Roman M. Stilling

Microbial genes, brain & behaviour – epigenetic regulation of the gut–brain axis

To date, there is rapidly increasing evidence for host–microbe interaction at virtually all levels of complexity, ranging from direct cell‐to‐cell communication to extensive systemic signalling, and involving various organs and organ systems, including the central nervous system. As such, the discovery that differential microbial composition is associated with alterations in behaviour and cognition has significantly contributed to establishing the microbiota–gut–brain axis as an extension of the well‐accepted gut–brain axis concept. Many efforts have been focused on delineating a role for this axis in health and disease, ranging from stress‐related disorders such as depression, anxiety and irritable bowel syndrome to neurodevelopmental disorders such as autism. There is also a growing appreciation of the role of epigenetic mechanisms in shaping brain and behaviour. However, the role of epigenetics in informing host–microbe interactions has received little attention to date. This is despite the fact that there are many plausible routes of interaction between epigenetic mechanisms and the host‐microbiota dialogue. From this new perspective we put forward novel, yet testable, hypotheses. Firstly, we suggest that gut‐microbial products can affect chromatin plasticity within their hosts brain that in turn leads to changes in neuronal transcription and eventually alters host behaviour. Secondly, we argue that the microbiota is an important mediator of gene‐environment interactions. Finally, we reason that the microbiota itself may be viewed as an epigenetic entity. In conclusion, the fields of (neuro)epigenetics and microbiology are converging at many levels and more interdisciplinary studies are necessary to unravel the full range of this interaction.

Minireview: Gut Microbiota: The Neglected Endocrine Organ

The concept that the gut microbiota serves as a virtual endocrine organ arises from a number of important observations. Evidence for a direct role arises from its metabolic capacity to produce and regulate multiple compounds that reach the circulation and act to influence the function of distal organs and systems. For example, metabolism of carbohydrates results in the production of short-chain fatty acids, such as butyrate and propionate, which provide an important source of nutrients as well as regulatory control of the host digestive system. This influence over host metabolism is also seen in the ability of the prebiotic inulin to influence production of relevant hormones such as glucagon-like peptide-1, peptide YY, ghrelin, and leptin. Moreover, the probiotic Lactobacillus rhamnosus PL60, which produces conjugated linoleic acid, has been shown to reduce body-weight gain and white adipose tissue without effects on food intake. Manipulating the microbial composition of the gastrointestinal tract modulates plasma concentrations of tryptophan, an essential amino acid and precursor to serotonin, a key neurotransmitter within both the enteric and central nervous systems. Indirectly and through as yet unknown mechanisms, the gut microbiota exerts control over the hypothalamic-pituitary-adrenal axis. This is clear from studies on animals raised in a germ-free environment, who show exaggerated responses to psychological stress, which normalizes after monocolonization by certain bacterial species including Bifidobacterium infantis. It is tempting to speculate that therapeutic targeting of the gut microbiota may be useful in treating stress-related disorders and metabolic diseases.

Regulation of prefrontal cortex myelination by the microbiota

The prefrontal cortex (PFC) is a key region implicated in a range of neuropsychiatric disorders such as depression, schizophrenia and autism. In parallel, the role of the gut microbiota in contributing to these disorders is emerging. Germ-free (GF) animals, microbiota-deficient throughout life, have been instrumental in elucidating the role of the microbiota in many aspects of physiology, especially the role of the microbiota in anxiety-related behaviours, impaired social cognition and stress responsivity. Here we aim to further elucidate the mechanisms of the microbial influence by investigating changes in the homeostatic regulation of neuronal transcription of GF mice within the PFC using a genome-wide transcriptome profiling approach. Our results reveal a marked, concerted upregulation of genes linked to myelination and myelin plasticity. This coincided with upregulation of neural activity-induced pathways, potentially driving myelin plasticity. Subsequent investigation at the ultrastructural level demonstrated the presence of hypermyelinated axons within the PFC of GF mice. Notably, these changes in myelin and activity-related gene expression could be reversed by colonization with a conventional microbiota following weaning. In summary, we believe we demonstrate for the first time that the microbiome is necessary for appropriate and dynamic regulation of myelin-related genes with clear implications for cortical myelination at an ultrastructural level. The microbiota is therefore a potential therapeutic target for psychiatric disorders involving dynamic myelination in the PFC.

The neuropharmacology of butyrate: The bread and butter of the microbiota-gut-brain axis?

