Gut microbiota, nitric oxide and microglia as pre-requisites for

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Gut microbiota, nitric oxide and microglia as pre-requisites for neurodegenerative disorders Joyce Ka Yu Tse ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00176 • Publication Date (Web): 22 Jun 2017 Downloaded from http://pubs.acs.org on June 24, 2017

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ACS Chemical Neuroscience 1

Gut microbiota, nitric oxide and microglia as pre-requisites for neurodegenerative disorders Tse, Joyce K.Y.* Affiliation: The Hong Kong Polytechnic University University Research Facility in Chemical and Environmental Analysis, and Department of Civil and Environmental Engineering *To whom correspondence should be addressed.

Email: [email protected]

Mailing address: The Hong Kong Polytechnic University, Room ZS972, 9/F, Department of Civil and Environmental Engineering, Hung Hom, Kowloon, Hong Kong No funding is required for this review FOR TABLE OF CONTENTS USE ONLY Graphical Table of Contents: (Size revised as specified)

Abstract Regulating fluctuating endogenous nitric oxide (NO) levels is necessary for proper physiological functions.

Aberrant NO pathways are implicated in a number of

neurological disorders, including Alzheimer’s Disease (AD) and Parkinson’s Disease. The mechanism of NO in oxidative and nitrosative stress with pathological consequences involves reactions with reactive oxygen species (e.g. superoxide) to form the highly reactive peroxynitrite, hydrogen peroxide, hypochloride ions and hydroxyl radical. NO levels are typically regulated by endogenous nitric oxide synthases (NOS), and inflammatory iNOS is implicated in the pathogenesis of neurodegenerative diseases, in which elevated NO mediates axonal degeneration and activates cyclooxygenases to provoke neuroinflammation. NO also instigates a down-regulated secretion of brain-derived neurotrophic factor, which is essential for neuronal survival, development and differentiation, synaptogenesis, and learning and memory. The brain-gut axis denotes communication between the enteric nervous system (ENS) of the GI tract and the central nervous system (CNS) of the brain, and the modes of communication include the vagus nerve, passive diffusion 1

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and carrier by oxyhemoglobin.

Amyloid precursor protein that forms amyloid beta

plaques in AD is normally expressed in the ENS by gut bacteria, but when amyloid beta accumulates, it compromises CNS functions. E. coli and S. enteric are amongst the many bacterial strains that express and secrete amyloid proteins and contribute to AD pathogenesis. Gut microbiota is essential for regulating microglia maturation and activation, and activated microglia secrete significant amounts of iNOS. Pharmacological interventions and lifestyle modifications to rectify aberrant NO signaling in AD include NOS inhibitors, NMDA receptor antagonists, potassium channel modulators, probiotics, diet and exercise. Keywords: gut microbiome, nitric oxide, microglia, neurodegeneration, gut-brain axis, amyloids

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Gut Microbiota The ubiquitous human microbiome resides in distinct ecosystems on and within the body, including the oral, nasal and aural cavities, the surface of the skin, the urogenital tract and the gastrointestinal (GI) tract (Hill et al. 2014). The dramatic ratios of the number of human microbes to human somatic cells and microbial genes to host genes are 100:1 and 150:1, respectively. In particular, the GI tract constitutes 95% of the human microbiome with an estimate of approximately 100 trillion bacteria and 1000 discrete bacterial species (Bhattacharjee & Lukiw 2013). More than 99% of the microbiota in the GI tract is anaerobic bacteria, and the remaining 1% comprises fungi, protozoa, archaebacteria and other microorganisms. The bacterial gut microbiome consists of mainly two major phyla: the Bacteroidetes and Firmicutes (Lankelma et al. 2015; Qin et al. 2010). The former constitutes approximately 17 to 60 percent of the gut microbiota, whereas the latter is about 35 to 80 percent (Qin et al. 2010; Huttenhower et al. 2012). Interestingly, the proportion of Bacteroidetes to Firmicutes in the gut microbiota is related to obesity (Ley et al. 2006). In particular, increased Firmicutes and decreased Bacteriodes have the propensity towards obesity. Proteobacteria, Actinobacteria, Fusobacteria and Verrucomicrobia phyla are in relatively low abundance in the gut. Colonization of gut microbiota begins at birth and is established by the first three years of life (Palmer et al. 2007). Gut Microbiome and Disease The connection between gut microbiome and stress response has been studied extensively. Gut pathogens, such as the B2 and D phylogenetic groups of Escherichia coli, possess virulence genes and can activate the hypothalamic-pituitary-adrenal (HPA) axis if they enter the gut (Katouli 2010). Germ-free mice have shown exaggerated HPA response to psychological stress, while mice treated with probiotics have had a blunted HPA response (Dinan & Cryan 2012). In fact, stress induces increased permeability of the gut, allowing bacteria and their antigens to cross the epithelial barrier and to activate and elicit an immune response, which alters the composition of the microbiome, leading to an enhanced HPA response. Notably, major depression and irritable bowel syndrome are induced by an increased gut permeability with coexisting alteration of the HPA. Furthermore, stress-induced neurological dysfunctions are associated with shifts in the gut microbiota (Bharwani et al. 2016). Additionally, the metabolomic profiles of plasma and liver in a murine model of depression contrast with those in the control group (Aoki-Yoshida et al. 2016).

