity appears to be complex. When transferred into host mice, SCFA-treated MOGspecific effector Th17 cells have greater EAE activity than similarly prepared control Th17 cells 18 , indicating that SCFAs can affect both the inflammatory and regulatory arms of the CNS immune system. More work is required to understand the effects of SCFAs on key immune and CNS cell types, such as T and B cells, ILCs, glial cells, neurons, macrophages, and dendritic cells.
Indole derivatives and certain environmental contaminants, such as the AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been studied for their regulatory effects on EAE activity. AhR activation by FICZ and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) (Trp-derived indole metabolites) suppressed EAE responses 114,115 , whereas AhR activation by TCDD exacerbated EAE pathogenesis 70 . Similarly, FICZ and TCDD differentially affected Th17 cells, Tregs, DCs, and astrocytes. The administration of dietary indoles was effective at suppressing EAE development 48 . Moreover, dietary indoles significantly ameliorated existing EAE 48,116 ; this protection is thought to be mediated by the AhR-mediated expansion of regulatory T cells. These Trp-derived metabolites appear to suppress CNS inflammation by decreasing proinflammatory cytokine expression and inflammatory monocyte infiltration 47,48,116 , which may involve activation of the type-I IFN pathway in astrocytes to limit CNS inflammation in an AhRdependent manner. Similarly, cruciferous plant-derived AhR ligands such as glucosinolates suppressed EAE pathogenesis 117 .

SIGNIFICANT ASSOCIATIONS BETWEEN MICROBIAL METABOLITES AND MS IN HUMANS
In humans, MS manifests in several ways with different patterns of disease progression and relapse; the common categorizations of MS include clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS) 118,119 . RRMS, with repeated relapses and remissions, is most common (~85%). Most people with RRMS progress to SPMS with steadily worsening symptoms with or without remissions and relapses. PPMS is rarer (~10%), with slowly worsening symptoms from disease onset without remissions and relapses.
Changes in gut luminal microbial composition have been observed along with the abnormal invasion of microbes in the brains of RRMS patients 120 . RRMS patients have increased levels of Actinobacteria but decreased levels of Firmicutes and Bacteroidetes in feces. Within the Firmicutes family, fourteen species within Clostridium clusters XIVa and IV were found to be decreased in MS patients 121 . A cohort study found decreased Prevotella_9 but increased Streptococcus species, along with reduced levels of fecal SCFAs, in MS patients. In general, Tregs and Th17 cells in the blood indicate tolerogenic and inflammatory activity, respectively 122 . A positive correlation between Streptococcus species and Tregs was reported, and an inverse correlation between Prevotella_9 and Th17 cells was observed. Increased numbers of microbial peptidoglycan-containing macrophages and dendritic cells were detected in the brains of MS patients. Thus, MS patients have both dysbiosis and microbial invasion of the brain 120,123 . CCR9 is the small intestine-homing receptor for lymphocytes 124,125 and is important for thymocyte localization in the thymic medulla 126,127 . Interestingly, CCR9 + CD4 + T cells were found in the cerebrospinal fluid (CSF) of MS patients. T cell migration into inflamed CNS tissues involves another chemokine receptor, CCR6, that is highly expressed by Th17 cells 128 . CCR9 + CD4 + T cells express RORγ and secrete IL-17 and IFN-γ in MS patients. Antibiotic treatment decreased these Th17 cells, suggesting that inflammatory CCR9 + CD4 + T cells are induced by microbes 129 . While further study is required to determine the origin of these T cells, they appear to be generated in the intestine following dysbiosis and neural inflammation. These data imply the active movement of microbes and immune cells from the gut to CNS tissues under inflammatory conditions.
Normally, antigen-induced EAE induction in animals requires the use of potent adjuvants to break immune tolerance to selfantigens 75 . Fecal microbial transfer (FMT) from MS patients to animals induced EAE development following immunization without any adjuvant. FMT decreased certain microbial species, such as those in the Sutterella genus, and decreased IL-10 expression by T cells 16,130 . The effect of SCFA-generating dietary fibers on MS activity has been studied. A diet rich in plant fibers increased the fecal presence of the Lachnospiraceae phylum in MS patients and increased the numbers of Tregs and IL-10-producing PD-L1 + monocytes in the blood. While a fiber-rich diet failed to change MS clinical activity, patients on a Western diet had increased MS clinical activity 131 .
