Gut-brain axis: the role of microbiota in gut and brain ageing
Creators: name: Altera, Annalisa
Publisher: name: Università di Siena
Year: 2022
Subjects: subject: BIO/16 ANATOMIA UMANA

description: In the last decade there has been a growing interest in the reciprocal impact occurring between the gut and the brain and this is well conceptualized in the gut-brain axis notion. The gut-brain axis is the bidirectional communication route between the “little brain” (gut) and the “big brain” (brain). There are several factors that play an important role in this axis but it has become more and more evident that the gut bacteria represent a key component. This has led to the new concept of the microbiota-gut-brain axis, emphasizing the importance of the gut microbiota in this axis. The gut has evolved with bacteria in a symbiotic way and the human gut hosts about 1014 bacterial cells. Researches in the last years have highlighted the importance of the microbiota not only for gut functions but also for the central nervous system (CNS) development, physiology and pathology. However, there are different factors that influence the composition of the gut microbiota (mode of delivery, diet, stress and ageing). In particular, the composition of the gut microbiota changes with ageing: in the adults the majority of taxa are Bacteroidetes and Firmicutes while the elderly has a different composition of the gut microbiota. Some studies have reported a decrease in Bifidobacteria and an increase in Escherichia, Enterobacteriaceae and Clostridium difficile in the elderly. Interestingly, the centenarians apparently have no changes in gut microbiota in comparison to adult, further highlighting the importance of gut bacteria in longevity. Ageing is a physiological process related to the loss of function in different body systems and also associated with a decline in cognitive functions. It has become more and more evident that events taking place in the gut play a major role in the ageing process and in age-related diseases. Faecal microbial transplant (FMT) is a technique that consists in the transfer of gut microbiota from a donor to a recipient (usually via an oral gavage in rodents or colonoscopy in humans) and allows to establish a donor-like microbiota in the gastro-intestinal tract of the recipient. FMT is used to treat recurrent Clostridium difficile infections but there are studies trying to test this technique in the treatment of other pathologies such as irritable bowel syndrome, inflammatory bowel disease and constipation. It is also worth noticing that the imbalance in the composition of the gut microbiota (dysbiosis) has been associated with a plethora of neurological disorders. In this context FMT is being investigated as a therapeutic option not only for treatment of gut disorders but also for diseases of the CNS. The present thesis illustrates a series of experiments by which we tested the impact of FMT from aged donor mice into young adult recipients. Controls were carried out operating FMT from young adult donor mice to age-matched recipients. Following transplantation, characterization of the microbiota and metabolomics profiles along with a series of cognitive and behavioural tests were carried out. Label-free quantitative proteomics was employed to evaluate protein expression in the hippocampus and gut after the transplant. In addition, in the attempt to elucidate the mechanisms underlying microbiota-host interactions within the framework of the gut-brain axis, we worked on setting up a procedure to tracking down and visualize bacterial metabolites (such as peptides and lipids) that are thought to play a role acting as signaling molecules. To this end, we used copper-catalysed azide-alkyne cycloaddition (CuAAC) click chemistry, a biorthogonal reaction of widespread utility throughout medical chemistry and chemical biology. We sought to optimize click-based protocols to detect the production of lipids in gut-bacteria to track the metabolism of active bacterial cells. This technique use click chemistry to stain synthetic (e.g., noncanonical) precursors incorporated into bacterial cell biomass. After incorporation, the artificial molecules can be fluorescently detected via azide-alkyne reaction and visualized by confocal microscopy. FMT from aged mice into adult recipients affected spatial learning and memory while we did not observe effects on locomotion and explorative behaviour. Alongside, there was an alteration in the expression of proteins related to synaptic plasticity and neurotransmission in the hippocampus which was not observed in controls. FMT from aged into young adult mice did not induce a significant increase in glial fibrillary acidic protein expression in hippocampal astrocytes suggesting the lack of an overt neuroinflammatory response. On the other hand, a significant increase in the expression of F4/80, a typical trait of the ageing brain, was observed in microglial cells resident in the fimbria. Gut permeability and levels of systemic and local (hippocampus, gut) cytokines were not affected. As regards click chemistry, we used Bacteroides thetaiotaomicron grown in minimal medium supplemented with palmitic acid alkyne (PAA) and stained this molecule using an azide-containing fluorescent dye. After palmitic acid staining, co-culture experiments were performed to assess the transfer of this bacterial product to eukaryotic cell lines (CaCo2 and SK-N-SH cell lines). The successful transfer to host cells was confirmed by confocal microscopy. Results obtained in FMT experiments highlighted the importance of the gut microbiota on protein expression and functions of CNS. These results support the key role of microbiota in gut-brain axis and it would be of great importance to get more insight into the restoration of a young microbiota in the elderly to try to improve cognitive functions and the quality of life. Click chemistry experiments demonstrate that this technique could be employed to track molecules produced by gut bacteria to unveil their role in host-microbe interactions.