Next Generation Probiotics Relevance to Neurodegenerative Diseases: Novel Therapeutic Perspective

1. Introduction

Age-dependent neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), are chronic and progressive neurological disorders with a range of causes and clinical presentations [1,2]. The prevalence of neurodegenerative diseases has increased worldwide in parallel with the rise in life expectancy, and effective treatments for neurodegenerative diseases are highly desired. Of note, the primary prodromal symptom of AD and PD, gastrointestinal dysfunction, is detected prior to the clinical diagnosis, suggesting that the gastrointestinal tract and its connection to the central nervous system (CNS) are involved in the disease aetiology [3]. Among other causes (genetic, immune system, etc.) attributing to AD and PD pathogenesis [4], gut dysbiosis is an emerging factor and receiving more and more attentions. Clinic treatments that target gut microbiota provide a new and promising approach for reducing the risk, modulating the symptoms, and delaying the neurodegenerative progression.

In this communication, we review recently progresses in the understanding of gut dysbiosis and neurodegeneration, particularly emphasize on the potential application of next-generation probiotics for AD and PD therapies.

2. Pathological Features Of Neurodegenerative Diseases

Neurodegenerative diseases are relatively common, progressive, and devastating neurological disorders. As the population aged, the incidence and prevalence of neurodegenerative diseases have risen rapidly in the past two decades. AD is the most prevalent neurodegenerative disease and the leading cause of dementia worldwide. Pathologically, AD is characterized by β-amyloid (Aβ)-containing extracellular plaques and tau-containing intracellular neurofibrillary tangles [2]. The presentation of AD with memory impairment is most common, but the difficulties in expressive speech, visuospatial processing and executive functions co-occur, ultimately with the classic clinical signs of dementia. PD apparently results from the complex interplay of α-synuclein aggregation, neuroinflammation, mitochondrial dysfunction, and abnormal synaptic transmission, leading to the gradual, irreversible loss of dopaminergic neurons in the substantia nigra and the resulting striatal dopamine depletion [5]. In addition to the cardinal motor symptoms such as resting tremor, rigidity, bradykinesia, PD patients also exhibit non-motor symptoms, including hyposmia, sleep disorders (rapid eye movement sleep behavior disorder), psychiatric symptoms (anxiety, depression), cognitive impairment, and gastrointestinal disturbances (constipation, delayed gastric emptying, dysphagia, sialorrhoea) [6].

In general, approximately 10-30 years before the onset of dementia, Aβ peptides begin to accumulate, which occurs in the early stages of AD and is detectable in the basal temporal and medial frontal regions [7]. The early stage of PD is difficult to recognize, and by the time when patients notice the motor symptoms, the disease has usually advanced with a long period. It has been shown that non-motor symptoms, particularly gastrointestinal dysfunction, frequently occur approximately 20 years before the neurodegeneration appear. Considering the high prevalence of gastrointestinal symptoms, it has been considered as the prodromal phase of neurodegenerative diseases [6]. Specifically, the pathology in the gastrointestinal tract shows the similarity with brain. The misfolded α-synuclein and Aβ confer prion-like properties and spread gradually from the enteric nervous system (ENS) to specific brain regions, subsequently seeding the toxic aggregates in recipient neurons [8,9]. More studies are needed to understand the relationship between gastrointestinal symptoms and disease progression, with the aim discovering new biomarkers for diagnosis. Furthermore, in light of the many recent developments in gastrointestinal dysfunction, the gut might be a gateway for the development of an urgently needed disease-modifying therapy.