Negative correlations have been found between SCFA levels and T cell activity in MS patients. Higher fecal and blood SCFA levels were associated with increased Treg and suppressed Th1 cell activity 122,131,132 . Compared with healthy individuals, MS patients have decreased levels of blood C4 and SCFA-producing microbial species 118 . In line with the dysbiosis and reduced levels of SCFAs in MS patients 118,133 , decreased levels of all major SCFAs, such as C2, C3, and C4, were found in the blood of SPMS patients compared with that of healthy controls 18 . In addition, gut SCFA levels were decreased in RRMS patients 122 . In this regard, C3 increased Treg activity and decreased episodes of MS relapse 134 . In another study, fecal C3 levels were decreased in both RRMS and SPMS patients 132 . The therapeutic effect of C3 in MS patients was recently reported 134 . Regardless of MS subtype, C3 supplementation alleviated the clinical symptoms of MS, and MS patients with increased levels of SCFA-producing gut bacteria showed limited inflammatory T cell activity. C3 also increased regulatory T cells that produce IL-10, altered the expression of many genes in T cells, and increased oxygen consumption in T cell mitochondria. However, increased levels of plasma C2 and increased numbers of circulating Th17 cells were observed in MS patients with severe disability 135,136 . Because C2 is also produced by host cells under inflammatory and infection conditions 137 , the increased levels of C2 could originate from the host rather than microbial cells. These data, while contradictory to other results, seem to be in line with the effect of C2 on Th17 cell generation 109 . Therefore, more studies are required to establish the possible associations between SCFA levels and MS subtypes. More data may enable the utilization of SCFAs as biomarkers for MS activity and gut microbial status in MS patients.
Oral Trp administration boosted serotonin but decreased plasma cortisol levels in patients with neurological diseases. Acute MS and other neurologic symptoms were alleviated 138,139 . Dietary Trp can be metabolized by gut microbes or host cells after enteric absorption. While gut microbes produce various indole metabolites, host cells produce Kyn, kynurenic acid (KA) and quinolinic acid (QA) via the kynurenine pathway. The levels of urinary Kyn were decreased in RRMS patients 140 . KA levels were increased in RRMS patients but decreased in PPMS patients, while the level of QA was increased in most MS patients 141 . Trp administration improved the cognition and memory function of MS patients 140,[142][143][144] . Pediatric MS is less frequent than adult MS, but relapses are more frequent and possibly more inflammatory in affected pediatric patients 145 . Increased Actinobacteria but decreased Clostridiales were observed in pediatric MS patients 146 . Decreased levels of Trp and indole lactate were also observed in pediatric MS patients. Interestingly, higher Kyn levels were associated with an increased relapse rate 142 . Many Trp metabolites, such as I3S, tryptamine (TA), IAA, Kyn and KA, function as AhR ligands 147,148 . KA also activates another host receptor, GPR35 149 . Trp metabolites increase AhR expression on microglia and astrocytes and control the inflammatory activities of these cells 116,150 . Secondary bile acids also regulate astrocytes by upregulating GPBAR1. Tauroursodeoxycholic acid (TUDCA), a taurine conjugate of ursodeoxycholic acid (UDCA), suppressed microglial and astrocytic polarization ( Table 2) 143 . Overall, multiple gut metabolites can modulate microglial cells and astrocytes to suppress MS pathogenesis (Fig. 1).

MICROBIAL METABOLITES AND PARKINSON'S DISEASE
Parkinson's disease (PD) is caused by the loss of dopaminergic neurons in the substantia nigra (SN), leading to dopamine deficiency in the midbrain; in this region, dopamine is important for the regulation of body movement 151 . A number of genes and their polymorphisms have been implicated in PD pathogenesis 152 . The presence of neurotoxic protein inclusions called Lewy bodies, which are composed of α-synuclein oligomers, in the midbrain is a pathological feature of PD 153,154 . Th1 and Th17 cell-driven inflammation, potentially induced by Lewy bodies, plays significant roles in PD pathogenesis 155,156 . Because of the inflammatory nature of PD and the potential roles of microbiota and gut metabolites in regulating PD, we also include a discussion of PD in this review (Tables 1, 2). Both genetics and environmental factors, such as diet and exposure to neurotoxic chemicals, play significant roles in PD pathogenesis. Genetic risk factors include autosomal dominant mutations in the alpha-synuclein (SNCA), leucine-rich repeat kinase 2 (LRRK2) and vacuolar protein sorting ortholog 35 (VPS35) genes as well as autosomal recessive mutations in the PTEN-induced kinase 1 (PINK1), DJ-1 (also known as PARK7) and PARKIN genes 157 . Interestingly, polymorphisms in the LRRK2 gene are commonly found in PD and inflammatory bowel disease patients 158,159 .
