When the microbiome helps the brain‐current evidence

Abstract The gut microbiota‐brain axis has been recognized as a network of connections that provides communication between the gut microflora and both central and autonomic nervous system. The gut microbiota alteration has been targeted for therapy in various neurodegenerative and psychiatric disbalances. Psychobiotics are probiotics that contribute beneficially to the brain function and the host mental health as a result of an interaction with the commensal gut bacteria, although their mechanism of action has not been completely revealed. In this state‐of‐art review, the findings about the potential therapeutic effects of the psychobiotics alone or in combination with conventional medicine in the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, as well as in some psychiatric diseases like depression, schizophrenia, and bipolar disorder, have been summarized. The evidence of the psychobiotics therapeutic outcomes obtained in preclinical and clinical trials have been given respectively for the observed neurodegenerative and psychiatric disorders.


| INTRODUC TI ON
The probiotics usage has been known much earlier than microbes were discovered. Fermented dairy products were painted on Egyptian hieroglyphs, and Tibetan peasants traditionally used fermented yak milk to preserve it during long journeys. In the early 19th century, scientists noticed the apparent health effects of fermented dairy products. Although Pasteur identified the responsible bacteria and yeasts for the fermentation process, no health effects had been attributed to the microbes. 1 In 1908, Metchnikoff associated the Bulgarians' long life span with the Lactobacillus species from regularly consuming fermented milk and their presence in the gut. 2 Tissier isolated Bifidobacterium species in infants and claimed that they could displace gut pathogens. 3 These findings have catalyzed research into health-promoting microbes and their role in disease prevention. In one of the earliest human studies, in 1922, Lactobacillus acidophilus was used in 30 patients with chronic constipation, diarrhea, and eczema, showing improvement in all three conditions. 4 Soon after, the beneficial effects of Lactobacillus acidophilus were confirmed in patients with constipation and mental illness. 5 Again, scientists brought to the light bidirectional communication between brain and gut, claiming that emotional state could modify gut function. Moreover, Canon 6 showed that certain parts of the gastrointestinal tract, as well as the urinary bladder, were susceptible to mental state.
In the 21st century, the growing scientific research highlighted the bond between the brain and the gut. 7,8 Alterations in the gastrointestinal microbiome, known as dysbiosis, have been observed during the onset and development of mental disorders. Numerous adverse psychiatric reactions to antibiotics have been reported, even in patients with no history of psychiatric illness. 9 To manage depression, Logan and Katzman (2005) 10 proposed the probiotics as concomitant therapy with antidepressants. Many studies were done to shed light on the interplay between the gut microbiome and the brain, and thus, the digestive system-brain axis concept was created. 11 Preclinical and clinical studies suggested that administration of beneficial microbes may reduce depression, anxiety, stress, neuroinflammation, neurodegeneration, eliminate panic attacks, hypochondriac behavior and somatization, and improve cognition. 12 Furthermore, early-life perturbations in the gastrointestinal microbiome influenced neurodevelopment, with emerging mental health issues years later. 13 Dinan et al. (2013) 14 coined the term psychobiotics, as probiotics, which, if taken in appropriate amounts, have positive mental health effects. Magnetic resonance spectroscopy revealed that the oral administration of Lactobacillus leads to increased levels of γ-aminobutyric acid (GABA), N-acetylaspartate, and glutamate in the brain, assuming that these neuroactive molecules were enrolled in the mechanism of Lactobacillus strain action. Sarkar et al. (2016) 15 suggested that the definition of psychobiotics could be expanded to any substance that confer positive alterations in the microbiome. The prebiotics could also be part of the psychobiotics, supporting the psychobiotic bacteria growth and thus contributing mental health. Afterward, similar terminology has emerged: paraprobiotics, also known as inactivated probiotics, and postbiotics, which are non-viable bacterial cells, their products, or metabolites of live probiotic microorganisms. Both of them showed physiological benefits to the host. 16 Recently, it has been suggested that the concept of postbiotics should be expanded to comprise paraprobiotics in the definition. 17 Although many details in the mechanism of action remain unraveled, today, it is believed that the intestinal microbiome affects the brain and human behavior.