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An altered gut metagenome is associated with various other disease states. Shotgun sequencing of the gut metagenome showed that the genus Collinsella was upregulated in patients with symptomatic atherosclerosis, which is associated with lipid accumulation and carotid arterial wall inflammation, leading to cerebrovascular events (Karlsson et al. 2012). On the other hand, Roseburia and Eubacterium were enriched in healthy controls. Furthermore, the gut microbiome contributes to carcinogenesis (Schwabe & Joblin 2013). Metagenomics and 16S rDNA sequencing correlate increased fecal Fusobacterium and Porphyromonas with colorectal cancer (Vogtmann et al. 2016). Proteobacteria Helicobacter pylori infection is the single strongest risk factor for gastric cancer (Brawner et al. 2014). Nitrate-reducing bacteria, mainly Proteobacteria, reduce gastric nitrate to nitrite, which enhances the generation of N-nitrosamines, a carcinogen in animals (Lundberg, et al., 2004; Archer, M.C., 1969). Prior to the 1990s, characterization of the gut microbiome was limited as only culturable bacteria could be identified. Only approximately less than 30 percent of the gut microbiota had been cultured at that time (Fraher et al. 2012). With the rising popularity of sequencing and genomic technologies, microbial communities and composition diversity, as well as the role they play in human physiology and pathology, can be elucidated. Sequencing full length 16S rRNA genes by Third Generation Sequencers, such as the PacBio Sequel, allows species identification, and the most recent development in metagenomics can facilitate the study of collective genomes from the environment and further expand our knowledge of the association between the gut microbiome and health and disease, helping to identify the causative mechanisms. Experimental studies using germ-free “gnotobiotic” mice indicate that commensal gut microbiota is obligatory for normal physiological functioning of the GI tract, including its role in maintaining action potentials of the enteric nervous system (ENS), which is part of the autonomic nervous system that is able to function independently of the central nervous system (CNS) (Rao et al 2016). The ENS neural circuit is embedded within the gut wall and comprises sensory neurons, motor neurons and interneurons. Nitric Oxide – Its Role and Source Nitric oxide (NO) acts as the principal neurotransmitter of the nonadrenergic noncholinergic (NANC) ENS in humans and animals (Salzman 1995). Its release is stimulated by the activation of NMDA receptors by glutamate (Figure 1). Potential 4


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sources of NO in the GI tract include intrinsic intestinal tissues (e.g. mast cells, epithelium, smooth muscle and neural plexus, namely released by inhibitory motor neurons), resident and infiltrating leukocytes (e.g. neutrophils and monocytes), and reduction of luminal gastric nitrate and nitrite and denitrification by commensal anaerobes. Exogenous NO mimics endogenous NO action. NO is originally identified as endothelium-derived relaxing factor (EDRF) mediating relaxation of blood vessels (Furchgott & Zawadzki 1980). It is a small, highly diffusible, gaseous and reactive molecule with a short half life that is synthesized by nitric oxide synthase (NOS) via enzymatic conversion of L-arginine to L-citrulline (Figure 2). Three distinct NOS genes are present: 1) neuronal NOS (nNOS, NOS1), 2) inducible NOS (iNOS, NOS2), and 3) endothelial NOS (eNOS, NOS3). eNOS and nNOS generate nanomolar concentrations of NO and confer a physiological neuroprotective function, whereas iNOS synthesizes micromolar concentrations of NO that are neurotoxic in response to proinflammatory stimuli (Steinert et al 2010). More specifically, NO S-nitrosylation of the NMDA (N-methyl-D-aspartate) receptor subunits (thus inhibiting aberrant excitatory toxicity) or the active sites of caspases (inhibiting cellular apoptosis and enhancing survival) dictates its neuroprotective function in regulating neuronal cell proliferation, survival and differentiation (Calabrese et al 2007). Furthermore, NO activates signaling molecules Akt and CREB (cyclic AMP-responsive-element-binding protein) in cerebellar granule cells to promote cell survival. On the other hand, excessive NO production becomes noxious and may undergo redox reactions to form toxic compounds known as reactive nitrogen species (RNS) that cause cellular damage, promote axonal degeneration and induce neurodegenerative disorders. nNOS is coupled to NMDA receptor activation; thus, excitotoxicity-related neuronal injury constitutes a nitrergic component . The well-known ecological nitrogen cycle involves the reduction of nitrogen oxides to ammonium, followed by oxidation of ammonia back to nitrites (NO2-) and nitrates (NO3-) by plants and bacteria. In recent years, considerable attention has focused on salivary bacterial reduction of nitrate to nitrite and then to nitric oxide and ammonium in humans, which has been known to be the major mode of nitrite formation in humans (Tannenbaum et al. 1974). Nitrate induces both nitrite and ammonia generation in cultured Escherichia coli and Lactobacillus plantarum grown at oxygen concentrations compatible with the content in the gastrointestinal tract (Tiso & Schechter 2015). Interestingly, exogenously supplied nitrite induces NO gas production independent of the added nitrate. Furthermore, nitrite can be converted to NO by E. coli even at neutral pH. Furthermore, Klebsiella strain of 5



