Metabolitos de la microbiota intestinal implicados en el trastorno del espectro autista Metabolites of the gut microbiota involved in the autism spectrum disorder

En los últimos años, ha habido un aumento de los estudios que buscan comprender la relación existente entre el microbiota intestinal (MI) con el trastorno del espectro autista (TEA), que debe producirse a través del eje microbiota-intestino-cerebro. A pesar de que los distintos autores señalan que los cambios encontrados en distintos filos, familias y géneros bacterianos están implicados en el TEA, no hay consenso científico a día de hoy. Algunos autores apuntan a la posible relación existente entre dichas poblaciones bacterianas con ciertos productos de excreción o metabolitos como el ácido propiónico ya que aparecen con frecuencia en niños con TEA. Aunque en los últimos años la MI comienza a acumular evidencia científica, en términos de neurociencia, el estudio de la metabolómica asociada a la misma y los mecanismos mediante los cuales estos metabolitos pueden influir en la aparición y desarrollo del TEA aún permanece en sus primeros estadios.


Introduction
Autism spectrum disorder (ASD) is a neurodevelopmental disorder that manifests during the first three years of life and persists throughout the individual's life.ASD is characterized by the presence of persistent deficits in social interaction and oral communication (communicative socio-emotional and non-verbal reciprocity in social interaction in multiple contexts); and behavior patterns restricted and repetitive that can be mild or severe [1].ASD affects one in every 68 people in USA and maybe one in 30 in Korea, being more prevalent in men (4:1) [2].In Spain the prevalence data of ASD are lower where it is estimated that 8 out of 10,000 children suffer from autism [3].In this sense, the scientific community tries to discover the Resumen: En los últimos años, ha habido un aumento de los estudios que buscan comprender la relación existente entre el microbiota intestinal (MI) con el trastorno del espectro autista (TEA), que debe producirse a través del eje microbiota-intestinocerebro.A pesar de que los distintos autores señalan que los cambios encontrados en distintos filos, familias y géneros bacterianos están implicados en el TEA, no hay consenso científico a día de hoy.Algunos autores apuntan a la posible relación existente entre dichas poblaciones bacterianas con ciertos productos de excreción o metabolitos como el ácido propiónico ya que aparecen con frecuencia en niños con TEA.Aunque en los últimos años la MI comienza a acumular evidencia científica, en términos de neurociencia, el estudio de la metabolómica asociada a la misma y los mecanismos mediante los cuales estos metabolitos pueden influir en la aparición y desarrollo del TEA aún permanece en sus primeros estadíos. .
Abstract: In recent years, there has been an increase in studies that seek to understand the relationship between gut microbiota (GM) with the behavior of people with autism spectrum disorders (ASD), which must occur through the microbiota-gut-brain axis.Although the different authors point out that the changes found in different phyla, families and bacterial genera are involved in ASD, there is no scientific consensus to date.Some authors point to the possible relationship between these bacterial populations with certain products of excretion or metabolites such as propionic acid since they frequently appear in children with ASD.Although in recent years the GM has begun to accumulate scientific evidence, in terms of neuroscience, the study of the metabolomics associated with it and the mechanisms by which these metabolites can influence the appearance and development of ASD remains in its first stages.origin of ASD and, apparently, the complex interactions between genetic, epigenetic and environmental factors can contribute to the development and expression of ASD [2].
At the brain level, one of the possible genetic mechanisms for the appearance of ASD has recently been elucidated in Canada.Specifically, a direct relationship between the mutation of the GRIN2B gene, the production of a protein, the formation of defective neurons and the manifestation of autism has been found [4].However, there is still much to be studied in terms of the mechanisms of interactions between the other factors that contribute to the development and expression of ASD.