| G UT MI CROB I OTA-B R AIN A XIS
A key step in understanding the psychobiotics mechanism lies in studying the ongoing communication between the gut microbiota and the brain. 15 In humans, substantial evidence of this interaction happened more than 25 years ago. Patients suffering from hepatic encephalopathy showed the dramatic improvements after oral antibiotics. 18 The intestinal flora interacts closely in a bidirectional manner, linking to the intestine permeability, immune system, and entero-endocrine signaling with the cognitive and emotional brain centers. 19 The gut microbiota-brain axis binds the neuroendocrine and neuroimmune systems, as well as the autonomic nervous system with the intestinal microbiota. [19][20][21] The hypothalamic-pituitary-adrenal axis (HPA) is considered to be the main part of the neuroendocrine system that provides an adequate hormonal response to the stress. 22 The stress hormones, glucocorticoids, disrupt gut barrier integrity through alterations in tight junctions, leading to a leaky gut and triggering inflammatory immune response (Figure 1). 23 It is recognized that early colonization of the intestinal microbiota affects certain aspects of brain function and behavior, including neuroendocrine responses to stress. Sudo et al. (2004) 24 showed that germ-free mice exerted enhanced physiological reactions to stress in comparison to control. However, following the gut microbiota recolonization with probiotics, these abnormal reactions were reversible. The manifestation of homeostatic effects of probiotics on neuroendocrine physiology is highly significant, and it indicates new therapeutic possibilities. 25 The development of the intestinal immune system largely depends on exposure to microorganisms. 26 Gut microbiome alteration is associated with aberrant immune response due to the overproduction of cytokines through the HPA axis modulation. 27 The elevation of circulating pro-inflammatory cytokines IL-6 and TNFα, with concomitant activation of microglia, brain resident macrophages, is involved in induced depressive and anxiety states, and other affective disorders. 28,29 Nonetheless, certain probiotics can reverse the microglia activation and decrease the levels of pro-inflammatory cytokines, mediating anti-inflammatory response. 15 The vagus nerve is pivotal in coordinating parasympathetic activity with afferent terminals under the intestinal epithelium. Gut microbiota signals, transported to the brain, alter host behavior and possibly cause lethargy, loss of appetite, depression, or anxiety state. 30 In several animal studies, it has been found that n. vagus mediated the interplay between psychobiotics and their psychophysiological effects, since vagotomy has eliminated the response to psychobiotic administration. [31][32][33] The enteric nervous system (ENS) is responsible for coordinating the various digestive functions by forward and backward brain signal propagation, all via the vagus nerve. 34 Myenteric neurons are close to the gut lumen, facilitating their contact with the microbiota. 8 In addition, plenty of evidence suggested that gut bacteria modulated the ENS, regulating electrophysiological thresholds of myenteric neurons. 32,[35][36][37] According to both preclinical and clinical evidences, changes in beneficial bacteria may have significant health consequences, while certain factors such as infection, drug use, diet, exercise, environment, social interactions, and stress can alter the microbiome. 12,38,39 This determined changes in motility and intestinal secretion, caused visceral hypersensitivity, and lead to modification of the enteroendocrine and immune system. 19

| PSYCHOB IOTIC S AND NEUROAC TIVE MOLECULE S
Gut microbiota produces a broad range of neuroactive molecules, signaling in the crosstalk between the gut microbiome and host metabolism ( Figure 1). Due to the chemical and functional resemblance, these metabolites act as human neurotransmitters or neuromodulators. Serotonin (5-HT), dopamine (DA), noradrenaline (NA), GABA, acetylcholine (Ach), as well as short-chain fatty acids (SCFAs), produced by the gut microbiota via the metabolism of indigestible fibers, are of special interest. [40][41][42][43] Altered levels of 5-HT and DA are implicated in several mental health and neurological diseases. 8,44 NA is able to modulate cognitive functions, learning, memory processes, and mood disturbances. 45 GABA and Ach are the main inhibitory/excitatory neurotransmitters. Muller et al. (2021) 46 demonstrated that SCFAs composition is associated with psychiatric and gastrointestinal symptoms in adults with affective or anxiety disorders. Therefore, psychobiotics control the neural excitatory-inhibitory balance and modulation of the host's response to anxiety and depression. Due to these effects of microbially produced neuroactive molecules, psychobiotics have been suggested as a promising alternative or supportive therapy in treating neuropsychiatric disorders. In Table 1 results of different psychobiotic treatments associated with neurodegenerative and psychiatric diseases are summarized.