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Proteobacteria phylum and Clostridiales strain of Firmicutes phylum found in the gut microbiota have been shown to be capable of nitrogen fixation in the human gut (Igai et al. 2016). Nitrogen fixation activity is determined by on metagenomic sequencing of the nifH gene in fecal samples. The nitrate-nitrite-NO pathway fuels the production of NO, and the gut is the primary site for the production of significant amounts of NO concentrations (Pereira et al 2013). A high nitrate intake increases both nitrite and NO levels in the vasculature and tissues. Gut lactobacteria, bifidobacteria and E. coli Nissle 1917 convert nitrate and nitrite into NO and can also stimulate host epithelial cells to form NO (Oleskin & Shenderov 2016). Changes in its abundance alters NO neurotransmitter levels in both ENS and CNS. Pediococcus acidilactici (strains S2 and S3) and Lactobacillus plantarum (strain T119) were isolated from fermented dairy items, pickled food and silage and were found to synthesize cytotoxic NO concentrations of about 50 uM (Gundogdu et al 2006; Oleskin et al 2016). Some bacterial NOSs (bNOSs) produce NO via the same host’s classical L-arginine pathway, which has been shown both in vitro and in vivo, and they are found in streptomycetes and bacilli that inhabit the human intestines (Oleskin & Shenderov 2016). Microbial NO production was examined in vitro using L-arginine or nitrate as substrates (Vermeiren et al. 2009). Interestingly, L-arginine did not influence microbial NO production; however, significant gastrointestinal NO was produced by fecal microbiota from nitrate by an 15N tracer experiment that reduced nitrate to ammonium by the dissimilatory nitrate reduction to ammonium (DNRA) pathway. In addition to the endogenous synthesis of NO by NOS, nitrate in the human intestinal system can also be sourced from diet rich in nitrate (Tannenbaum et al. 1978; Lidder et al. 2013). The main dietary source of nitrate is vegetables, which account for approximately 60 to 80 percent of the daily nitrate intake with a typical western diet (Lundberg et al. 2004). Nitrite is also found in meat as a food additive to prevent botulism and to enhance its appearance. Other environmental sources of nitrate and nitrite include cigarette smoke and car exhausts. Nitrate is absorbed from the proximal small intestine in healthy individuals, but a considerable one-third of a fraction could reach the lower intestine. By bacterial respiratory denitrification, nitrite is converted into nitrogen gas via nitric oxide and nitrous oxide, in the specified order (Tiso & Schechter et al. 2015). Most nitrate is excreted in the urine, but some is excreted via saliva, sweat or feces. Nitric Oxide in the Central Nervous System 6


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Aberrant NO pathways are implicated in a number of neurological disorders, including the most prevalent dementia, Alzheimer’s disease (AD), and the second most prevalent, Parkinson’s disease (PD) (Togo et al. 2004). Anomalous overproduction of NO leads to neuroinflammation due to its free radical properties, which compromise cellular integrity and viability and result in mitochondrial dysfunction through mitochondrial fission. NO binds to aconitase and complexes I and II, resulting in glycolysis and electron transfer chain inhibition, respectively, and disrupting mitochondrial energy production (Dawson et al. 1992). Damaged DNA by NO could lead to poly ADP-ribose polymerase (PARP) over-activation, causing neuronal ATP depletion and death (Ha & Snyder, 1999). NO has long been known to be part of the neurotoxic insult induced by neuroinflammation in AD. NOS was first identified in the brain, but it is also widely expressed in the peripheral nervous system (e.g. NANC neurons in the GI tract) and skeletal muscle (Steinert et al 2010). In the brain, it is present in the cerebral cortex, the ventral endopiriform nucleus, the claustrum, the olfactory bulb, the olfactory nuclei, the nucleus accumbens, the striatum, the amygdale, the hippocampus (particularly the CA1 region and the dentate gyrus), the hypothalamus (the supraoptic and paraventricular nuclei), the thalamus, the lateral dorsal and pedunculopontine tegmental nuclei, the trapezoid body, the raphe magnus, the nucleus of solitary tract and the cerebellum (Calabrese et al 2007). Within the CNS, nNOS is also present in astrocytes and cerebral blood vessels. There are four splice variants of nNOS, namely alpha, beta, gamma and mu. In the brain, eNOS is expressed in the cerebral endothelial cells, and it regulates cerebral blood flow by the pyramidal neurons of the CA1, CA2 and CA3 subfields in the hippocampus and by the granule cells of the dentate gyrus. In the periphery, eNOS is present in the vascular endothelium for blood flow control. iNOS levels in the CNS are low, but it can be induced in astrocytes or microglial cells post inflammation, viral infection or trauma. A lack of eNOS and iNOS is well tolerated, but nNOS deficiency results in apoptotic cell death of the spinal cord neurons (Steinert et al 2010). eNOS and nNOS are necessary in the nitrergic mechanism of long-term potentiation (LTP) in the hippocampus that is responsible for memory and learning. In particular, eNOS provides the basal level of NO and nNOS functions on an activity-dependent manner. It has been shown that knockout of either eNOS or nNOS blocks NO-dependent potentiation of LTP. NO in the telecephalon and cerebellum also confers synaptic plasticity that is involved in cognitive processes such as memory. Ischemic hypoxia causes glutamate release in cerebrovascular disease, leading to overactivation of NMDA receptor and excessive calcium influx, which in turn 7