Several lines of evidence suggest that brain function and behaviour are influenced by microbial metabolites. Key products of the microbiota are short-chain fatty acids (SCFAs), including butyric acid. Butyrate is a functionally versatile molecule that is produced in the mammalian gut by fermentation of dietary fibre and is enriched in butter and other dairy products. Butyrate along with other fermentation-derived SCFAs (e.g. acetate, propionate) and the structurally related ketone bodies (e.g. acetoacetate and d-β-hydroxybutyrate) show promising effects in various diseases including obesity, diabetes, inflammatory (bowel) diseases, and colorectal cancer as well as neurological disorders. Indeed, it is clear that host energy metabolism and immune functions critically depend on butyrate as a potent regulator, highlighting butyrate as a key mediator of host-microbe crosstalk. In addition to specific receptors (GPR43/FFAR2; GPR41/FFAR3; GPR109a/HCAR2) and transporters (MCT1/SLC16A1; SMCT1/SLC5A8), its effects are mediated by utilisation as an energy source via the β-oxidation pathway and as an inhibitor of histone deacetylases (HDACs), promoting histone acetylation and stimulation of gene expression in host cells. The latter has also led to the use of butyrate as an experimental drug in models for neurological disorders ranging from depression to neurodegenerative diseases and cognitive impairment. Here we provide a critical review of the literature on butyrate and its effects on multiple aspects of host physiology with a focus on brain function and behaviour. We find fundamental differences in natural butyrate at physiological concentrations and its use as a neuropharmacological agent at rather high, supraphysiological doses in brain research. Finally, we hypothesise that butyrate and other volatile SCFAs produced by microbes may be involved in regulating the impact of the microbiome on behaviour including social communication.

Friends with social benefits: host-microbe interactions as a driver of brain evolution and development?

The tight association of the human body with trillions of colonizing microbes that we observe today is the result of a long evolutionary history. Only very recently have we started to understand how this symbiosis also affects brain function and behavior. In this hypothesis and theory article, we propose how host-microbe associations potentially influenced mammalian brain evolution and development. In particular, we explore the integration of human brain development with evolution, symbiosis, and RNA biology, which together represent a “social triangle” that drives human social behavior and cognition. We argue that, in order to understand how inter-kingdom communication can affect brain adaptation and plasticity, it is inevitable to consider epigenetic mechanisms as important mediators of genome-microbiome interactions on an individual as well as a transgenerational time scale. Finally, we unite these interpretations with the hologenome theory of evolution. Taken together, we propose a tighter integration of neuroscience fields with host-associated microbiology by taking an evolutionary perspective.

Microbes & neurodevelopment – Absence of microbiota during early life increases activity-related transcriptional pathways in the amygdala

The mammalian amygdala is a key emotional brain region for eliciting social behaviour, critically involved in anxiety and fear-related behaviours, and hence a focus of research on neurodevelopmental and stress-related disorders such as autism and anxiety. Recently, increasing evidence implicates host-microbe interactions in the aetiology of these conditions. Germ-free (GF) mice, devoid of any microbiota throughout organismal maturation, are a well-established tool to study the effects of absence of the microbiota on host physiology. A growing body of independently replicated findings confirm that GF animals demonstrate altered anxiety-related behaviour and impaired social behaviour. However, the underlying mechanisms of this interaction and the nature of the pathways involved are only insufficiently understood. To further elucidate the molecular underpinnings of microbe-brain interaction, we therefore exploited unbiased genome-wide transcriptional profiling to determine gene expression in the amygdala of GF and GF mice that have been colonised after weaning. Using RNA-sequencing and a comprehensive downstream analysis pipeline we studied the amygdala transcriptome and found significant differences at the levels of differential gene expression, exon usage and RNA-editing. Most surprisingly, we noticed upregulation of several immediate early response genes such as Fos, Fosb, Egr2 or Nr4a1 in association with increased CREB signalling in GF mice. In addition, we found differential expression and recoding of several genes implicated in brain physiology processes such as neurotransmission, neuronal plasticity, metabolism and morphology. In conclusion, our data suggest altered baseline neuronal activity in the amygdala of germ-free animals, which is established during early life and may have implications for understanding development and treatment of neurodevelopmental disorders.