SCFA-producing bacteria and decreased microbial carbohydrate processing activity were detected in a group of PD patients with reduced mobility [165][166][167] . Among the LRRK2 gene mutations associated with PD, the G2019S mutation that affects the kinase domain is the most frequent 168 . The immunological characteristics of transgenic rats expressing human LRRK2 G2019S were examined 169 . Defective myelopoiesis and a decreased Th17 cell frequency were observed in these rats when they were challenged with colitis-inducing agents, such as 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dextran sulfate sodium (DSS). Interestingly, we observed increased levels of Bacteroidetes in this animal model 169 . The MitoPark (MP) mouse model was created by deleting the mitochondrial transcription factor A (TFAM) gene in dopaminergic neurons 170 . Decreased gastrointestinal tract motility and gut dysbiosis were observed in this model. Moreover, increased levels of Prevotella were observed in this model and in PD patients. Young rats fed permethrin, a pesticide, developed PD-like symptoms and increased gut permeability. These animals also developed dysbiosis and showed decreased SCFA production.
The protective effects of valproic acid (a SCFA-related HDAC inhibitor) and C4 were observed in a model of PD induced with the chemicals manganese and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine 160,163,[171][172][173][174] . Interestingly, a synthetic FFAR3 (GPR41) agonist (AR420626) corrected the movement disorder and neuronal loss in a 6-hydroxydopamine (OHDA, a neurotoxin)induced PD mouse model 163 . In contrast, the microbiota and SCFAs were reported to promote PD pathogenesis in α-synucleintransgenic mice 71 . Other researchers have also reported that SCFA administration exacerbates PD pathogenesis in another animal model 175 . The pathogenic role of SCFAs is unexpected but corresponds with the role of these metabolites in promoting the generation of inflammatory Th1 and Th17 cells, as reported previously by our group 109,176 . While these animal models provide insight into PD pathogenesis, the experimental conditions and pathogenesis in these models differ greatly from each other and from PD patients. Thus, the effects of SCFAs on PD pathogenesis are unclear, and more studies are required.
A previous study reported potential changes in bile acid metabolism with increased levels of secondary bile acids, such as LCA and DCA 165 . In a latent PD model in which α-syn fibrils were injected into the olfactory bulb to induce PD-like pathogenesis, the levels of bile acids such as ω-muricholic acid, TUDCA and UDCA were decreased in the blood 177 . Beneficial roles of TUDCA in acute, progressive and nigral transplant animal models of PD have been reported [178][179][180] . TUDCA ameliorated the decrease in dopaminergic fibers, mitochondrial dysfunction and neuroinflammation in an MPTP-induced animal model 179,181,182 . Moreover, TUDCA suppressed α-synuclein-induced oxidative stress via the activation of Nrf2, JNK and AKT 179,182 . In addition, UDCA decreased mitochondrial dysfunction in skin fibroblasts from patients harboring the LRRK2 G2019S mutation 183 . Thus, secondary bile acids have protective effects against PD pathogenesis.

MICROBIAL METABOLITES AND ALZHEIMER'S DISEASE
AD is a progressive neural disease induced by the accumulation of toxic misfolded amyloid β-protein (Aβ) plaques in the brain. In this disease, extracellular β-amyloid plaques form in the basal, temporal, and orbitofrontal neocortex. In severe AD cases, the plaques spread to the hippocampus 184 . Dysbiosis is a risk factor for AD development. Mice humanized with stool from AD patients showed defects in cognitive function 185 . In addition to the gut bacterial composition, the diversity of gut fungal species (mycobiome) in patients with mild cognitive disorder was lower than that in control subjects. Dietary intervention with a Mediterranean ketogenic diet enhanced fungal microbiome diversity and decreased the levels of AD biomarkers in cerebral spinal fluid 186 . Similar to MS and PD patients, AD patients have decreased levels of SCFAs [186][187][188][189] . In particular, AD patients with brain amyloid accumulation and endothelial dysfunction had decreased blood levels of C4 187 . AD pathology is associated with increased blood LPS and inflammatory cytokines. Interestingly, blood C4 and IL-10, but not acetate and valerate, were negatively associated with AD pathology, suggesting a role for SCFAs in AD pathogenesis. Aging and oxidative stress have been suggested to alter the gut microbial community and thus reduce the levels of SCFAs. Caloric restriction and dietary antioxidant supplementation can diminish AD development, in part by modulating microbial composition [190][191][192] . Dietary fiber and SCFAs have been reported to have beneficial effects on AD pathogenesis [193][194][195] . SCFA treatment decreased the polymerization of Aβ1-40 or Aβ1-42 peptides into neurotoxic multimeric Aβ forms 188 . Probiotic supplementation increased the hippocampal concentration of SCFAs and reduced anxiety-like behavior in a humanized AD mouse model 73 . The microbiota has been shown to regulate AD pathogenesis in several animal models. For example, amyloidosis with defective long-term synaptic potentiation develops in the APP/PS1 mouse model, created by overexpressing amyloid precursor protein (APP) and a mutant form of presellin 1 (PS1) [196][197][198] . Fecal microbiota transplantation into APP/PS1 mice increased C4 levels in the colon and increased the expression of synaptic plasticity-related proteins (PSD-95 and synapsin I) in the brain, indicating a decrease in synaptic disorder. The administration of C4-producing bacteria suppressed β-amyloid deposition and effector cytokine release from microglial cells 73,195,199,200 . In AD patients, the blood Kyn/Trp ratio was found to be increased due to decreased Trp levels, indicating an abnormality in Trp metabolism 201 . The levels of secondary bile acids such as DCA, TCA, 3-DCA, and UDCA were increased in AD patients 202,203 . Thus, AD patients have an imbalance in the levels of metabolites that regulate AD pathogenesis. SCFAs have both protective and pathogenic effects, Trp metabolites have protective effects, and bile acid metabolites have overall pathogenic effects on AD (Table 2).