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activates NO synthesis (Dawson, D.A., 1994; Zhang & Snyder, 1995). NMDA receptor activation and NO-mediated excitotoxicity are also observed in Huntington’s disease (Meldrum & Garthwaite, 1990). Additionally, the human immunodeficiency virus in Acquired Immune Deficiency Syndrome (AIDS) induces NO production by iNOS via the glutamate pathway (Adamson, et al. 1999; Corasaniti, et al. 1998). The mechanism of NO in neurotoxicity is not well established. The overaugmentation of NO excitability via modulation of voltage-gated potassium channel activity, namely Kv7 and Kv2, is one of the proposed mechanisms (Balez & Ooi 2016). Another potential mechanism of NO and AD is with regards to the ability of NO to increase the excitability of presynaptic neurons to promote neurotransmitter release. Specifically, M-channels are voltage-gated outward potassium channels that remain open at the resting membrane potential of neurons. Increases in M-current reduce neuronal excitability, while inhibition of M-current increases action potential firing. In sensory neurons, NO is a potent neuromodulator that increases excitability by inhibiting M-current (Gamper & Ooi 2015; Ooi et al, 2013). Additionally, NO-induced changes in excitability were tested in the mouse hippocampus with resultant modulated outward potassium current in which potentiation of Kv2 and Kv3 currents promoted sustained action potential firing (Steinert et al. 2010). Aberrant Nitric Oxide Signalling The mechanism of NO in oxidative and nitrosative stress leading to pathological consequences involves reaction with reactive oxygen species, such as superoxide anions (O2-), to form the highly reactive peroxynitrite ion (ONOO-), which is responsible for protein nitrotyrosination (aka addition of NO2 group to the tyrosine residue of the protein), S-nitrosylation (aka addition of NO2 group to a sulphur molecule on an amino acid residue) and inhibition of mitochondrial respiration, resulting in neurodegeneration such as a nitrotyrosination and misfolding of Tau protein in AD and nitrotyrosination of alpha-synuclein in PD (Steinert et al 2010). Nitrotyrosine is a marker of nitrosative stress. The formation of hydrogen peroxide (H2O2), and subsequent formation of hypochloride ions (OCl-) and the highly reactive hydroxyl radical (OH) leads to lipid peroxidation (aka degradation) and DNA damage. The inflammatory iNOS is strongly implicated in the pathogenesis of neurodegenerative diseases. It has been shown that elevated NO induces axonal degeneration (Steinert et al 2010). NO also activates both the constitutive and inducible isoforms of cyclooxygenase, leading to neuroinflammation via the release of free radicals and formation of prostaglandins (Calabrese et al 2007). Inducible cyclooxygenase is upregulated in patients with AD and is considered a biomarker of 8


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the progression of AD.