3. Gut microbiome alterations in neurodegenerative diseases

A healthy human gut harbors a microbiome of 200-400 species and trillions of microbial cells. This gut microbiome (GM) is dynamic, and the microbial composition and the abundances of species are affected by environment, diet, age, feeding mode, application of antibiotics, etc. [10]. Notably, the GMs of AD and PD patients displayed different features from healthy GMs, which might be involved in the pathogenesis of neurodegeneration by regulating gut barrier integrity, neuroinflammation, immune response, and neurotransmitter activity [11,12,13]. The GMs of fecal samples from AD patients showed increased abundances of Ruminococcaceae [13], Enterococcus [14], Streptococcus [15], Alistipes [16], Dorea [15], Collinsella [13,14], and Eggerthella [14], while decreased abundances of Faecalibacterium [14], Lachnospira [13,17], Roseburia [14], and Coprococcus [14]. In addition to AD, more and more evidences showed that the GM dysbiosis was implicated in PD-related pathology. Profiling of the GMs of PD patients reported many alterations compared to healthy controls, including the increased abundances of Alistipes [18], Streptococcus [19], Ruminococcus [20,21], Enterobacter [19,22], Enterococcus [19,23], Verrucomicrobium [20,24], Desulfovibrio [25], and Anaetroncus [26], whereas decreased in Faecalibacterium [19,24], Prevotella [11,20,22,27], Blautia [19,24,28], Lachnospira [24,25], and Roseburia [11,24,28]. Summarizing the current studies indicated that AD and PD shared common GM dynamics. i.e., increased abundances of Streptococcus, Ruminococcaceae, and Alistipes, and decreased abundances of Faecalibacterium, Lachnospira, and Roseburia (Table 1). It was speculated that AD and PD might be correlated with GM-induced inflammation and neurodegeneration. While an increasing number of studies revealed the association of GM dysbiosis with neurodegenerative diseases, further causative studies are still needed to reveal the mechanisms and the potential relevance to clinical manifestations.

4. Microbiota-gut-brain axis

The microbiota-gut-brain axis is well accepted that the GM exerts considerable influence on brain function. The GM communicates with the brain via the activation of vagus nerve, stimulation of enterochromaffin cells, immune system and direct transport of metabolites from the circulation into the brain [29]. In regard to the neuronal pathways for gut-brain connections, vagus nerve is the most direct and well-studied pathway. In autism spectrum disorder (ASD) mice, Lactobacillus reuteri has been reported to rescue social dysfunction in a vagus nerve-dependent manner [30]. It was found that microbial production of indole from tryptophan was more likely to develop host anxiety and depression, because bacterial indole would activate vagal neurons and negatively impact emotional behaviors [31]. The propagation of Aβ and α-synuclein in the gastrointestinal tract were transmitted via vagus nerve to brain [32,33]. Colonic enterochromaffin cells express receptors for various GM-derived metabolites, such as short-chain fatty acids (SCFAs), aromatic amino acids and secondary bile acids [34,35]. Furthermore, enterochromaffin cell production of serotonin has the potential to influence brain function directly or indirectly [36]. For immune-mediated routes, SCFAs interact closely with the immune system through activation of G protein-coupled receptor (GPCR) and inhibition of histone deacetylase (HDAC) activity, leading to the decreased neuroinflammation [37,38]. On the other direction, bacterial endotoxin lipopolysaccharide (LPS) has been shown to aggravate the neuroinflammation via directly entering the brain or activating immune response [39].

5. Linking gut microbiome dysbiosis and neurodegenerative diseases

5.1. Gut microbiome interacts with hosts Subsection

GM and host interaction is an important direction to understand the regulation of health and disease. As previously reported, the GMs from patients with bipolar disorder depression were sufficient to induce depression-like behavior in mice, which was attributed to the elevated expression of tetratricopeptide repeat and ankyrin repeat containing 1 (TRANK1), a robust risk gene of bipolar disorder [40]. Inspiringly, the interplay between GM and host was investigated in PD. Intracellular protein aggregates primarily composed of α-synuclein in Lewy bodies serve as the neuropathological hallmark of PD. α-Synuclein is encoded by SNCA gene, of which mutations lead to the drastic overexpression of α-synuclein and cause Mendelian autosomal dominant PD [41]. Considering an overabundance of opportunistic pathogens in PD, whether these pathogens are triggers of the neurodegeneration is being investigated and there is likely a connection to SNCA variants. Recently, Wallen et al. [42] reported the association of three opportunistic pathogens with PD, which is dependent on SNCA genetic variations. The candidate interacting SNCA genetic variants for Corynebacterium, Porphyromonas, and Prevotella are rs356229, rs10029694, and rs6856813, respectively. Of which, the Porphyromonas interacting genetic variant is also associated with PD risk. These findings indicate that the increased abundance of opportunistic pathogens in PD gut might be modulated by host genotype. In this sense, the genetic susceptibility to disease and the GM dysbiosis are interacted (Figure 1). Nonetheless, the power of single gene in explaining host gene and GM interaction is limited, and the conclusions may be partial and misleading [43]. Further studies to integrate multiple genetic variations in experimental models and humans will be needed to tease out these interactions.