IMPACTS ON AUTISM SPECTRUM DISORDER
Autism spectrum disorder (ASD) is a group of developmental disabilities that involve social, behavioral and communication challenges. Both genetic and environmental factors have been identified as risk factors for the development of ASD, which is heritable. For example, chromosome 16p11.2 deletion and 15q11-q13 duplication are risk factors for ASD development. In addition, environmental factors such as maternal age, health status, infection/inflammation, perinatal hypoxia, medication, nutrition, and toxic exposure during fetal or early development are associated with ASD 204,205 . Pathogenic maternal immune activation of the IL-17A pathway was shown to promote ASD development in offspring 206 . The injection of synthetic dsRNA into pregnant dams, mimicking viral infection during pregnancy, induced placental IL-17A expression and fetal brain IL-17 receptor expression in animal models 206 . Th17 inflammation is associated with abnormal cortical development in animal models.
While ASD is different from MS, PD and AD in that it primarily involves CNS developmental issues rather than an ongoing inflammatory disease, a significant body of literature indicates an active role of dysbiosis in ASD. Some ASD patients report GI problems, such as abdominal pain, diarrhea, and food sensitivities 207,208 . Decreased levels of Firmicutes, Fusobacteria and Verrucomicrobia but increased levels of Bacteroidetes have been reported in ASD patients 209 . Elevated levels of Th17-inducing gut microbial species, such as segmented filamentous bacteria (SFB), Bifidobacterium adolescentis, and certain E. coli isolates, during pregnancy were shown to increase ASD development in offspring 210 . Intestinal CD11c + DCs, which recognize microbial TLR3 ligands and produce IL-1β, IL-23, and IL-6, can induce pathogenic Th17 responses 206,210 . The significance of the microbial community in the onset of ASD has been validated in an FMT model 72 . When germ-free mice were humanized with microbes from ASD patient stool, their offspring showed behavioral disorders and decreased locomotion and interactions with other animals. The abundance of Bacteroides spp. and P. merdae was correlated with increased social behavior. Increased E. tayi, however, was associated with decreased social interaction in offspring 72 . Thus, dysbiosis can affect ASD at both the developmental and effector phases.
An altered microbial composition can impact metabolite production. It appears that microbial metabolites can bidirectionally regulate ASD activity. Decreased levels of fecal SCFAs (C2, C3, and C5) were observed in ASD patients 211,212 . Moreover, C4producing bacteria and fecal SCFA levels were reported to be increased in ASD patients compared with unaffected individuals ( Table 2). In a rat ASD model, C3 administration altered the patterning of human neural stem cells, leading to gliosis, disturbed neurocircuitry, inflammatory responses in the hippocampus and increased ASD-like behavior 213,214 . On the other hand, C4 administration improved social behavior and regulated autism-related genes related to the excitatory/inhibitory balance in the prefrontal cortex of mice 215 (Table 1). Thus, the effect of SCFAs on ASD is currently equivocal. It is interesting that different SCFAs exert different effects on ASD development. Offspring from female animals that received an FMT from human ASD patients had lower levels of 5-aminovaleric acid (5AV) and taurine in the colon; these metabolites are gamma-aminobutyric acid (GABA) receptor agonists. When 5AV and taurine were administered to a mouse model of ASD (i.e., BTBR T+ tf/J mice), ASD-related behavior was suppressed 72 . Thus, microbial metabolites have significant regulatory effects on ASD activity.