One of the most studied hypotheses, which can include most of the factors responsible for the appearance and development of ASD, and many other diseases, postulates that a dysbiosis of the gut microbiota (GM), together with the metabolites that it produces, can be responsible for the origin of ASD [5].This concept is not new since there is enough scientific evidence regarding the relationship between a dysbiotic GM and the development of different diseases such as intestinal inflammation associated with the pathogenesis of obesity, type 2 diabetes mellitus [6], and other psychological disorders where ASD is included [7].A proven example of the relationship between GM and ASD occurred in Chicago (USA) at the beginning of the present millennium, where a child suffering from a serious ear infection was given broad-spectrum antibiotics as a treatment.After 3-4 months of treatment the child began to show the typical symptoms of autism, being diagnosed as autistic later.The explanation given to the appearance of ASD is based on the fact that antibiotics eradicated most of the GM, except those resistant microorganisms capable of forming spores and secreting toxic metabolites such as those of the genus Clostridium.After the application of specific antibiotics for Clostridium such as vancomycin or metronidazole, the autistic symptoms of the child improved in a very remarkable way, contrasted with studies performed on more ASD children later [8,9].However, chronic treatment with antibiotics is still under debate by the scientific community.
The GM encompasses the set of bacteria that inhabit the intestine, being in a symbiotic relationship both commensal and mutualism.GM represents about 99% of the complete human microbiome [10], weighs approximately 1 kg (about 9.9 million bacterial genes) [11], and is composed of: 92.9% bacteria, 0.5% eukaryotes, 0.8% archaea and 5.8% viruses [12].Bacteria can play an important role in the production of several metabolites such as short-chain fatty acids (SCFA), free amino acids (FAA) and gaseous molecules.These metabolites can induce effects on the gut, brain and behavior [2] through the so-called microbiota-gut-brain axis whose character is bidirectional.In the brain-GM direction, the central nervous system (CNS) exerts control over the composition of the GM through secreted peptides at the time of satiety and, therefore, affect the availability of nutrients.The hypothalamic-pituitary-adrenal axis releases cortisol that regulates intestinal motility and integrity.Intestinal epithelial cells secrete mucin that is regulated by immune and neuronal pathways, exerting control over microbial populations within the intestine.In the GM-brain direction, GM has been shown to control CNS activities through a variety of neuronal, endocrine, immune, and metabolic mechanisms [13,14].Specifically, certain metabolites activate immune cells in the brain called microglia and astrocytes, which mediate responses to inflammation in the CNS in mice with autoimmune disease [15].They can also produce their effects through biochemical, immunological and neuroendocrine mechanisms that involve endogenous and microbial modulators and transmitters [10].
One of the most studied metabolites produced by GM is propionic acid (PPA).The PPA is found in the intestine along with other SCFAs such as acetate and butyrate.These SCFAs are produced by enteric bacteria such as Clostridia, Desulfovibrio, and Bacteroidetes after the fermentation of carbohydrates and some dietary amino acids in the intestinal lumen.PPA is a weak organic acid that can be found in ionized and non-ionized forms at physiological pH, which allows it to easily cross the intestinal barrier, enter the bloodstream and be metabolized, mainly, in the liver.It is also able to cross the blood-brain barrier and reach the CNS.Specifically, the PPA accesses the CNS through transporters of monocarboxylates in the intestinal lumen and in the cerebrovascular endothelium, which actively transport many carboxylic acids, particularly PPA and ketones.PPA is also a specific ligand of many SCFA receptors coupled to G proteins (GPR41, 43).PPA and other SCFAs are absorbed by glial and, to a lesser extent, by neurons once they reach the CNS, where they are thought to comprise the major source of cellular metabolism energy, particularly during early brain development.APP and other SCFAs affect various physiological processes such as cell signaling, synthesis and release of neurotransmitters, production of free radicals, mitochondrial function, lipid metabolism, immune function, gap junction synchronization, maintenance of intracellular pH, and modulation of gene expression through histone phosphorylation and acetylation [16].
The relationship between ASD and PPA has recently been demonstrated using an animal model, where PPA was injected directly into the brain of rats, inducing repetitive and antisocial behaviors that were reversible after approximately 30 min.The potential mechanisms for these rapidly inducible and reversible behaviors are complex and include SCFAs-mediated effects such as improvement in the release of calciumdependent glutamate in astrocytes, release of serotonin and dopamine, inhibition of neurotransmitter receptors, gamma-aminobutyric acid, activation of receptors coupled to specific G proteins of SCFAs, increased sensitivity of the glutamate receptor, increased catecholamine synthesis, intracellular acidification, mitochondrial dysfunction and gap junction closure [16].

Objective
The objectives of the present work are to provide current information on the correlation between the metabolites produced by the gut microbiota, mainly short chain fatty acids, found in feces of individuals with ASD and current treatments of autism that take into account the modification of the intestinal microbiota.