5.2. Gut microbiome-mitochondria connection

As the endosymbiosis hypothesis demonstrated, the mitochondria are descendants of primordial aerobic pleomorphic bacteria (likely Rickettsia), which develops into a mutualistic partnership with ancient anaerobic microbes (likely Archaea) [44]. As a consequence, a stable symbiosis is established to provide energy for the host. Bacterial peptidoglycan muropeptides, the unique component of bacterial cell walls in both Gram-positive and Gram-negative species, accumulate in host intestinal mitochondria, which can maintain the mitochondrial homeostasis and suppress host mitochondrial oxidative stress [45]. Moreover, intestinal infection with Gram-negative bacteria Citrobacter rodentium can trigger mitochondrial antigen presentation and elicit the mitochondria-specific autoreactive CD8⁺ T cells to kill dopaminergic neurons, thereby causing the transient motor dysfunction resembling in PD (Figure 1) [46]. β-N-methylamino-L-alanine (BMAA), a natural neurotoxin produced by cyanobacteria or other microbes, has been shown to be involved in the neurodegeneration. BMAA can cross the blood-brain barrier (BBB), which was previously detected in the brains of patients with neurodegenerative disease [47]. Mechanistically, BMAA elicits mitochondrial dysfunction and AD features in cortical neurons with increased tau phosphorylation and Aβ peptides deposition [48]. Recently, Esteves et al. [49] reported that BMAA triggered a chain of events including mitochondrial dysfunction and innate immunity activation, which recapitulates the PD pathology from the gut to the brain. Although there is still no evidence that gut microbiota can produce BMAA, chronic exposure to environmental microbial neurotoxins is sufficient to induce mitochondrial dysfunction, which has been advanced as a potential cause for the neurodegeneration [50].

5.3. Defective autophagy

Autophagy, in a broad sense, refers to a cellular homeostatic mechanism delivering cytoplasmic constituents to lysosomes for degradation. Initially described as a “self-eating” survival pathway that enables nutrient recycling during starvation, autophagy can also respond to a range of inputs, including microbial products commonly as pathogen-associated molecular patterns (PAMPs) [51]. One of the best-appreciated manifestations of autophagy is to defend against microbial invasion through direct elimination of intracellular pathogens [52]. Autophagy degrades invading pathogens (e.g., Salmonella and Escherichia/Shigella), modulates the release of proinflammatory cytokines, and participates in the antigen presentation. In the intestinal epithelial cells, autophagy enhances tight junction barrier function owing to the reduced permeability of ions and small molecules by lysosomal degradation of claudin-2 [53]. Furthermore, autophagy in colonic epithelial cells has been reported to protect against colitis by the maintenance of antimicrobial peptides and secretion of mucins that act as a mucosal barrier against bacterial invasion [54,55]. The disruption of autophagy in the intestinal epithelium cells induces the alterations in the composition of gut microbiota and reduces α-diversity. In autophagy deficient mice, the abundance of Candidatus Arthromitus and Pasteurellaceae is increased, whereas Akkermansia muciniphila and Lachnospiraceae are found to be reduced (Figure 1) [55]. Indeed, both AD and PD are accompanied by the defective autophagy, leading to the failure of eliminating protein aggregates or damaged mitochondria. Given the impact of autophagy dysfunction in gastrointestinal homeostasis, there is therapeutic interest in activating autophagy to eliminate pathogenic bacteria and the spread of toxic misfolded protein aggregates, thus halting the progression of neurodegenerative diseases.

