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The Role of Gut Microbes in Autism

The Role of Gut Microbes in Autism

By Elisa Hill-Yardin & Ashley Franks

Gut microbes can modify our mood and even change our behaviour. They’ve now been implicated in a neuronal mutation found in the gut and brain of autistic patients.

Autism and gut problems go hand-in-hand. As well as having impaired social and communication skills plus repetitive and/or restricted behaviours, patients with autism are four times more likely to be admitted to hospital with gastrointestinal issues. Gut problems include constipation that often alternates with diarrhoea, abdominal pain, bloating and vomiting. It remains a mystery why gut dysfunction is so common in autism.

Neuronal Genes in Autism

While hundreds of genes contribute to autism, no single gene is implicated in more than 2–3% of autism cases. Many of the gene mutations implicated in autism help neurons communicate via synapses between brain cells.

Mice engineered to express these gene mutations have been helpful in progressing research into autism. When mutations found in human patients are expressed in mice, the mice also show autism-like behaviours. Many autism mouse models show altered brain activity that resembles what is seen in some human autistic patients due to changes at the synapse.

We are investigating whether faulty connections between neurons cause gut issues in autism involving changes in microbes.

A Second Brain: The Nervous System of the Gut

The gut has its own brain. The enteric nervous system is made up of two mesh-like networks of neurons sandwiched between muscle layers in the gut wall. These large neuronal networks extend along the length of the gastrointestinal tract from the mouth to the anus. The neurons regulate gut motility and the secretion of water and electrolytes into the gut lumen. Similar to the brain, where different areas vary in neuron types and connections, gut neurons also differ depending on their location.

Gut neurons release more than 20 neurotransmitters including acetylcholine, serotonin and nitric oxide. Gut neurons come in different shapes and sizes, contain different combinations of neurochemicals, and vary in the way they communicate in the enteric network. Although the brain and gut communicate to fine-tune activity throughout life, the gut can function independently from the central nervous system.

Many of the synapse genes mutated in autism are also expressed in gut neurons. We have been studying mice expressing a mutation in the gene for a synaptic protein called Neuroligin 3. This mutation has been identified in autism patients with gastro­intestinal issues. The Neuroligin 3 protein maintains stable synapses and neuronal communication in the brain, and the mice we are studying have altered brain activity and behaviours relevant to autism.

Because Neuroligin 3 is also found in the gut, we reasoned that some gut problems in autism could be due to wiring glitches in the enteric nervous system of the gastrointestinal tract. To look at this we dissected the colons of mice with and without the mutation and analysed their colonic contractions. Under some conditions, colons from mutant mice had fewer contractions than colons from those without the mutation. This tells us that the Neuroligin 3 mutation changes gut function. Because neurons regulate these contractions, the Neuroligin 3 mutation probably disrupts neural communication in the gut of the mice.

How Do Gut Microbes Fit In?

Our gut contains about 2.5 kg of microorganisms. Like an ecosystem, these bacteria, fungi and archaea live in an exquisitely balanced community. Our microbes help digest food, stimulate the body’s response to infection, and release metabolites that act on neurons and our immune systems to alter mood. An imbalance in the gut microbial community, known as dysbiosis, can both cause disease and be caused by disease. Dysbiosis can change gut function and lead to alterations in mood and behaviour.

Gut microbes are essential for the development of our immune and nervous systems. Altering the balance of microbes that colonise the gastrointestinal tract in infants can affect enteric neurons and gut function in early development and even throughout life.

Early in life, microbes help ensure that our nervous system develops normally and train our immune system to respond to infection. In fact, infants with reduced exposure to microbes during development are more likely to develop allergies. This interplay between microbes and a fully functional immune system sets the scene for normal development and healthy outcomes.

Microbes Are Shaped by the Environment

Altered microbes in the gastrointestinal tract might be to blame for gut problems in autism patients. Unfortunately, studies of microbes in autism so far have not yielded a clear answer. This is partly because most studies have been poorly designed, with too few patients included and a lack of consideration of environmental effects like diet and medications. However, differences in the metabolites that microbes utilise as their energy source have been noted in autistic patients. There are also reports of altered microbial community structure, such as the ratio of the bacterial phyla Firmicutes and Bacteroides.