| Alzheimer's disease
AD is a chronic neurodegenerative disease with a progressive decline in cognitive and memory function. Recently, poor gut microbiota diversity in AD patients has gained an ongoing interest followed by finding an additional molecular pathogenesis for AD.
Novel findings suggest that autoimmune and autoinflammatory mechanisms are engaged in AD. 99 The data suggested that SCFAs, such as butyric, propionic, acetic acids, and microbial metabolites in colon reduced the AD neuropathological features and other neurodegenerative diseases by providing alternative energy sources to the brain. 100 Moreover, selected SCFAs may modulate neuroinflammation, a significant pathomechanism of the early and preclinical course of AD. Amyloid β abnormality, tau phosphorylation, neurotransmitter dysregulation, and oxidative stress in AD followed the derangement in the gut microbiota composition. 47,101 In addition, several probiotic strains, such as Lactobacillus plantarum and Bifidobacterium infantis, enhance gut barrier function via upregulation of tight junction expression and production of SCFAs. 102 Bearing in mind the richness of the gut milieu with endotoxins and amyloid β, the maintenance of secure gut barrier is requisite to avoid inflammation.

| Preclinical probiotic supplementation in AD
Research on rodents showed that memory storage and cognition began to decline with age and were severely damaged in AD. 103 Germ-free mice displayed a decreased level of tight junction protein and decreased level of brain-derived neurotrophic factor (BDNF) and N-methyl-D-aspartate (NMDA) receptor expression in the cortex and hippocampus. 104 BDNF and NMDA are proven to play a pivotal role in neuroplasticity, which loss is a significant F I G U R E 1 Gut microbiota-brain axis. Brain acts on the gut microbiome through the HPA axis and vagus nerve. In turn, gut dysbiosis acts on the brain through the vagus mechanism and immune endocrine pathway to cause neuronal degeneration and behavioral abnormalities. indicator of the AD etiology. 105 GABA and glutamine levels, brain metabolites, are enhanced by long-term administration of Lactobacillus spp. and Bifidobacterium spp. to aging rats, improving task-specific memory. 48  to restore synaptic plasticity, followed by a decrease in microglial activation and increased BDNF, improving cognitive function and spatial learning. 48 Another study found that the L.acidophilus, L.fermentum, B.lactis, and B. longum mixture attenuated the learning deficits and oxidative stress and improved spatial memory. 52 L.
plantarum was also shown to regulate microglia activation, reduced amyloid β load via suppressing NF-κB activation in AD mice. 53 These findings indicated a possible role of specific probiotic strains in enhancing memory and cognitive function.

| Clinical probiotic supplementation in AD
The Firmicutes/Bacteroidetes ratio has emerged as an indicator of intestinal microbiota health. It is shown that AD patients have decreased Firmicutes level along with increased Bacteroidetes level. 107

| Parkinson's disease
Parkinson's disease (PD) is a neurodegenerative disorder associated with the progressive loss of dopaminergic neurons in substantia nigra and abnormal intracellular aggregation of α-synuclein. 110 Motor impairment and characteristic brain pathology do not appear as symptoms until a fairly advanced stage of the disease. On the other hand, intestinal dysfunction, such as bloating, delayed gastric emptying, constipation, prolonged intestinal transit time with incomplete defecation, occurs years before the initiation of motor impairment. 111,112 Dysbiosis in gut microbiota has also been reported in PD. Decrease of Prevotellaceae in stool samples of PD patients, 113 along with increased Lactobacilliceae, are found to be associated with lower ghrelin level, a gut hormone that maintains normal dopamine function. 114 A study observed an antiinflammatory bacteria depletion, such as genus Blautia, Roseburia, and Coprococcus in stool samples of PD patients, along with a reduction in Lactococcus bacteria. 115 It is assumed that this shift from predominantly antiinflammatory phenotype toward the proinflammatory phenotype of the gut microbiota contributes the increased gut permeability and decreased dopamine function. 116 Furthermore, a significantly reduced abundance of SCFA (butyrate, acetate, propionate)-producing bacteria was found in PD patients.

| Clinical probiotic supplementation in PD
Several clinical studies reported beneficial probiotics effects in PD patients, especially in combating constipation, a common symptom in PD with a prevalence of up to 70%. 62,63,118 The first clinical trial conducted in 2011 highlighted that fermented milk with Lactobacillus casei administration improved stool consistency, defecation, decreased bloating, and abdominal pain in PD patients suffering from constipation. 64 Delayed gastric emptying, also common in PD patients, was found to be accelerated after the Lactobacillus reuteri administration. 119 In the randomized, double-blind, placebo-controlled clinical study, L. acidophilus, L. fermentum, and B. bifidum supplementation for 12 weeks reduced the expression of proinflammatory cytokines (IL-1, TNFα) and oxidative markers, and increased the antiinflammatory factors expression (TGFβ, PPARγ). 65 According to Barichella et al. (2016), 66 the number of spontaneous and total bowel movements were improved in PD patients treated with probiotics for 4 weeks.