6. Next-generation of probiotics in neurodegenerative diseases

6.1. Clostridium butyricum

Clostridium butyricum, a butyrate-producing, spore-forming anaerobic bacterium, is found in a wide variety of environments, including soil, milk, and vegetables. C. butyricum was detected in 10-20% of the human gastrointestinal tract, and is one of the earliest colonizers in infants [56]. Traditionally, C. butyricum have been used as a potent probiotic owing to the beneficial effects on host health. Because of the increased butyrate production, C. butyricum is able to enhance the thickness of the mucosal layer and strengthen the gut barrier integrity via increasing the expression of tight junction proteins (e.g., occludin and ZO-1). In addition, C. butyricum plays a protective role in gastrointestinal infections and regulate the host immune system [57]. C. butyricum is effective against Clostoridioides difficile, the causative pathogen of nosocomial infections; Helicobacter pylori, the causative pathogen of gastric cancer; antibiotic-resistant Escherichia coli, as well as Staphylococcus aureus and Vibrio cholerae infection [58,59,60]. It has been shown that C. butyricum can also upregulate protectin D1, an anti-inflammatory lipid metabolite, in colon tissue under antibiotic therapy to alleviate systemic inflammation [61].

C. butyricum exerts neuroprotective effects in various neurodegenerative diseases. In PD mice model, oral administration of C. butyricum can improve motor deficits, dopaminergic neuron loss, synaptic dysfunction and microglia activation. These neuroprotective effects may be related to the increased levels of colonic glucagon-like peptide-1 (GLP-1) and cerebral GLP-1 receptor, eventually restoring gut microbiota homeostasis [62]. Moreover, the anti-depressive effects of C. butyricum in chronic unpredictable mild stress-induced depressive-like behavior may be resulted from the stimulation of intestinal GLP-1 secretion [63]. In AD models, administration of C. butyricum for 4 weeks prevents cognitive impairment, Aβ deposits, and neuroinflammation, which is mediated by the restoration of gut microbiota and butyrate (Figure 2) [64]. In vascular dementia mice, C. butyricum significantly alleviated the cognitive dysfunction and histopathological changes via anti-apoptotic properties and the consequent activation of PI3K/Akt pathway [65]. Treatment with C. butyricum defends against cerebral ischemia/reperfusion injury through anti-oxidant and anti-apoptotic mechanisms, which may be partially attributed to the increased butyrate contents in the brain [66]. Consistently, C. butyricum treatment has been shown to improve the neurological dysfunction and neurodegeneration in a mouse model of traumatic brain injury [67]. Although the neuroprotective effects of probiotic C. butyricum appears well-established, additional human randomized controlled trials would further provide valuable clinical data related to the strains’ utility as an intervention in the neurodegenerative diseases.

6.2. Akkermansia muciniphila

Akkermansia muciniphila, a gram-negative, anaerobic bacterium first identified in 2004 [68], is considered as a promising candidate as a “next-generation probiotics” [69]. The benefits of A. muciniphila are not limited to protecting the mucosal barrier integrity, improving the host metabolic functions and immune responses, A. muciniphila also possesses a value in modulating brain function. In this case, the critical role of A. muciniphila has been demonstrated in neurodegenerative diseases. Several studies have consistently reported that Akkermansia is highly effective in distinguishing PD or serves as a potential early biomarker for PD diagnosis [24,27,70]. It is noteworthy that the abundance of A. muciniphila is also found to be increased in PD patients [71]. One of the possible explanations is that the compensation of increased A. muciniphila in PD aims to fight and prevent disease progression. Although treatment with A. muciniphila has been reported to improve cognitive deficits and reduce Aβ levels in AD mice model [72], whether A. muciniphila can alleviate the neurodegeneration in PD patients remains unknown (Figure 2). There are also opposite results demonstrating that A. muciniphila-conditioned medium can initiate α-synuclein aggregation in enteroendocrine cells [73]. To date, few studies have explored the direct impact of A. muciniphila on the nervous system, the likelihood of beneficial effects exerted by A. muciniphila should be addressed. Further characterization of the relevance for the neurodegeneration will be fundamental to unveil the consequences of A. muciniphila dysbiosis.