Furthermore, NO instigates a rapid down-regulation of

brain-derived secretion of neurotrophic factor (BDNF) in cultured hippocampal neurons via a cGMP/protein kinase G pathway to prevent calcium release (Canossa 2002). BDNF is a secreted neurotrophic factor that is essential in neuronal survival, development and differentiation, synaptogenesis and synaptic plasticity in cognitive functions including memory and learning (Hill et al 2014). The alternative function of NO in causing smooth muscle relaxation is mediated by soluble guanylyl cyclase and cyclic GMP to reduce intracellular calcium concentration and to activate calcium-dependent potassium channels in order to hyperpolarize the neuron (Bolotina et al 1994). The Gut-Brain Axis Recent studies denote bidirectional communication between the ENS and the CNS known as the gut-brain axis (GBA). It is well known that gut peptides released from enteroendocrine cells can act directly on the brain (Lyte & Cryan 2014). Gut peptides orexin, galanin, ghrelin, gastrin and leptin modulate feeding behavior, energy homeostasis, circadian rhythm, sexual behavior, arousal and anxiety (Figure 3). Probiotic studies show that changes in the gut microbiota composition alter gut hormone release. Levels of neuropeptide Y are sensitive to microbiota manipulations, and neuropeptide Y itself is involved in gut-brain interactions and functions as both a neurotransmitter and endocrine messenger analogous to NO. The most propelling evidence of microbiome-brain interaction came more than twenty years ago when administration of oral antibiotics dramatically improved the morbidity of patients with hepatic encelphalopathy (Carabotti et al 2015). The abundant gram-positive facultative anaerobic Lactobacillus and Bifidobacterium species in the GI tract are capable of metabolizing glutamate to produce gamma-amino butyric acid (GABA), which is the major inhibitory neurotransmitter in the CNS (Bhattacharjee & Lukiw 2013). Studies show that increased GI tract GABA correlates with increased CNS GABA levels (Hill et al 2014). Dysfunctions in GABA-mediated signaling have been shown to be concomitant with defects in synaptogenesis and cognitive impairment in AD. Furthermore, cyanobacteria from the GI tract correlates with PD. Such pathological consequence occurs more frequently in the elderly when the intestinal epithelial barrier of the GI tract becomes more permeable to bacteria-derived neurotoxins. The ENS and CNS have many common neurotransmitters, signaling pathways and anatomical properties. As a result, the pathophysiological processes that underlie CNS diseases often correlate with enteric manifestations. For example, degeneration of nigrostriatal dopaminergic neurons in patients with PD may correlate with the function of 9



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dopaminergic neurons in the ENS as animal models reveal that the ENS is vulnerable to degeneration in PD.

Mechanism of neuronal death induced by NO has been

exemplified above. Furthermore, the amyloid precursor protein (APP) that forms amyloid beta plaques in AD is normally expressed in the ENS by gut bacteria, in addition to nitrotyrosination of the Tau protein by NO. Other than AD and PD, aberrant NO signaling contributes to a wide variety of neurodegenerative disorders, including stroke, excitotoxicity and multiple sclerosis. Serotonin (5-HT), similar to NO, is a neurotransmitter within the CNS and ENS. Despite approximately 95% of 5-HT within the body is produced by gut mucosal enterochromaffin cells and ENS neurons, it is one of the major neurotransmitters in the brain. In the brain, 5-HT signaling regulates mood and cognition, whereas in the periphery, it regulates GI secretion, motility and pain perception (McLean et al. 2007). Gut synthesis of 5-HT has been shown to be regulated by the gut microbiota, in which indigenous spore-forming bacteria from mouse and human promote 5-HT synthesis from colonic enterochromaffin cells (Yano et al. 2015). This suggests that gut microbiota modulation of 5-HT levels in the gut also contributes to its levels in the brain. Similarly, NO synthesis in the gut by gut microbiota can impact NO levels in the brain, possibly leading to neurodegenerative disorders. One of the hypotheses for disease spread from the ENS to the CNS is about the nerves that interconnect the two systems, especially the vagus nerve, which imparts direct innervation. The vagus nerve is proposed to be the most important neural pathway for bidirectional communication between the gut microbiota and the brain (Forsythe et al. 2012; Forsythe et al. 2014; Goehler 2006). The importance of the vagus nerve in GBA communication can be demonstrated by the following. Chronic treatment with probiotic Lactobacillus rhamnosus resulted in alterations in GABA receptor expression in the CNS, accompanied by reduced anxiety, depression and stress response only in the presence of an intact vagus nerve (Bravo et al. 2011). Similarly, the anxiolytic effect of probiotic Bifidobacterium longum in a colitis model was absent in vagotomised mice (Bercik et al. 2011). Furthermore, neurochemical and behavioral effects in altered gut microbiota were not observed in vagotomized mice, supporting the role of the vagus nerve as a major communication pathway between the gut microbiota and the brain (Carabotti et al 2015). As aforementioned, NO has a recognized role as a neurotransmitter in inhibiting neural transmission, which suggests a possible link from the ENS to the CNS, via the vagus afferent nerve, as it is expressed in both systems. On a slight deviation, it has been recently suggested that luminal nutrients and bacteria stimulate luminal epithelial 10


