The gut-brain-axis: how does the gut microbiota affect the function of the brain?

Secondary Supervisor(s): Professor Willem Van Schaik

University of Registration: University of Birmingham

BBSRC Research Themes: Integrated Understanding of Health (Ageing, Diet and Health)

Project Outline

The gut microbiota is an important contributor to overall health of the central nervous system (CNS), with a bidirectional communication (gut-brain-axis). In humans, there is evidence of association between the gut microbiome and mental health disorders, however, causality and target validation are still needed. Gut microbiota perturbations, specifically a depletion of anti-inflammatory butyrate-producing bacteria and an enrichment of pro-inflammatory bacteria, are associated with depression, bipolar disorder, schizophrenia, and anxiety. A disruption of the gut microbiome, known as dysbiosis, can predispose to mental health disorders whilst diet-mediated changes in the gut microbiota have been associated with poor mental health.

Gut bacteria can produce neurotransmitters such as serotonin, dopamine, noradrenaline and can also modulate tryptophan metabolism. All of these molecules have profound effects on the brain and alter mood, memory, behaviour and more. Inflammation of the gut makes the lining of the gut “leaky” and this is communicated to the brain and can cause several mental illnesses including anxiety and depression. Probiotics have the ability to restore normal microbial balance, and therefore may have a potential role in the treatment and prevention of anxiety and depression. We hypothesise that changes in the gut microbiota in rodents over their lifespan lead to inflammation in the brain that causes mental illness development and that restoring the normal gut microbiota will alleviate inflammation in the brain and subsequent mental illnesses.

In this project we will use rodent models throughout their lifespan to understand the impact of the microbiome on the normal postnatal CNS development and potential changes in the brain over time that may explain the occurrence of mental health problems. In rodents, we will first define the “normal” gut microbiota by analysing stool samples; convenient, inexpensive and can be repeatably sampled. We will then challenge the gut microbiota (e.g. by changing the diet to reduce fibre intake and increase fatty acids and monosaccharides) and determine circulating levels of proinflammatory cytokines and neurotransmitters in blood as fluid biomarkers for potential changes. Brain tissues will then be subjected to analysis by immunohistochemistry (IHC) to determine microglial and astrocyte activation and changes in proinflammatory cytokines, using a panel of specific antibodies. Attention will be paid to proinflammatory cytokines associated with mental illnesses such as interleuikin-6, which is associated with for example, depression and psychosis in general. Blood brain barrier (BBB) permeability will be assessed in depth using Evans Blue dye extravasation into the brain parenchyma and measurement of tight junction proteins such as claudin 5, CCLN and ZO-1. All of these changes will be correlated with behavioural changes in rodents which test for depression, anxiety and aggression amongst others, that are routinely used in the Ahmed lab. Finally, we will perform neuropathological assessments to determine changes in specific regions of the brain associated with mental illnesses. For example, we will measure gray matter volume loss, which correlates with mental illnesses.

This project will combine the expertise of two world-leading researchers in microbiology & infection and neuroscience to lead to a deeper understanding of the gut-brain-axis and how changes in the gut microbiota may bring about the development of mental illnesses.

The role of AMIGO3 in the formation and repair of myelination the central nervous system

Secondary Supervisor(s): Dr Daniel Fulton

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Neuroscience and Behaviour, Systems Biology)

Project Outline

Oligodendrocytes are key cells responsible for making new myelin. Oligodendrocytes arise from a class of CNS progenitors, termed oligodendrocyte progenitor cells (OPC), that proliferate and migrate widely throughout the developing CNS. Once OPCs reach sites of myelination they begin to wrap target axons and undergo a series of molecular and morphological changes that culminate in the production of a terminally differentiated myelinating oligodendrocyte. These dramatic molecular and morphological changes are mediated, in part, through the actin cytoskeleton, with cell surface receptors, such as leucine-rich repeat and immunoglobulin (Ig)-like domain-1 (LINGO-1), providing a link connecting extrinsic ligands to intracellular actin-regulating pathways (e.g. RhoA/ROCK). Interestingly, genetic deletion and antibody-mediated antagonism of LINGO-1 was found to enhance OPC differentiation whilst disruption of LINGO-1 lead to impairments in myelin formation and repair. However, disappointing results and failure to reach the primary endpoint in clinical trials of anti-LINGO-1 antibodies in myelin diseases such as multiple sclerosis and optic neuritis, led to us and others to question the suitability of LINGO1 as a therapeutic target. In particular, our group, and others, have shown through various methods including subcellular fractionation that LINGO-1 is not present in the membrane fraction, and instead resides mostly within the cytoplasm. These results may help to explain the failure of the LINGO-1 trials since cytoplasmically localised protein would not be expected to interact with therapeutic antibodies in the extracellular space.

As an alternative to LINGO-1, our group has explored the function of a structurally related molecule, amphoterin-induced open reading frame-3 (AMIGO-3). Our unpublished work shows that AMIGO-3 is present at higher levels in the CNS than LINGO-1, and that it is expressed in myelinating regions of the CNS where it is downregulated prior to the onset of myelination. Moreover, our in vitro data identify AMIGO-3 as negative regulator of OPC differentiation, with siRNA knockdown of AMIGO-3 leading to an increase in OPC differentiation. Importantly, AMIGO-3 is localised primarily in the plasma membrane, marking it as a likely player in extracellular receptor complex signalling events, and in line with this finding, stimulation of the AMIGO-3 receptor complex in myelinating cell cultures triggered a reduction in OPC maturation. Taken together, our data identify AMIGO-3 as a negative regulator of OPC differentiation, and a promising target for therapeutic antibodies designed to promote myelin formation and repair.

This project will study a new AMIGO3 knockout (KO) mouse which we have recently established in Birmingham. The project will examine the hypothesis that AMIGO3 antagonism/knockdown will promote oligodendrocyte differentiation and myelination in vivo. We will document OPC differentiation and myelination in white matter tracts of AMIGO3 KO mice to understand if it is accelerated compared to wild type mice. We will also culture OPC from AMIGO3 KO mice and determine changes in their differentiation compared to wild type littermates. Because the onset of myelination in normal mouse development typically occurs around postnatal day (P) 5, we will also examine myelination in the brain and spinal cord at ages spanning this age by electron microscopy to further assess if myelination is accelerated in the AMIGO-3 KOs. A major ambition will be to explore the therapeutic potential of AMIGO-3 in myelin disease. Therefore, we will explore OPC differentiation and myelin repair in a spinal cord myelin injury model. Here, the myelin toxin Lysolecithin (LPC) will be injected into the spinal cord of KO and control mice to create focal demyelination. We will then ask if AMIGO3 KO mice can repair the damage through accelerated OPC differentiation and remyelination.