6.3. Faecalibacterium prausnitzii

Faecalibacterium prausnitzii, an anaerobic Gram-positive bacterium, belongs to the Firmicutes phylum and the Ruminococcaceae family, also known as Clostridium cluster IV. The interest in F. prausnitzii is related to its capacity to produce beneficial metabolites, such as fructose, formic acid, and d-lactate and it is the most important butyrate-producing bacteria [74]. In addition, a 15 kDa protein with anti-inflammatory properties produced by F. prausnitzii can inhibit the nuclear factor-κB (NF-κB) pathway in intestinal epithelial cells and prevent colitis in animal models [75]. Consistently, several other studies in mice also clarify a protective role of F. prausnitzii in experimentally induced colitis. Intragastric administration of either F. prausnitzii or its culture supernatant can significantly decrease the severity of colitis by down-regulating pro-inflammatory cytokines [76]. Butyrate produced by F. prausnitzii modulates Th17/Treg balance and exerts anti-inflammatory effects in colorectal colitis rat model [77]. In terms of neurodegenerative diseases, the abundance of F. prausnitzii is decreased in the mild cognitive impairment (MCI) group compared with the healthy control, which is correlates with cognitive scores [78]. Two isolated F. prausnitzii strains from the healthy group have been shown to improve cognitive impairment in AD mice model [78]. So far, studies on the potential effects of F. prausnitzii on PD have not been reported (Figure 2). Additional researches are needed further prove the beneficial role of F. prausnitzii in the remediation of neurodegenerative diseases.

6.4. Bacteroides fragilis

Bacteroides fragilis is another promising probiotic, which is a commensal, Gram negative, non-spore forming obligatory anaerobic bacterium abundant in the human gastrointestinal tract. Traditionally, B. fragilis can interfere with other microbes via inhibiting their growth or translocation. As previously reported, B. fragilis treatment prevents Clostrioides difficile infection possibly by resisting pathogen colonization, enhancing the relative abundance of A. muciniphila and improving gut barrier integrity [79]. Indeed, B. fragilis can be classified into two subgroups: non-enterotoxigenic and enterotoxigenic B. fragilis [80]. Enterotoxin-containing B. fragilis secretes an unusually complex mixture of neurotoxins including the pro-inflammatory LPS. In this sense, enterotoxigenic B. fragilis exposure to human primary brain cells is an exceptionally potent inducer of the inflammatory pathway, driving pro-inflammatory degenerative neuropathology in the AD brain [81]. Conversely, non-enterotoxigenic B. fragilis strains exert the beneficial effects owing to an anti-inflammatory and immunomodulatory activity. Oral administration of B. fragilis has been found to increase gut microbiota diversity and beneficial commensal bacteria, thereby improving gut tight junction integrity and reducing inflammatory cytokines [82]. The fecal bacterial assessment by 16S rRNA amplicon sequencing shows that the abundance of B. fragilis is lower in PD patients than that of healthy control [83]. Considering that B. fragilis is one of the main hydrogen-producing intestinal bacteria, the decreased B. fragilis may be accounted for the lower amount of intestinal hydrogen level in PD (Figure 2) [84]. Although B. fragilis are potentially interesting as the next-generation probiotic, the role of B. fragilis in relation to the neuropathology is contrasting, thus the specific strains should be carefully evaluated for the safety and efficacy in the neurodegenerative diseases.

7. Perspectives and conclusions

Mounting information presents compelling evidence that gastrointestinal dysfunction serves as a prodromal symptom preceding the clinical manifestations of neurodegenerative diseases. Lack of understanding the mechanisms and pathophysiology of gastrointestinal manifestations hampers the diagnosis and clinical treatment. The GM, gastrointestinal tract and brain have historically been studied independently, but a growing appreciation for their interaction via gut-brain axis advocates the idea of targeting GM as a strategy for clinical intervention. Currently, there is a growing interest toward next-generation probiotics as the potential therapeutic agents by altering GM. Therefore, the safety and tolerability of these novel probiotics need to be validated in both animal models and human trials, so as to develop a personalized application. Another challenge is the storage due to the strict anaerobic conditions during microbial collection and freeze-drying. Even so, the current state of the next-generation probiotics remains largely promising in the context of neurodegenerative diseases, for the purpose of slowing down or preventing the neurodegeneration, as well as developing effective therapeutic interventions.