If it is now acknowledged the gut microbiota plays a role in the pathophysiology of depression, the specific biochemical mechanisms underlying this connection are hard to unpick and poorly understood.

Using a multi-omics approach, scientists in Spain have now identified high levels of proline as a metabolite that is directly linked to depression severity, and shown that variations in levels of circulating proline are dependent on microbiome composition.

When mice are given a fecal microbiota transplant from humans with depression, the mice experience depression and there is increased expression of proline transporter genes in the brains of the mice.

Similarly, giving mice proline supplements exacerbates depression, while the knockdown of proline confers protection against depression-like states in drosophila.

The results highlight a potential causative role of the microbiome in depression, through the disruption of glutamate and gamma-aminobutyric acid (GABA) neurotransmitter homeostasis, via alteration of proline metabolism.

While it has recently been shown the microbiota-gut-brain axis is involved in control of inhibitory behavior and cognitive function, previous studies examining the gut microbiome in depression have thrown up inconsistent results, due to small numbers of subjects and a lack of fine details about the species composition of individual microbiomes.

The new research, published in Cell Metabolism on May 3, 2022, describes how the link between proline levels and depression was identified by looking beyond bacterial taxonomy to analyses of microbial function and metabolomics, and by carrying out shotgun metagenomics sequencing, a technique that makes it possible to sequence thousands of organisms in parallel.

The influence of diet and any antidepressive drugs also was factored into the research.

By applying this integrated 'omics approach to three different groups of patients, the researchers were able to discern molecular mechanisms underlying the microbiome-gut-brain axis in depression.

They first assessed how bacterial composition and functionality was related to depression using the Patient Health Questionnaire-9 (PHQ-9), a standard test that is used for screening, diagnosing, monitoring and measuring the severity of depression. The test was applied to the Ironmet cohort of patients, who the researchers, led by Jordi Mayneris-Perxachs at the Institut d'Investigacio Biomedica de Girona (IDIBGI, Institute of Biomedical Research, Girona, Spain), had recruited to an earlier study.

Applying PHQ-9 to the Ironmet cohort, 44 were not depressed, 47 were mildly depressed and 25 had major depression.

Non-depressed subjects were found to have greater species diversity in their microbiomes than depressed participants, while the researchers identified 30 bacterial species that were significantly associated with depression.

Patients with higher PHQ-9 scores had higher levels of Parabacteriodes and Acidaminococcus species, but lower levels of Bifidobacterium pseudolongum and species from the butyrate-producing Lachnospiraceae family.

There was no difference in microbial profiles between patients taking or not taking antidepressants or anxiolytics.

Gut dysbiosis triggers a proinflammatory state in the host, facilitating translocation of bacterial antigens into the circulation. Increased intestinal barrier permeability previously has been associated with altered gut microbiota in depression. In line with this, the researchers found two biomarkers of gastrointestinal tract barrier integrity correlated positively with higher PHQ-9 scores.

When these amino acids break down they drive the production of glutamate and subsequently of GABA neurotransmitter. The researchers consistently found significant associations of bacterial glutamate metabolism, glutamatergic synapse and GABAergic synapse, with host PHQ-9 scores.

Overall, the researchers identified 327 out of 5,714 bacterial functions that were significantly associated with depression scores.

Some of the bacterial functions with the strongest associations with depression scores were involved in the tricarboxylic acid cycle (TCA, or Krebs cycle), the main source of energy for cells.

Having uncovered these associations, the researchers further explored microbiome functionality by metabolomics analyses of plasma and fecal samples. That threw up several metabolites that were linked to PHQ-9 scores, and in line with the previous observations, several of these were involved in the TCA cycle.

The most consistent finding was the strong positive association of circulating proline with depression scores. It was also shown that metabolites from proline, and from histidine and arginine degradation pathways that converge into glutamate and the GABA shunt (a closed loop process for producing and conserving supplies of GABA), are associated with depression.

Recognizing that proline levels are related to dietary intake, the researchers next investigated the association of diet with PHQ-9 scores in the Ironmet cohort, finding individuals with high dietary intake of proline and circulating proline above the median, had the highest depression scores.

"We were surprised that what was most associated with depression, assessed through [PHQ-9], was the consumption of proline," said Jose Manuel Fernández-Real, of the Nutrition, Eumetabolism and Health research group at IDIBGI.

Adding further weight to the link between proline and depression, the researchers also found circulating levels of proline were related to a microbial signature that was similar to that associated with high PHQ-9 scores.

Individuals who despite consuming high proline levels had low circulating proline levels, had a microbiota composition consistent with that associated with lower depression scores, and their microbiota were enriched with bacterial functions involved in the transport and metabolism of proline.

"The microbiota of patients with high proline consumption but low plasma proline levels was similar to the microbiota associated with low levels of depression and was enriched in bacterial genes involved in the transport and metabolism of proline," said Mayneris-Perxachs.

To test their 'omics generated data pointing to a causal role of the microbiota in depression, the researchers did fecal transplants from 20 human donors with different plasma proline levels, into antibiotic-treated mice.

They found that an "emotionally impaired" phenotype emerged in mice receiving microbiota from donors with higher depression scores.

In addition, the human donor's PHQ-9 score was associated with a recipient mouse microbiome signature very similar to that related to depression in humans.

In another experiment, it was shown that a decrease in the expression of proline and GABA transporters confers protection against depression-like states in drosophila.

"These results show the importance of proline and its influence on people's depressive mood, an element that has not been taken into account until now," said Fernandez-Real. The work also opens the door to new studies to find possible diet-based treatments for depression.

And as the researchers note, all classical depression drugs target the brain, mostly with limited effectiveness.

"Understanding how the microbiome links proline to glutamate and GABA metabolism is thus crucial to design new effective treatments for depression," they conclude.