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biosensors called enteroendocrine cells, which communicate in a paracrine fashion with ENS neurons innervating the small intestine and colon to reach the connected CNS neurons (Bohorquez et al. 2015). A second hypothesis for disease spread via the GBA is based on the fact that NO can easily diffuse; it is unconstrained by cell membranes and can act across a broad volume (Steinert et al 2010). It is suggested that spillover of NO from eNOS and iNOS from other compartments into the nervous system as a result of diffusion is a potential pathological source of NO with neurological implications. Moreover, GI-produced NO can be scavenged by hemoglobin in red blood cells, thereby entering the peripheral vasculature and crossing the blood-brain-barrier (BBB) into the cerebral vasculature (Banks 2008). It is also possible for GI tract microbiome secretory products to translocate to the brain via the lymphatic system. Other modes of communication between the microbiota and the brain include gut hormone signaling, the immune system, tryptophan metabolism and microbial metabolites such as SCFA (Foster et al. 2017). Microglia and Nitric Oxide Microglial cells represent a major cell typeinvolved in NO-induced neuropathogenesis. Microglia are resident macrophages in the innate immune system of the brain, constituting approximately 10% of the cells in the nervous system (Solito and Sastre 2012). Microglia are considered as the macrophages of the CNS and are crucial for scavenging of pathogens, dying cells, as well as synaptic pruning and remodeling during development. In neurodegenerative conditions and aging, microglia enter a hyperactive state, in which they produce abundant neurotoxic pro-inflammatory mediators, including iNOS (which results in excessive NO), cytokines and chemokines (e.g. IL-1beta, IL-6, TNFalpha, IL-8, TGFbeta and macrophage inflammatory protein-1alpha), prostaglandins, cyclooxygenase and free radicals, that serve as a positive feedback mechanism in the inflammatory response. Despitetheir principal physiological role to engulf dead cells by phagocytosis and to restore homeostasis, complications often arise, resulting in exacerbation of existing damage and further detrimental effects. Additionally, the state of activity of microglia is represented by their morphologies, either amoeboid or ramified; the former represents developing microglia and the latter represents mature microglia (Mosher & Wyss-Coray 2015). Note that basal NO is impertinent in stabilizing microglia that are juxtaposed to neurons (Stefano et al. 2004). Nitrite, a metabolite of the free radical NO derived from L-arginine, was detected in abundant levels in 11



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activated microglial/neuronal cell co-cultures and in purified microglial cultures, but not in purified astrocyte or neuronal cell cultures, suggesting that microglia were the principal source of NO (Chao et al. 1992). Interestingly, NO-synthesizing neurons are resistant to NO-induced neurotoxicity, such that they are spared in patients with AD, leading to further accumulation of NO and neurotoxicity of other neurons (Hyman et al. 1992). Gut Microbiome, Microglia, Amyloids, Nitric Oxide and Alzheimer’s Disease The coining of the term “microbial endocrinology” has been attributed to the combined fields of microbiology and neurobiology as microorganisms are capable of producing the same neurochemicals as the host during periods of stress, leading to the pathogenesis of neurological diseases. It is proposed that neurochemicals produced by microorganisms can mimic those of the host, and these neurochemicals can impact on the host’s neuroimmunological functions, such as inflammation, as well as the host’s neurophysiological functions, such as behavior (Lyte 2016). The rise of microbial endocrinology is based on the observation that stress could affect susceptibility to infectious diseases (Lyte 2016). Notably, this has gained acceptance following the introduction of neuroendocrine-bacterial interactions. Animal models have shown that intestinal microbiota affects behavior, learning abilities and memory, anxiety and depression levels, reaction to emotional stimuli and stress resistance (Averina & Danilenko 2017). Recently, it has been shown that gut microbiota is essential for regulating microglia maturation and activation. RNA sequencing of microglia obtained from germ-free and specific pathogen-free (SPF) mice showed a robust difference in the transcriptional profiles, of which are correlated with the microglia morphology and state of activity. Gut microbiota secretes significant amounts of amyloids and lipopolysaccharides that activate microglia (Pistollato et al 2016). Dysbiosis of gut microbiome composition contributes to AD and PD (Figure 4). Bacterial amyloids activate microglia as aforementioned, and lipopolysaccharides enhance inflammatory responses to cerebral accumulation of amyloid beta that is produced from amyloids and APP. When amyloid beta accumulates and exceeds a certain threshold, it self-propagates and compromises CNS functions. Escherichia coli, Salmonella enteric, Salmonella typhinurium, Bacillus subtilis, Mycobacterium tuberculosis and Staphylococcus aureus are some of the many bacterial strains that express and secrete amyloid proteins. Additionally, E. coli endotoxin potentiates the formation of amyloid beta in vitro, thereby facilitating the pathogenesis of AD. Furthermore, overexpression of APP is associated with upregulation of peroxynitrite, 12