| Depression
Although the depression is a complex chronic mood disorder associated with various etiology factors, there is a growing scientific evidence that the intestinal microbiota derangement is implicated in its pathophysiology. 120 Luo et al. (2018) 121 showed that changes in the gut microbiota composition altered mice behavior toward anxiety, depression, and even autism. The fecal microbiota transplantation performed from depression suffering patients into rats with entire microbiota previously removed, resulted in depression-like behavior of the animals, suggesting that the transplantation of the abnormal microbiome could lead to the depression "transmission." 122 Clinical studies indicated that the gut microbiota of depressed patients differs significantly from healthy subjects. 8 Antibiotics can also destroy important intestinal microorganisms and thus induce digestive-brain dysfunction, which may cause an increased incidence of various diseases, including mental disorders like depression. 123 The signal dysfunction of GABA, a major inhibitory neurotransmitter in the CNS, is associated with anxiety and depression. Lactobacillus and Bifidobacterium are capable of metabolizing glutamate to GABA.
An in vivo experiment with L. rhamnosus reported alterations in GABA receptor expression in brain regions related to stress. 8 In addition, pathogens and their metabolites may induce brain inflammation through circulation and cytokine cascade reactions, which further affect the various brain processes involved in mood and behavior. 124

| Clinical probiotic supplementation in depression
The clinical trials' results regarding the effects of different probiotic strains in mitigating depressive-like behavior are inconsistent. Three clinical trials demonstrated that the probiotic supplementation did not reverse behavioral deficits in patients with depression. [79][80][81] In contrast, several studies showed beneficial neurobehavioral effects of probiotics and prebiotics in combination. 126,127 Moreover, heat-killed Lactobacillus paracasei were efficient in maintaining the desirable mood state in healthy adults. 82 Heat-killed Lactobacillus gasseri reduced anxiety and altered the gut microbiota composition in young adult students preparing for the national examination. 83 In concomitant therapy with antidepressants, probiotics exerted improvement in overall mood. 84 Ghorbani et al. (2018) 85 reported the improved depression clinical symptoms when specific probiotics and FOS were applied as an adjuvant therapy to the antidepressant drug fluoxetine.

| Schizophrenia
Schizophrenia is a debilitating psychiatric disorder, with symptoms characterized as positive (aberrant flow of thoughts, delusions, hallucinations), and negative (social withdrawal, lack of motivation, apathy). 128 Recently, dysbiosis has also been noticed in schizophrenic patients. Shen 97 indicated that probiotic consumption alleviated the severity of mania and depression over time. Significant improvements to attention and psychomotor processing speed were achieved after 1 and 3 months of probiotics treatment, respectively, indicating the potential beneficial effects of probiotics in improving cognitive function. 98 The lower rehospitalization rate was reported after probiotic supplementation as add-on therapy in bipolar disorder patients . 150

| CON CLUS ION
"Let food be thy medicine and medicine be thy food" is one of the antient quote that is so important nowadays. 151 Recent studies have justified it considering a modified version: "Let food for your microbes be medicine for your brain." 11 Abnormal microbiota is undoubtedly involved in the etiology and pathophysiology of neurodegenerative disorders, behavioral and mood alterations, and it is likely to be the target of future therapies. However, in line with differences in food preferences, environment, and lifestyle, an outstanding issue is to define the normal gut microbiota, due to interindividual and geographic differences in gut microbiota composition.
Studying the whole ecosystem, gut microbiota and probiotics, is requisite in understanding the interplay between the gut and host health. The novel class of probiotics -psychobiotics -has shown to be helpful in improving CNS functions, such as neuronal degeneration, memory, depression, anxiety, and mood, modulating the HPA, inflammation, and neurochemical production. The usage of psychobiotics alone or in combination with conventional medicine could contribute to the development of new therapeutic strategies; however, more research is needed due to the scarcity of the clinical data.
Not all psychobiotics are probiotics, and vice versa, not all probiotics have psychobiotic potential. An increasing number of preclinical and clinical research reported these promising psychobiotics effects, with no side effects. At present, the discrepancies between the results from laboratories occur due to the rapid microbiome testing commercialization leading to the misinterpretation of the results and the lack of consensus in general. Therefore, multi-omics approach would enable comprehensive insight into the linkage between the gut microbiota composition and related diseases, as a promising tool for further research.

ACK N OWLED G M ENT
This work was supported by the Autonomous Province of Vojvodina, Republic of Serbia, Grants No: 142-451-3120/2022-01.

CO N FLI C T O F I NTE R E S T
The authors declare they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.