IMPLICATIONS AND CONCLUDING REMARKS
The gut is functionally linked to the CNS by immunological mediators, lymphocytes, neurotransmitters, microbes and microbial metabolites. Indeed, it is apparent that most organs and tissues in the body are regulated by gut microbiota and their metabolites, and the CNS is no exception. Importantly, because of the unique (i.e., neurological) functions of the CNS, the impact of gut microbes and their metabolites is manifested quite uniquely in this system. However, the effects of the microbiota and metabolites on different CNS diseases appear to be heterogeneous. Overall, the microbiota and their metabolites influence and strengthen CNS function in the healthy state; for example, SCFAs strengthen the BBB to maintain CNS integrity and protect the CNS from toxic and immunological damage. Various microbial metabolites regulate both inflammatory responses and neuronal activity in the CNS. Gut microbial metabolites promote the maturation of key CNS cells, such as oligodendrocytes, microglial cells and astrocytes, and regulate synapse formation. In addition, the gut microbial metabolites serotonin, dopamine, 5AV and taurine regulate neurotransmission. The production of these metabolites by microbes partly explains the changes in neurological activity following FMT from patients to experimental animals. Certain microbial metabolites, such as SCFAs, secondary bile acids and Trp metabolites, promote immune tolerance by increasing the expression of IL-10 and IL-22 to prevent inflammatory diseases. Under the highly variable pathological conditions of different diseases and hosts, SCFAs and other metabolites induce the generation of effector cells that produce neuroinflammatory cytokines. In this regard, microbial metabolites have important functions in regulating T cells, antigen-presenting cells, microglial cells and astrocytes.
We discussed the effects of microbiota and their metabolites on the pathogenesis of four different CNS diseases or disorders. A common pattern of regulation emerging from the discussed information is that disease pathogenesis is accelerated under conditions of dysbiosis (Fig. 2). While dysbiosis alone would not cause overt inflammatory responses in the CNS in normal hosts, it appears to increase pathological processes in hosts with underlying disease activity or risk factors. Changes in microbial metabolites are significant modifiers of pathogenesis in the CNS; Fig. 2 The common regulatory network of microbial metabolites, inflammatory diseases and CNS disorders. The common initiating factors for the four neurological diseases are genetic predisposition and environmental factors, which include diet and lifestyle. Under pathogenic conditions, the intestinal barrier can be breached, and systemic inflammatory responses can occur. These changes can be followed by dysbiosis (i.e., decreased gut microbial diversity leading to decreased levels of beneficial microbes). For example, consumption of a diet high in calories and fat but low in dietary fiber can accelerate pathogenic dysbiosis. As a result, decreased levels of beneficial gut microbial metabolites such as SCFAs, Trp metabolites and phytochemicals are produced, and simultaneously, the production of harmful metabolites such as long-chain fatty acids (LCFAs), certain bile acid metabolites, and toxic microbial metabolites increases, thereby affecting immune and tissue cells in both the intestine and CNS. These changes can decrease immune tolerance, which is important for preventing autoimmune diseases, and exacerbate pathogenic immune responses mediated by inflammatory cells such as Th17 and Th1 cells. These pathogenic inflammatory responses can contribute to tissue damage (demyelination in MS), neuronal cell death (PD and AD), and neuronal synapse development (ASD). Moreover, certain microbial metabolites regulate neurotransmission and, therefore, can directly affect neurological activity. these changes either positively or negatively affect disease onset and pathogenesis. Moreover, the altered production of microbederived neurotransmitters in dysbiosis appears to significantly affect neurological activity.
Despite research progress in this field, significant controversies remain regarding the functions of microbiota and their metabolites in regulating various CNS diseases. We still lack an understanding of the detailed functions of microbial metabolites. Importantly, microbes and microbial metabolites have both proand anti-inflammatory roles. What underlies the apparent bidirectional activities of gut microbial metabolites? SCFAs, which are largely anti-inflammatory and disease-suppressing, are sometimes associated with increased CNS disease activity. While more information is needed to elucidate the mechanisms underlying the apparent bidirectional regulation, it is clear that gut microbiota and their metabolites are significant modifiers rather than primary inducers of CNS diseases. Their effects on CNS pathogenesis can be altered depending on host factors, such as the type of inflammatory responses and stage of pathogenesis. Thus, an improved understanding of CNS regulation by microbial metabolites is required. The precise mechanisms by which these metabolites regulate immune responses individually or in combination to suppress or increase pathogenesis in the CNS have yet to be elucidated, particularly for relatively understudied diseases such as PD, AD, and ASD.