The microbiome regulates amygdala-dependent fear recall

The amygdala is a key brain region that is critically involved in the processing and expression of anxiety and fear-related signals. In parallel, a growing number of preclinical and human studies have implicated the microbiome–gut–brain in regulating anxiety and stress-related responses. However, the role of the microbiome in fear-related behaviours is unclear. To this end we investigated the importance of the host microbiome on amygdala-dependent behavioural readouts using the cued fear conditioning paradigm. We also assessed changes in neuronal transcription and post-transcriptional regulation in the amygdala of naive and stimulated germ-free (GF) mice, using a genome-wide transcriptome profiling approach. Our results reveal that GF mice display reduced freezing during the cued memory retention test. Moreover, we demonstrate that under baseline conditions, GF mice display altered transcriptional profile with a marked increase in immediate-early genes (for example, Fos, Egr2, Fosb, Arc) as well as genes implicated in neural activity, synaptic transmission and nervous system development. We also found a predicted interaction between mRNA and specific microRNAs that are differentially regulated in GF mice. Interestingly, colonized GF mice (ex-GF) were behaviourally comparable to conventionally raised (CON) mice. Together, our data demonstrates a unique transcriptional response in GF animals, likely because of already elevated levels of immediate-early gene expression and the potentially underlying neuronal hyperactivity that in turn primes the amygdala for a different transcriptional response. Thus, we demonstrate for what is to our knowledge the first time that the presence of the host microbiome is crucial for the appropriate behavioural response during amygdala-dependent memory retention.

Early-life stress-induced visceral hypersensitivity and anxiety behavior is reversed by histone deacetylase inhibition

Stressful life events, especially in childhood, can have detrimental effects on health and are associated with a host of psychiatric and gastrointestinal disorders including irritable bowel syndrome (IBS). Early‐life stress can be recapitulated in animals using the maternal separation (MS) model, exhibiting many key phenotypic outcomes including visceral hypersensitivity and anxiety‐like behaviors. The molecular mechanisms of MS are unclear, but recent studies point to a role for epigenetics. Histone acetylation is a key epigenetic mark that is altered in numerous stress‐related disease states. Here, we investigated the role of histone acetylation in early‐life stress‐induced visceral hypersensitivity. Interestingly, increased number of pain behaviors and reduced threshold of visceral sensation were associated with alterations in histone acetylation in the lumbosacral spinal cord, a key region in visceral pain processing. Moreover, we also investigated whether the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), could reverse early‐life stress‐induced visceral hypersensitivity and stress‐induced fecal pellet output in the MS model. Significantly, SAHA reversed both of these parameters. Taken together, these data describe, for the first time, a key role of histone acetylation in the pathophysiology of early‐life stress‐induced visceral hypersensitivity in a well‐established model of IBS. These findings will inform new research aimed at the development of novel pharmaceutical approaches targeting the epigenetic machinery for novel anti‐IBS drugs.

The microbiome and childhood diseases: Focus on brain‐gut axis

Many childhood diseases such as autism spectrum disorders, allergic disease, and obesity are on the increase. Although environmental factors are thought to play a role in this increase. The mechanisms at play are unclear but increasing evidence points to an interaction with the gastrointestinal microbiota as being potentially important. Recently this community of bacteria and perturbation of its colonization in early life has been linked to a number of diseases. Many factors are capable of influencing this colonization and ultimately leading to an altered gut microbiota which is known to affect key systems within the body. The impact of the microbial composition of our gastrointestinal tract on systems outside the gut is also becoming apparent. Here we highlight the factors that are capable of impacting on microbiota colonization in early-life and the developing systems that are affected and finally how this may be involved in the manifestation of childhood diseases.

Probiotic modulation of the microbiota-gut-brain axis and behaviour in zebrafish

The gut microbiota plays a crucial role in the bi-directional gut–brain axis, a communication that integrates the gut and central nervous system (CNS) activities. Animal studies reveal that gut bacteria influence behaviour, Brain-Derived Neurotrophic Factor (BDNF) levels and serotonin metabolism. In the present study, we report for the first time an analysis of the microbiota–gut–brain axis in zebrafish (Danio rerio). After 28 days of dietary administration with the probiotic Lactobacillus rhamnosus IMC 501, we found differences in shoaling behaviour, brain expression levels of bdnf and of genes involved in serotonin signalling/metabolism between control and treated zebrafish group. In addition, in microbiota we found a significant increase of Firmicutes and a trending reduction of Proteobacteria. This study demonstrates that selected microbes can be used to modulate endogenous neuroactive molecules in zebrafish.