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so that NO triggers amyloid beta deposition (Asiimwe et al 2016). Active microglia also produce APP in response to exhausted neuronal excitation by NO and APP from the gut, which further contributes to AD pathogenesis. Amyloid and leukocyte dissemination from the gut to the brain is more prominent in aging, when the GI epithelium and blood-brain-barrier (BBB) are more permeable to small molecules (with altered tight junctions). Amyloid permeation across the BBB is mediated by the receptor for advanced glycosylation products (RAGE), chaperones and apolipoproteins E and J, and amyloid clearance is mediated by low-density lipoprotein receptor-related protein 1, and such mechanisms are altered in AD. Furthermore, the gut microbiota changes during aging, with Bacteroidetes outnumbering Firmicutes and Bifidobacteria. Additionally, bacterial lipopolysaccharide (LPS) induced production of NO and increased the expression of surface marker of microglia CD11b in mouse BV-2 microglial cells and primary microglia (Roy et al. 2006). Furthermore, NO production is abrogated by Dehydroepiandrosterone (DHEA), a multifunctional steroid involved in many CNS activities, and inhibition of LPS-stimulated BV2 microglial cells (Wang et al. 2001). The term “microgliopathy” means microglia dysfunction is the primary disease-causing condition. Note that in addition to NO, neuromediators such as biogenic amines, amino acids, peptides and short and long chain fatty acids also partake in microbial-host communication (Oleskin et al. 2016; Averina & Danilenko 2017). Germ-free mice displayed defects in microglia with an altered gene expression profile, an altered expression of maturation marker and an impaired cellular morphology (Erny et al. 2015). Temporal abolishment of host microbiota also changed the properties of microglia. A limited microbiota complexity resulted in defective microglia as well. On the other hand, re-colonization with complex microbiota partially restored microglia features and functions. In fact, short-chain fatty acids (SCFA), derived from microbiota as a fermentation product, regulate microbiota homeostasis. Germ-free animals showed a decreased expression of brain-derived neurotrophic factor (BDNF) in the hippocampus and cerebral cortex, which is one of the principle factors involved in memory (Carabotti et al 2015). Antimicrobials such as neomycin, bacitracin and pimaricin in SPF mice increased hippocampal expression of BDNF. Short-chain fatty acids (SCFA) such as butyric acid, propionic acid and acetic acid are products of bacterial metabolism that can also influence memory and the learning 13



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process (Stilling et al. 2013). Hippocampal neurogenesis has been shown to be regulated by the microbiome (Ogbonnaya et al. 2015). Apolipoprotein E (ApoE) is a major cholesterol carrier that supports lipid transport and injury repair in the CNS (Liu et al. 2013). ApoE polymorphic alleles are the main genetic determinants of AD risk, in which individuals carrying the ε4 allele are at increased risk of AD compared with those carrying the more common ε3 allele, whereas the ε2 allele is protective. ApoE4 transgenic mice produced significantly more NO than ApoE3 mice, which was attributed to an increase in arginine transport in microglia from ApoE4 mice (Colton et al. 2002). Interestingly, iNOS mRNA and protein levels are not significantly different between ApoE3 and ApoE4 mice, suggesting that induction of iNOS is not the primary cause of the increased NO production in ApoE4 mice and that iNOS is normally unsaturated. Taken together, this suggests increased arginine transport allows increased arginine substrate binding to NOS (at constant level of expression), leading to increased NO production. Germ-free Swiss-Webster mice display a short-term impaired recognition memory and working memory compared with the control mice (Gareau et al. 2011) In addition, normal gut flora plus Citrobacter rodentium in C57BL/6 mice led to short-term impairment in recognition and working memory after stress induction (Gareau et al. 2011). On the contrary, normal gut flora plus Bifidobacterium lactis and Lactobacillus fermentum results in improved spatial memory and ameliorated memory impairment in diabetes-like Wister rats (Davari et al. 2013). Furthermore, beta amyloid production is increased upon nitrotyrosination of presenilin-1 in aging rat hippocampal neurons, which parallels the situation observed with PSEN1 mutations associated with familial AD (Guix et al, 2012). In addition, prolonged NO exposure can induce the formation of cytoplasmic tau oligomers in SH-SY5Y cells, corroborating the potential mechanism underlying tau neuropathogenesis in AD (Takahashi et al. 2012). Gut microbiome varies between the sexes. Transfer of gut microbiota from male mice to female mice altered the recipient’s microbiota, resulting in elevated testosterone and metabolomic changes (Markle et al 2013). Females have a higher incidence and severity of many major autoimmune diseases, including the neurodegenerative disease multiple sclerosis, and it has been shown that androgens are protective (Fish 2008; Yurkovetskiy et al 2013; Harbo et al 2013). Whether sexual differences on gut microbiome have any effects on other neurodegenerative 14