The microbiome responds to a multitude of factors, including diet, environment and genetics, and these can produce conflicting results in patient studies. People who live in the same household, work together or even just watch a movie together have microbial signatures that become more alike over time. Similarly, the characteristic signatures of faecal microbial populations merge when mice are housed in the same cage.

When designing experiments in mice it is therefore important to remove or control for environmental differences before we attempt to understand the influence of genetics.

Microbial Clues to Understanding Autism

To find out whether microbes might play a role in gut problems in autism, we analysed faeces from control and mutant Neuroligin 3 mice. These mice were housed together in the same cages since weaning. By co-housing mice we are able to remove environmental variables and isolate the genetic differences between the mice as the cause of any changes to the microbiomes.

When control and Neuroligin 3 mutant mice were housed together, their faecal microbial communities still differed, both in terms of microbial types and their function. We collected mouse faeces over several weeks and saw different microbial populations in juvenile mice that were 5 weeks old. Adult mice at 9 weeks of age still showed effects on microbe function as a community.

These findings suggest that the Neuroligin 3 mutation found in autism patients influences the composition of gut microbes and affects neural function in the mice. While diet, exercise and other external factors had previously been implicated in these changes, this was the first time that a specific gene could cause such significant changes in the microbiome.

Probing Faeces for Microbes

Mouse faeces contain a huge diversity of microbial species. To investigate this diversity we can examine which microbes are present and which metabolites these microbes are consuming or producing. If we find differences we can then study why this might occur.

To find out about how microbes function in different samples, we measured the ability of the microbe community to consume carbon sources. Microbial communities can contain the same microbes yet function differently, and vice versa. These findings tell us that the same bacteria can perform different functions in different conditions.

Utilisation of carbon sources also gives us insights into events within the gut. For example, if the utilisation of an amino acid is reduced, this amino acid may be more readily available for metabolic reactions in the gastrointestinal tract. This might then affect overall gut function. Many bacteria also produce neurotransmitter molecules, so altering microbial communities could also disrupt gut neuron function.

To find how the microbes differ at a community level, we first need to extract DNA from all of the bacteria in the faeces so that we could estimate microbial diversity in the gut. We found differences in faecal microbes shortly after weaning, but were unable to identify the altered microbial types. What we could demonstrate was that changes to the microbiome were due specifically to the Neuroligin 3 mutation. High throughput 16S rRNA gene sequencing then revealed that microbes of the Firmicutes, Clostridia and Candidate phyla were changed in the mice.

Future Directions: Microbes and Our Minds

The microbiome is incredibly diverse and performs many different functions. By examining both the function and the structure of the microbial community, researchers can predict possible effects on gut function.

We want to understand how microbes interact with the enteric nervous system in autism. Our next step is to recruit the immune system to target and restore the balance of gut microbes in the Neuroligin 3 mice in an effort to lessen their gut issues. To do this we will use polyclonal antibodies to educate the mouse immune system so that the microbiome can become more like healthy individuals. Like a clinical trial, we will treat control and mutant mice with a commercial compound that stimulates the immune system, and compare our findings to mice given a placebo.

If changes in gut contractions are caused by faulty synapses in Neuroligin 3 mice, the pattern of microbes along the length of the gut could be disrupted. As part of this study we will sample microbes from different regions of the gut to determine if these are different in mice expressing the mutation identified in autism patients. Ultimately, we also want to know if changing the microbiome can influence behaviour in these mice.

How gut microbes modify mood and even change our behaviour is not well understood, but connections between the gut and the brain are involved. By modulating gut microbes we hope that we will find new ways to relieve gut disorders and potentially other traits such as anxiety and sleep issues in autism. This study is a step along the road towards discovering the mechanisms of this interaction.

Elisa Hill is an ARC Future Fellow in Physiology at The University of Melbourne. Ashley Franks is Head of Applied and Environmental Microbiology at La Trobe University.