Gut microbiota and short chain fatty acids
Although there is increasing scientific papers studying the metabolites produced by GM and its relationship with ASD, these are still rare in humans so that data can be obtained from these studies they are also scarce.Specifically, a review study conducted by the authors of the present work (not yet published) has identified 18 articles, published in the last 6 years, that study the GM of autistic individuals compared to their respective control groups.Of the 18 articles found, 11 of them studied some types of metabolites or expression of genes related to the GM, both in feces or intestinal epithelium and in blood or urine, such as calprotectin, oxytocin, serotonin, isopropanol or p-cresol.However, only one of them found correlations between SCFA and GM in feces of ASD people.
It is about the study carried out by De Angelis, Piccolo [17] where both GM and some metabolites that this produces [FAA and 82 volatile compounds] in the faeces of 10 ASD children was compared to both GM and some metabolites that this produces of the faeces of 10 healthy children, with an age between 4 and 10 years.Specifically, an increase of bacteria of the genus Clostridium was found in ASD children that was positively correlated with methyl esters of acetic, methyl, butanoic and pentanoic acids, together with some indoles (r = 1.0, p <0.05).In this sense, it is known that bacteria of the family Clostridiaceae are a group of bacteria that also synthesize phenols, p-cresol, isopropanol or certain indole derivatives that are potentially toxic to humans and they have also been related to ASD.
In this same study, bacteria of the genera Faecalibacterium, Ruminococcus and Bifidobacterium were less abundant, generally, in ASD children that were positively correlated with total SCFAs (r = 1.0; p <0.05).Among the bacteria of the genus Faecalibacterium, Faecalibacterium prausnitzii, belonging to the family Ruminococcaceae, is one of the largest producers of butyric acid.The high prevalence of bacteria of the genera Lactobacillus and Bifidobacterium is a biological marker of a healthy GM in breastfeeding babies.Both bacterial genera fulfill important probiotic functions in the gut, including the inhibition of pathogenic bacteria by competitive exclusion and/or the production of antimicrobial agents.Bacteria of the genus Bifidobacterium appear shortly after birth and, in a matter of weeks, represent the dominant microorganism in the gut.It has been shown that probiotics improve the symptoms of irritable bowel syndrome (IBS), abdominal swelling, abdominal pain and flatulence, and are suggested as a possible intervention to improve the behavioral problems associated with ASD since they improve gastrointestinal discomfort.
Finally, it was also found that the prevalence of Bacteroides was greater in ASD children which was positively correlated with total FAA, ammonia and PPA (r = 1.0; p <0.05).In general, a greater number of Bacteroidetes is found in faecal samples of children affected by gastrointestinal inflammatory diseases and ASD.In this regard, it is also known that Bacteroides fragilis is producing APP with other SCFA and, as has been discussed above, the APP shows neurobiological effects in rodents.In addition, this bacterium also synthesizes large amount of lipopolysaccharides, this being an important factor of bacterial virulence.

Present and future perspectives
The existence of marked cultural and dietary differences with respect to the composition of the GM around the world [18] should be taken into consideration when carrying out scientific studies mainly because the definition of a normalized GM is a complicated task and the comparative data between different populations should be taken with caution.
Regarding the possible treatments of ASD, there is an increasing scientific consensus that treatments based on the GM restoration of people with autism are the lines of research that should be followed.These treatments are diverse among those that can be mentioned: the treatment with antibiotics and/or antifungals to reduce specific components of the GM; the transfer of GM from healthy donors to individuals with ASD since it has been shown to improve gastrointestinal and autistic symptoms; treatment with helminths because modern civilization is thought to have caused them to disappear from the gut causing changes in the immune system; treatment with probiotics to favor the growth of beneficial bacteria; and dietary treatments, including ingestion of unprocessed and fermented food, treatment with digestive enzymes or administration of vitamins [5].Regarding dietary treatments, the recommendations aim to obtain the necessary carbohydrates from raw vegetables, with a high inulin content such as artichoke [19], instead of foods rich in refined carbohydrates.
Finally, the best understanding of the relationship between GM and ASD necessarily involves the study of the biochemical mechanisms that occur at the mitochondrial level.In this sense, future studies should look for possible correlations between: GM, metabolites in feces, metabolites in urine, metabolites in blood and gene expressions of the main known metabolic pathways, both in animal and human models.