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diseases such as AD and PD remains to be elucidated. Pharmacological Interventions in Aberrant Nitric Oxide Signaling As there is excess gut and brain NO in AD, NOS inhibitors can serve as pharmacological neuroprotective agent against neuronal toxicity elicited by APP (Esplugues 2002). In co-cultures of immunostimulated microglia and cerebellar granule neurons, neurotoxicity was blocked by an inhibitor of NOS (Boje & Arora 1992). All three isoforms of NOS are elevated in AD; isoform-specific NOS inhibitors can be tailored to maintain physiological concentrations of eNOS and nNOS and to minimize pathological iNOS. Increased NO levels along with glutamate-mediated neuroexcitotoxicity upon NMDA receptor activation can be inhibited by NMDA receptor antagonist Memantine (Precoma et al 2016). Microglial-induced neurotoxicity was also partially attenuated by two NMDA receptor antaongists, MK-801 and 2-amino-5-phosphovalerate (APV) (Boje & Arora 1992). Modulating gut microbiota by probiotics and prebiotics to control NO-producing bacteria can also regulate NO levels in the gut and brain. Probiotics have been found to decrease proinflammatory cytokines, such as IL-5, IL-6, IL-1beta, IL-8 and TNFalpha, which are upregulated in the elderly and AD (Pistollato et al 2016). Probiotics can also significantly upregulate BDNF and restore tight junction integrity to protect the intestinal barrier so that it is less permeable to NO and other proinflammatory mediators (Pistollato et al 2016; Carabotti et al 2015). Antioxidants can reduce reactive oxygen species and reactive nitrogen species that lead to neurotoxicity (Asiimwe et al 2016). In fact, diet is one of the most important modifying factors of the microbiome and affects the GBA (Foster et al. 2017). Diet has a profound effect on the diversity of gut microbiota. There are significant differences in the gut microbiome profiles between individuals who consume Western-style diet and those who consume Asian diet (Filippo et al. 2010). Calcium channel blockers can also minimize excitotoxicity. Additionally, M-channel modulators and other modifiers of potassium channel activity and neuronal excitability could be potential drug candidates (Balez & Ooi, 2016). Exercise is reported to enhance hippocampal neurogenesis via release of BDNF, reduction of free radicals and energy restriction in the brain (Precoma et al 2016).

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27



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Triggers: Commensal anaerobes Leukocytes Mast cells Epithelium Smooth muscles Neural plexus

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Glutamate

Extracellular

Intracellular

NO

Figure 1. Schematic diagram of a NMDA receptor. Triggers lead to extracellular glutamate activation of the NMDA receptor, followed by intracellular nitric oxide (NO) release.


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A) Physiological NO L-arginine --> L-citrulline --> NO --> Akt

CREB --> Cell survival

B) Excess NO

Cellular damage

redox --> RNS

Axonal degeneration Neurodegenerative disorders

NO-

NO + ROS (O2-) --> ONOO- --> protein-tyrosine-NO2 + protein-sulphur-NO2 H2O2 --> OCl- + OH- --> lipid peroxidation + DNA damage

Figure 2. Nitric oxide signalling - physiological and pathological. Expression of physiological nitric oxide (NO) by nitric oxide synthase, leading to activation of cell signalling molecules with important role in cell survival (A). In aberrant conditions of excess NO during pathogenesis, reactive nitrogen species (RNS) and reactive oxygen species (ROS) lead to modification of protein residues with subsequent DNA damage with observed axonal degeneration in neurodegenerative disorders.



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A)

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ENS

CNS

Gut peptides (orexin, galanin, ghrelin, gastrin, leptin)

Feeding behaviour Energy homeostasis Circadian rhythm Sexual behaviour Arousal Anxiety

------->

Gut bacteria Gram positive anaerobic ----------------------------> Produce GABA inhibitory neurotransmitter (lactobacillus, bifidobacterium) (cyanobacterium) ------------------------------------> Parkinson’s Disease Gut amyloid precursor protein (APP)

-----------> Amyloid beta plaques in Alzheimer’s Disease

B)

1 2 Passive diffusion

3 RBC carrier mediated

GUT

CNS

Legend: RBC NO

Figure 3. Gut-brain-axis. Components of the enteric nervous system communicating with the central nervous system (A). The gut communicates with the brain by diffierent modes: via the vagus nerve (1), by passive diffusion (2) or by red blood cell (RBC) carrier mediated (3) (B).


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E. coli (nitrate --> nitrite --> NO) S. enteric S. typhinurium B. subtilis, M. tuberculosis S. aureus Gut microbiota NORMAL secretes APP

activates

iNOS Cytokines Chemokines (IL-1β, IL-6, TNFα, IL-8, TGFβ) releases PGs COXs APP APP APP

DISEASE

APP APP APP APP APP APP

APP APP APP APP

Increase APP

Increase NO Figure 4. Microglia, nitric oxide, gut microbiome and amyloids. Pathway of gut microbiome leading to increased nitric oxide (NO) levels via activation of microglia, secretion of iNOS, cytokines, chemokines, prostaglands (PGs), cyclooxygenases (COX) and release of amyloid precursor protein (APP).



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Graphical Table of Contents

Gut microbiome Nitric oxide production

Neurodegenerative disorders

Microglia actviation


Gut microbiota, nitric oxide and microglia as pre-requisites for

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